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Type III secretion system translocator has a molten globule conformation both in its free and chaperone-bound forms
The FEBS Journal
Abstract
Type III secretion systems of Gram-negative pathogenic bacteria allow the injection of effector proteins into the cytosol of host eukaryotic cells. Crossing of the eukaryotic plasma membrane is facilitated by a translocon, an oligomeric structure made up of two bacterial proteins inserted into the host membrane during infection. In Pseudomonas aeruginosa, a major human opportunistic pathogen, these proteins are PopB and PopD. Their interactions with their common chaperone PcrH in the cytosol of the bacteria are essential for the proper function of the injection system. The interaction region between PopD and PcrH was identified using limited proteolysis, revealing that the putative PopD transmembrane fragment is buried within the PopD/PcrH complex. In addition, structural features of PopD and PcrH, either individually or within the binary complex, were characterized using spectroscopic methods and 1D NMR. Whereas PcrH possesses the characteristics of a folded protein, PopD is in a molten globule state either alone or in the PopD/PcrH complex. The molten globule state is known to enable the membrane insertion of translocation/pore-forming domains of bacterial toxins. Therefore, within the bacterial cytoplasm, PopD is preserved in a state that is favorable to secretion and insertion into cell membranes.
... One half of the translocon is embedded in the host membrane while the other half is protruding in the host cytoplasm ( Figure 18) (Park et al., 2018). Inside bacteria, SctB and SctE interact with their cognate chaperone responsible for their stability and prevention of self-oligomerization (Discola et al., 2014;Faudry et al., 2007). Unfortunately, unlike the hydrophilic tip complex SctA, the uncovering of the exact structure of the pore is still challenging because the hydrophobic structure makes it unstable in vitro, leading to rapid aggregation or degradation. ...
... On the other hand, no structure of the 1TD translocator SctB is available. The N-terminus of SctB is predicted to present a coiled-coil structure and was shown to be involved in the interaction with the cytoplasmic chaperone (Faudry et al., 2007;Job et al., 2010;Schreiner and Niemann, 2012). SctB contains only one transmembrane helix and unlike the 2TD translocator, the 1TD translocator could be detached from liposome, suggesting that this protein superficially binds to bilayer lipid membrane (Discola et al., 2014). ...
... In similar experiments with the translocator chaperone PcrH bound or not to its cargos PopB and PopD, the Kds of the ATPase and the complex PcrH-PopB, 4.9 µM, or PcrH-PopD, 4.8 µM, were 6 fold-lower than the one of PscN and the chaperone PcrH alone (Kd = 31µM) ( Figure 3C). Unfortunately, it is not possible to assess the Kd of PscN and PopB or PopD alone because these proteins are not stable in vitro without their cognate chaperone PcrH (Faudry et al., 2007;Wager et al., 2013). Nevertheless, taken together these results indicate that PscN would rather bind to cargo or complex proteins than to the chaperone protein alone. ...
Characterization and inhibition of Pseudomonas aeruginosa Type III Secretion SystemPseudomonas aeruginosa is a Gram-negative opportunistic pathogen that causes nosocomial diseases and infects cystic fibrosis patients. The Type III Secretion System (T3SS) is one of its most important virulence factors, allowing the direct injection of four exotoxins into the target eukaryotic cells. An important protein of T3SS is the conserved ATPase, named PscN that is involved in the assembly and functioning of this system. In this work, we demonstrated the interaction of PscN with T3SS secreted cargo proteins and with their chaperons in complex or alone using ELISA, HTRF and MST assays. Of important, MST (Microscale Thermophoresis) allowed us to determine the dissociation constants (Kd) of these proteins and PscN, showing the interaction preference of this enzyme for the cargo or complex proteins rather than for the corresponding chaperons alone. This confirm the hypothesis that the chaperons are released in the bacterial cytoplasm after the complex dissociation. Otherwise, we assess the Kd between the effector, translocator or needle complexes and PscN bound or not to the gate-keeper complex which is described as a regulator of substrate sorting for the secretion. The results showed that the binding of the gate-keeper to PscN dramatically increases its relative affinity for the needle complex, thus revealing a new role of the gate-keeper in the loading of the needle complex to the ATPase for the control of substrate hierarchical secretion in P. aeruginosa.In parallel, committed to the anti-virulence strategy, we take the opportunity to characterize the ex vivo and in vivo effects of compounds identified by a previous in vitro screening to inhibit the interaction of PscE and PscG. These proteins are the two cognate chaperons of the T3SS needle protein PscF in the bacterial cytoplasm. This interaction had been shown to be a valid anti-virulence target because single or double point mutations introduced within the binding site between PscE and PscG lead to a decrease of P. aeruginosa virulence. This work points out two best leads which belong to the structural hybrid cluster combining hits from two different chemical libraries. The two compounds inhibit the cell damages caused by T3SS positive P. aeruginosa strains, are non-toxic for eukaryotic cell and have minimal effect on bacterial fitness. They were also shown to be specific for T3SS and could protect Galleria mellonnela against P. aeruginosa infection.Key words: Pseudomonas aeruginosa, Type III Secretion System, ATPase, Anti-virulence
... Les translocateurs PopB/YopB et PopD/YopD sont des protéines avec, respectivement, deux et une région transmembranaire putative(Francis et al., 2001 ;Faudry et al., 2007). L'association entre PopD et PcrH se fait au niveau du domaine N terminal de PopD et une partie de son domaine hydrophobe conduisant à l'enfouissement de ce dernier dans la chaperone(Faudry et al., 2007). ...
... Les translocateurs PopB/YopB et PopD/YopD sont des protéines avec, respectivement, deux et une région transmembranaire putative(Francis et al., 2001 ;Faudry et al., 2007). L'association entre PopD et PcrH se fait au niveau du domaine N terminal de PopD et une partie de son domaine hydrophobe conduisant à l'enfouissement de ce dernier dans la chaperone(Faudry et al., 2007). Ces interactions sont donc nécessaires à la stabilisation des translocateurs hydrophobes qui sont maintenus sous forme monomérique. ...
... Ces interactions sont donc nécessaires à la stabilisation des translocateurs hydrophobes qui sont maintenus sous forme monomérique. De plus, l'interaction PcrH-PopD maintient la protéine PopD dans une conformation particulière partiellement dépliée(Faudry et al., 2007) ce qui pourrait augmenter l'efficacité de sa sécrétion. La conservation entre les opérons lcrVGHyopBD et pcrVGHpopBD est si importante que PcrH et LcrH sont fonctionnellement interchangeables. ...
Pseudomonas aeruginosa can cause severe infections in immunocompromised patients, and Yersinia pestis is the causative agent of plague. During acute infections both bacteria rely on numerous virulence factors including a common type III secretion system (T3SS). T3SS is composed of a basal body anchored in the bacterial bilayer membrane and a hollow needle assembled at the cell surface. The effector molecules are directly injected into the target cells via a translocation pore inserted into the host cell membrane. The aim of this study is to determine the interactome of the protective V antigens PcrV in P. aeruginosa and LcrV in Y. pestis. Both are localised at the tip of type III needles being essential for the translocation process. Biochemical studies in vitro showed that V proteins are able to form oligomeric ring like structures which were characterised by size exclusion chromatography, native gel, native mass spectrometry and TEM. Moreover, the multimerization depends on the hydrophobic residues within the α12 C terminal helix. The fact that the strains expressing the mutant proteins incapable to oligomerize are non-cytotoxic toward macrophages supports the idea that oligomerization is required for the proper function of the V antigens.
Next, direct interaction between PcrV and the needle forming subunit, PscF, was demonstrated by co-purification in vitro. Moreover, two PscF mutants exhibiting a translocation defect were shown as being incapable to form a stable translocation complex. Last, the fact that the C terminal α helix of PscF can be exchanged by the C terminal α helix of PcrV resulting in an hybrid protein that can polymerise, suggests that this helix may be necessary for the complex formation. Taken together, these studies show that the assembly of the multimeric V proteins as well as their tip location are essential for their functions with the α12 C terminal helix of PcrV playing a major role. These conclusions could be of great importance for the future development of new vaccines and antimicrobials.
... Although extensive proteolysis was also observed for a PopB sample trypsinized in the absence of liposomes (not shown), as expected for a molten globule protein (46), for the PopB sample in the lipid-bound form proteolysis was restricted to the N-terminal region (Fig. 2, B and D). After trypsin treatment, most of the protein still ran on SDS-PAGE with a molecular weight similar to that of the non-digested sample (Fig. 2B); N-terminal sequencing of the five minor bands in the sample treated with trypsin for 2 h revealed the same sequence for all of them (ESEEAAKE). ...
... Notably, molten globule forms are folded proteins with extensive native-like secondary structures but that lack tertiary structures and are often characterized by spectra with extremely broad peaks with poor chemical shift dispersions. Thus, this is in agreement with studies that suggested that Pop proteins are molten globules (46), which could be a require-FIGURE 3. CCF2 and nitrocefin assays used to determine the orientation of the C terminus of PopB on membranes. PopB could potentially be inserted in host cell membranes with its N and C termini pointing toward the inside (panel A) or the outside (panel B) of the infected cell. ...
... The N Terminus of PopB Binds to the Concave Region of PcrH-After having mapped the architecture of PopB on bilayers, we thus set out to investigate its cytoplasmic form; that is, in a complex with its chaperone PcrH. We had previously shown through limited proteolysis that the 95 N-terminal residues of the minor translocator PopD could associate to PcrH with 1:1 stoichiometry (26,46). By solving the high resolution crystal structure of PcrH, we had also shown that it folds into seven TPR motifs that generate a concave region that is capable of binding PopD residues 49 -56 (8). ...
The Type III secretion system is a widespread apparatus used by pathogenic bacteria to inject effectors directly into the cytoplasm of eukaryotic cells. A key component of this highly conserved system is the translocon, a pore formed in the host membrane that is essential for toxins to bypass this last physical barrier. In Pseudomonas aeruginosa the translocon is composed of PopB and PopD, both of which prior to secretion are stabilized within the bacterial cytoplasm by a common chaperone, PcrH. In this work, we characterize PopB, the major translocator, in both membrane-associated and PcrH-bound forms. By combining sucrose gradient centrifugation experiments, limited proteolysis, 1D NMR, and β-lactamase reporter assays on eukaryotic cells, we show that PopB is stably inserted into bilayers with its flexible N-terminal domain and C-terminal tail exposed to the outside. In addition, we also report the crystal structure of the complex between PcrH and an N-terminal region of PopB (residues 51-59), which reveals that PopB lies within the concave face of PcrH, employing mostly backbone residues for contact. PcrH is thus the first chaperone whose structure has been solved in complex with both T3SS translocators, revealing that both molecules employ the same surface for binding and excluding the possibility of formation of a ternary complex. The characterization of the major T3SS translocon component in both membrane-bound and chaperone-bound forms is a key step for the eventual development of antibacterials that block translocon assembly.
... The secretion defect of the PopD(A290K) mutant furthermore suggests that this interaction is important for C-TES function. This result was somewhat surprising, given the fact that prior analysis of the PopD-PcrH interaction by pull-downs and limited proteolysis had been unable to detect an interaction between the C-terminus of PopD and PcrH (Faudry et al., 2007). However, we were similarly unable to detect an interaction between the C-terminal half of PopD alone and PcrH by two-hybrid analysis (Fig. S1A), and none of our TES mutants interfered with the PcrH-PopD interaction when assayed in our two-hybrid system (Fig. S1B), indicating that the interaction is weak. ...
... Indeed, residues adjacent to the SycE binding site of the Y. pseudotuberculosis effector YopE were recently shown to be important for directing this effector for export via the T3SS (Rodgers et al., 2010). While the chaperone binding domain of PopD was initially determined to lie between residues 28-95 by limited proteolysis (Faudry et al., 2007), subsequent crystallographic analysis of the interaction demonstrated that the core chaperone binding domain of PopD resides at residues 49-54 (Job et al., 2010). To probe whether residues flanking the chaperone binding site of PopD are important for export of the translocator, we assayed the export of a series of amino-terminally truncated PopD proteins fused to ExoS(1-51) akin to the fusion proteins assayed in the preceding section. ...
... At the time, these deletions were not, however, linked to establishing the translocator-effector secretion hierarchy. Previous attempts to demonstrate an interaction of the C-terminus of PopD with PcrH by pull-down were unsuccessful (Faudry et al., 2007). As noted above, we were similarly not able to detect an interaction of the C-terminus of PopD with PcrH by two-hybrid analysis and none of the TES mutants diminished the PopD-PcrH interaction in our two-hybrid system. ...
Type III secretion systems are used by many Gram-negative pathogens to directly deliver effector proteins into the cytoplasm of host cells. To accomplish this, bacteria secrete translocator proteins that form a pore in the host-cell membrane through which the effector proteins are then introduced into the host cell. Evidence from multiple systems indicates that the pore-forming translocator proteins are exported before effectors, but how this secretion hierarchy is established is unclear. Here we used the Pseudomonas aeruginosa translocator protein PopD as a model to identify its export signals. The N-terminal secretion signal and chaperone, PcrH, are required for export under all conditions. Two novel signals in PopD, one proximal to the chaperone binding site and one at the very C-terminus of the protein, are required for export of PopD before effector proteins. These novel export signals establish the translocator-effector secretion hierarchy, which in turn, is critical for the delivery of effectors into host cells.
... YopB and YopD could not be expressed and purified recombinantly. PopB and PopD showed bactericidal effect and localized in the inclusion bodies, when recombinantly expressed in E. coli [20,26,35,46]. Induction of IpaB could be detected by western blot but the protein could not be recovered in the soluble fraction. ...
... By chemical crosslinking, we have detected YspC-SycB complex with a stoichiometry of 1:1 (Fig. 4g). Similar translocator protein complexes like PopD-PcrH of P. aeruginosa, IpaC-IpgC of S. flexneri also formed 1:1 stochiometric, monomeric complexes [3,20,46]. PopB-PcrH complex of P. aeruginosa and AopB-AcrH complex of Aeromonas hydrophila showed two different forms during SEC analysis, corresponding to a oligomeric complex and a monomeric 1:1 complex, like YspC-SycB [20,46,47]. YspC and DSycB was found to interact, however, the yield was low and needed further optimization (data to be published later). ...
... Similar translocator protein complexes like PopD-PcrH of P. aeruginosa, IpaC-IpgC of S. flexneri also formed 1:1 stochiometric, monomeric complexes [3,20,46]. PopB-PcrH complex of P. aeruginosa and AopB-AcrH complex of Aeromonas hydrophila showed two different forms during SEC analysis, corresponding to a oligomeric complex and a monomeric 1:1 complex, like YspC-SycB [20,46,47]. YspC and DSycB was found to interact, however, the yield was low and needed further optimization (data to be published later). ...
In Yersinia enterocolitica biovar 1B, a genome encoded TTSS designated as Ysa-Ysp system is used for virulence. SycB is an annotated chaperone to this system. SycB is soluble in presence of translocator YspC. SycB and its truncated form (∆SycB((1-114))) exist as dimers. YspC forms a 1:1 complex with SycB. Homology model of SycB shows a flexible N-terminal may be required for solubility and dimerization; and concave core formed by antiparallel helices of TPRs. Far UV CD spectra confirm that SycB is predominantly alpha helical. Near UV CD spectra show that SycB has tertiary structure at pH 7.2 (native folded protein), which disappears at pH 5 (molten globule) and SycB releases YspC at pH 5. SycB has a cooperative melting behavior. At pH 7.2, SycB shows solvent accessible hydrophobic patches. Concave core in the model exhibits ANS binding within FRET distance of tyrosines in the TPR, allowing a range of interaction of SycB with its ligand.
... The four 57-bp primers were designed to introduce, with a single PCR, either three mutations E50R, N52A, and R55E (PopD-RAE mutant), or four mutations V49D, L51D, A53D, and P54D (PopD-4D mutant). The same primers were used with both vectors used in this work: pETDuet-6His-pcrH-popD (27) used for in vitro assays, and pIApG-pcrHpopBD (20) used for all the in vivo experiments. The presence of mutations was confirmed by DNA sequencing. ...
... PcrH Palm Recognizes a Specific PopD Sequence-Interaction between PcrH and minor translocator PopD generates a 1:1 complex, thus blocking PcrH in monomeric form (Fig. 1B), but no structural information regarding this type of complex was available. Our earlier study of the PopD-PcrH complex had identified that PopD displays molten globule characteristics both in the absence and presence of PcrH, and limited proteolysis of the PopD: PcrH 1:1 complex revealed that PopD residues 28 -95 were able to associate with PcrH and still maintain a 1:1 stoichiometry (27). Subsequently, Lunelli et al. (17) identified a short region in the Shigella major translocator protein IpaB ( 65 PELKAP 70 ) that specifically recognizes its type II chaperone, IpgC. ...
... Subsequently, Lunelli et al. (17) identified a short region in the Shigella major translocator protein IpaB ( 65 PELKAP 70 ) that specifically recognizes its type II chaperone, IpgC. By analyzing sequences of minor translocator proteins from T3SS of the Ysc family (8) and employing the IpaB peptide as a comparison criterium, we identified a potentially minimal PopD-PcrH interacting region (PopD residues 49 -54, VELNAP) that maps to the PopD region identified by proteolysis (Fig. 3A) (27). ...
The type III secretion system (T3SS) is a complex nanomachine employed by many Gram-negative pathogens, including the nosocomial
agent Pseudomonas aeruginosa, to inject toxins directly into the cytoplasm of eukaryotic cells. A key component of all T3SS is the translocon, a proteinaceous
channel that is inserted into the target membrane, which allows passage of toxins into target cells. In most bacterial species,
two distinct membrane proteins (the “translocators”) are involved in translocon formation, whereas in the bacterial cytoplasm,
however, they remain associated to a common chaperone. To date, the strategy employed by a single chaperone to recognize two
distinct translocators is unknown. Here, we report the crystal structure of a complex between the Pseudomonas translocator chaperone PcrH and a short region from the minor translocator PopD. PcrH displays a 7-helical tetratricopeptide
repeat fold that harbors the PopD peptide within its concave region, originally believed to be involved in recognition of
the major translocator, PopB. Point mutations introduced into the PcrH-interacting region of PopD impede translocator-chaperone
recognition in vitro and lead to impairment of bacterial cytotoxicity toward macrophages in vivo. These results indicate that T3SS translocator chaperones form binary complexes with their partner molecules, and the stability
of their interaction regions must be strictly maintained to guarantee bacterial infectivity. The PcrH-PopD complex displays
homologs among a number of pathogenic strains and could represent a novel, potential target for antibiotic development.
... Les translocateurs PopB/YopB et PopD/YopD sont des protéines avec, respectivement, deux et une région transmembranaire putative(Francis et al., 2001 ;Faudry et al., 2007). L'association entre PopD et PcrH se fait au niveau du domaine N terminal de PopD et une partie de son domaine hydrophobe conduisant à l'enfouissement de ce dernier dans la chaperone(Faudry et al., 2007). ...
... Les translocateurs PopB/YopB et PopD/YopD sont des protéines avec, respectivement, deux et une région transmembranaire putative(Francis et al., 2001 ;Faudry et al., 2007). L'association entre PopD et PcrH se fait au niveau du domaine N terminal de PopD et une partie de son domaine hydrophobe conduisant à l'enfouissement de ce dernier dans la chaperone(Faudry et al., 2007). Ces interactions sont donc nécessaires à la stabilisation des translocateurs hydrophobes qui sont maintenus sous forme monomérique. ...
... Ces interactions sont donc nécessaires à la stabilisation des translocateurs hydrophobes qui sont maintenus sous forme monomérique. De plus, l'interaction PcrH-PopD maintient la protéine PopD dans une conformation particulière partiellement dépliée(Faudry et al., 2007) ce qui pourrait augmenter l'efficacité de sa sécrétion. La conservation entre les opérons lcrVGHyopBD et pcrVGHpopBD est si importante que PcrH et LcrH sont fonctionnellement interchangeables. ...
Pseudomonas aeruginosa et Yersinia pestis sont responsables d'infections graves chez les individus immunodéprimés et de la peste, respectivement. Leur pathogénicité repose sur de nombreux facteurs de virulence dont le système de sécrétion de type III (SST3) qui a une action prépondérante lors d'infections aiguës. Le SST3 est composé d'une base ancrée dans la double membrane bactérienne, d'une aiguille creuse érigée à la surface et d'un pore de translocation inséré dans la membrane de la cellule hôte permettant à la bactérie d'y injecter des toxines. L'objet de cette thèse est l'étude de l'interactome de l'antigène protecteur V, PcrV chez P. aeruginosa et LcrV chez Y. pestis. Celui-ci est situé au sommet de l'aiguille et est nécessaire au processus de translocation des toxines. L'étude des propriétés biochimiques de la protéine in vitro nous a permis de mettre en évidence sa capacité à former des oligomères présentant une structure en forme d'anneaux. Les multimères ont été observés par chromatographie d'exclusion de taille, gel natif, spectrométrie de masse native et MET. Leur formation est dépendante de la présence de l'hélice α12 C terminale de PcrV et de l'intégrité de ses résidus hydrophobes. Le processus d'assemblage de la protéine est nécessaire à sa fonction in vivo : des mutants qui sont incapables d'oligomériser perdent leur cytotoxicité envers les cellules eucaryotes.
Puis, l'interaction directe entre PcrV et la sous unité formant l'aiguille, PscF, a été mise en évidence in vitro par co-purification. De plus, deux mutants ponctuels de PscF dont le phénotype présente un défaut de translocation se sont montrés défectueux pour la liaison avec PcrV. Enfin, l'hélice C terminale de PscF est échangeable avec l'hélice α12 C terminale de PcrV comme l'atteste la capacité de polymérisation d'un hybride créé entre ces deux protéines, suggérant un rôle de celle-ci dans la formation du complexe F-V. L'ensemble de ces études montre que l'assemblage multimérique des antigènes V ainsi que leur position au sommet de l'aiguille sont des éléments essentiels à leur fonction, avec un rôle prépondérant de l'hélice α12 C terminale de PcrV. Ces conclusions pourraient permettre de mieux cibler les développements futurs de nouveaux vaccins ou agents antimicrobiens.
... This transition is facilitated by the structural flexibility of SctB and SctE, shifting from a molten globule conformation in an aqueous solution to a ring-like structure composed of 6-8 subunits of each protein, embedded in the host membrane. 6,[25][26][27] At present, the sequence of events that enable SctB and SctE to shift from the soluble state into the oligomeric membrane-embedded confirmation is unclear. ...
... EspB wt -His is monomeric under soluble and membrane-simulating conditions The T3SS translocon complex was shown to comprise multiple subunits of SctB and SctE proteins. 8,25,27,[30][31][32] To investigate whether the EPEC SctB protein (EspB) oligomerizes on its own, we expressed EspB labeled with a His tag at its C-terminus (EspB wt -His) in a EPEC DespB strain, grew the transformed strain under T3SS-induction conditions, and purified the protein from the culture medium ( Figure 1A). The purified protein was then analyzed by size-exclusion chromatography (SEC) to determine its ability to form oligomers. ...
Many bacterial pathogens employ the type III secretion system (T3SS), a specialized complex that transports effector proteins that manipulate various cellular processes. The T3SS forms a translocon pore within the host-cell membrane consisting of two secreted proteins that transition from a soluble state into a transmembrane complex. Still, the exact sequence of events leading to the formation of a membranous functional pore remains uncertain. Here, we utilized the translocon proteins of enteropathogenic E. coli (EPEC) to investigate the sequence of those steps leading to translocon assembly, including self-oligomerization, hetero-oligomerization, interprotein interaction, and membrane insertion. We found that in EPEC, EspD (SctE) plays a dominant role in pore formation as it assembles into an oligomeric state, regardless of pH, membrane contact, or the presence of EspB (SctB). Subsequently, EspB subunits integrate into EspD homo-oligomers to create EspB-EspD hetero-oligomers that adopt a transmembrane orientation to create a functional pore complex.
... The translocators (typically two different secreted proteins) are mostly α-helical but do not adopt a stable water-soluble monomeric state. Individually they adopt a molten globular conformation and have a tendency to aggregate in solution (Faudry et al., 2007). In the cytosol of the bacterium, they exist as a complex with a cognate chaperone (Discola et al., 2014). ...
... A typical example of these proteins is the T3S system translocators (Faudry et al., 2007). These proteins need to be co-expressed with their cognate chaperone, and can only be purified as a complex with the chaperone (Faudry et al., 2006;Romano et al., 2011). ...
PFPs (Pore-forming proteins) perforate cellular membranes to create an aqueous pore and allow the passage of ions and polar molecules. The molecular mechanisms for many of these PFPs have been elucidated by combining high resolution structural information of these proteins with biochemical and biophysical approaches. However, some PFPs do not adopt stable conformations and are difficult to study in vitro. An example of these proteins are the bacterial Type 3 Secretion (T3S) translocators. The translocators are secreted by the bacterium and insert into the target cell membrane to form a translocon pore providing a portal for the passage of T3S toxins into eukaryotic cells. Given the important role that the T3S systems play in pathogenesis, methods to study these translocon pores in cellular membranes are needed. Using a combination of protein modifications and methods to selectively permeate and solubilized eukaryotic membranes, we have established an experimental procedure to analyze the topology of the Pseudomonas aeruginosa T3S translocon using P. aeruginosa strain variants and HeLa cell lines.
... https://doi.org/10. 1101/2020 Four classes of T3SS complexes were selected: i) the effector ExoU and its chaperone SpcU whose crystallographic structure was solved (Gendrin et al., 2012), ii) the translocators PopB and PopD and their cognate chaperone PcrH that have been extensively characterized (Discola et al., 2014;Faudry et al., 2007;Goure et al., 2004;Job et al., 2010); iii) the PscF (SctF) needle subunit that associates with the co-chaperones PscE and PscG (Ple et al., 2010;Quinaud et al., 2005Quinaud et al., , 2007; iv) The gate-keeper complex is composed of the gate-keeper PopN (SctW) associated with the co-regulator Pcr1 (TyeA in Yersinia spp.) and the two chaperones Pcr2 and PscB Yang et al., 2007). MS-based proteomic characterization of the proteins co-purified with 6His-tagged PopN indicated that it was effectively associated with its three partners (Table 2). ...
... In similar experiments with the translocator chaperone PcrH bound or not to its cargos PopB and PopD, the KDs between the ATPase and the complex PcrH-PopB, 4.9 µM, or PcrH-PopD, 4.8 µM, were 6 foldlower than the one between PscN and the chaperone PcrH alone (KD = 31µM) ( Figure 3C). Unfortunately, it is not possible to assess the KD between PscN and PopB or PopD alone because these proteins are not stable in vitro without their cognate chaperone PcrH (Faudry et al., 2007;Wager et al., 2013). Nevertheless, taken together these results indicate that PscN would rather bind to cargo or complex proteins than to the chaperone protein alone. ...
Pseudomonas aeruginosa is an opportunistic bacterium of which the main virulence factor is the Type III Secretion System. The ATPase of this machinery, PscN (SctN), is thought to be localized at the base of the secretion apparatus and to participate in the recognition, chaperone dissociation and unfolding of exported T3SS proteins. In this work, a protein-protein interaction ELISA revealed the interaction of PscN with a wide range of exported T3SS proteins including the needle, translocator, gate-keeper and effector. These interactions were further confirmed by Microscale Thermophoresis that also indicated a preferential interaction of PscN with secreted proteins or protein-chaperone complex rather than with chaperones alone, in line with the release of the chaperones in the bacterial cytoplasm after the dissociation from their exported proteins. Moreover, we suggest a new role of the gate-keeper complex and the ATPase in the regulation of early substrates recognition by the T3SS. This finding sheds a new light on the mechanism of secretion switching from early to middle substrates in P. aeruginosa .
Highlights
T3SS substrates are secreted sequentially but information on the switches are missing
Interaction of the T3SS ATPase with secreted proteins were investigated by different approaches
Microscale Thermophoresis revealed a lower affinity for chaperones alone compared to complexes
The Gate-keeper complex binds to the ATPase and increases its affinity for the needle complex
A new role of the Gate-keeper complex is proposed, directly acting on the T3SS ATPase
... The translocators adopt a molten globular state in aqueous solvents and they contain one or more hydrophobic segments in their primary structure; therefore, it is not unexpected that they are prone to form aggregates when not associated with membranes (Faudry et al. 2007). In the bacterial cytosol, these two proteins are kept in a secretion-competent state by association with a single cognate chaperone. ...
... Since the translocators are secreted through a protein-based needle in an unfolded conformation, later studies used denaturants such as guanidinium chloride or urea to replicate in vitro the initial unfolded state of these proteins (De Geyter et al. 1997;Dey et al. 2012;Faudry et al. 2007;Romano et al. 2011;Wager et al. 2015). These denaturing/refolding protocols have been optimized to allow a more efficient insertion of translocators into model membranes and subsequent biophysical characterization of the membrane-assembled complexes (Romano et al. 2011(Romano et al. , 2016. ...
The type 3 secretion (T3S) system is a syringe-like proteinaceous apparatus used by several Gram-negative bacteria to inject toxic/effector proteins into eukaryotic cells. Three proteins are essential for protein translocation into the host cell. One of these proteins forms a tip complex at the end of a needle that extends from the bacterial surface and plays a role in regulating secretion in response to host cell contact. The other two proteins, or translocators, insert into the target membrane and form a pore through which proteins are injected. Compared to other components of the T3S machinery, the needle tip and translocators show low levels of sequence identity among different T3S families, which suggests that these essential components adapted to the specific needs of the bacteria that use these T3S systems.
... The translocators adopt a molten globular state in aqueous solvents and they contain one or more hydrophobic segments in their primary structure, therefore it is not unexpected that they are prone to form aggregates when not associated with membranes (Faudry et al. 2007). In the bacterial cytosol, these two proteins are kept in a secretion-competent state by association with a single cognate chaperone. ...
... Since the translocators are secreted through a protein-based needle in an unfolded conformation, later studies used denaturants such as guanidinium chloride or urea to replicate in vitro the initial unfolded state of these proteins (De Geyter et al. 1997;Dey et al. 2012;Faudry et al. 2007;Romano et al. 2011;Wager et al. 2015). These denaturing/refolding protocols have been optimized to allow a more efficient insertion of translocators into model membranes and subsequent biophysical characterization of the membrane-assembled complexes (Romano et al. 2011;Romano et al. 2016). ...
The type 3 secretion (T3S) system is a syringe-like proteinaceous apparatus used by several Gram-negative bacteria to inject toxic/effector proteins into eukaryotic cells. Three proteins are essential for protein translocation into the host cell. One of these proteins forms a tip complex at the end of a needle that extends from the bacterial surface and plays a role in regulating secretion in response to host cell contact. The other two proteins, or translocators, insert into the target membrane and form a pore through which proteins are injected. Compared to other components of the T3S machinery, the needle tip and translocators show low levels of sequence identity among different T3S families, which suggests that these essential components adapted to the specific needs of the bacteria that use these T3S systems.
... This relaxed form may facilitate membrane insertion (38). The exposure of hydrophobic surfaces by molten globular states at neutral pH favor protein aggregation in solution, (as in the case of PopB and PopD 12,13,39). However, the increase of the net positive charge of the protein at low pH would increase the electrostatic repulsion between proteins monomers and favor the electrostatic interaction with negatively charged membranes. ...
... The T3S translocators do not belong to any of these groups. They are secreted through the needle in an unfolded conformation (61), they do not adopt a well define folded structure in aqueous solution (39,62), and no proteinaceous machinery has been identified for insertion and assembly into the target membrane. Therefore, the spontaneous assembly of unfolded proteins into large heterocomplexes with a defined stoichiometry constitutes a novel paradigm among membrane inserted proteins. ...
A type 3 secretion system is used by many bacterial pathogens to inject proteins into eukaryotic cells. Pathogens insert a
translocon complex into the target eukaryotic membrane by secreting two proteins known as translocators. How these translocators
form a translocon in the lipid bilayer and why both proteins are required remains elusive. P. aeruginosa translocators PopB
and PopD insert pores into membranes forming homo- or hetero-complexes of undetermined stoichiometry. Single-molecule fluorescence
photobleaching experiments revealed that PopD formed mostly hexameric structures in membranes, while PopB displayed a bi-modal
distribution with six and twelve subunits peaks. However, in isolation the proteins are not functional for effector translocation.
We have found that when added together, the translocators formed distinct hetero-complexes containing eight PopB and eight
PopD molecules. Thus, the interaction between PopB and PopD guide the assembly of a unique hetero-oligomer in membranes.
... The first proteins proposed to be exported through the needle are PcrV, PopB and PopD, playing an essential role in toxin translocation across the plasma membrane of target cell [8,9]. As a majority of T3S substrates, the three translocators are kept in bacterial cytoplasm in complexes with their cognate chaperons, PcrG for PcrV, and PcrH shared by PopB and PopD [10,11,12,13]. PcrV belongs to a family of surface-exposed, soluble antigens, some of them shown to oligomerize on the secretion needle extremity [14,15,16,17]. ...
... There is still little structural information on PopB/D proteins [9], probably due to their hydrophobic characteristics, molten globule conformation and/or oligomerization capacity [12,19]. PopB shares secondary fold with its Salmonella orthologue SipB, for which exhaustive proteolysis experiments showed that it is deeply inserted into lipid bilayer, with the N-terminal region being exposed and cleaved off [46]. ...
The pathogenesis of Pseudomonas aeruginosa (P.a) implies multiple virulence factors among which the type III secretion system (T3SS). This multiprotein complex is composed of a needle through which four exotoxins are exported. The protein PopB and PopD form an oligomeric structure (translocon) at the end of the needle that inserts into the host cell membrane and translocates the exotoxins into the cytoplasm. Synthesis and toxin secretion is induced on contact with eukaryotic cell. In this work, we examined the influence of host cell elements on exotoxin translocation efficiency. The delivery of T3SS toxins was investigated using a CCF2/β-lactamase fluorescent reporter system In parallel, the association of translocon proteins with host plasma membranes was evaluated by immunodetection of PopB/D following sucrose gradient fractionation of membranes. Promyelocytic HL-60 cells and promonocytic U937 cells were found to be resistant to toxin injection even though PopB/D associated with host cell plasma membranes. Differentiation of these cells to neutrophil- or macrophage-like cells resulted in an injection-sensitive phenotype without any significant change in the level of membrane-inserted translocon proteins. Treatment of sensitive HL-60 cells with a cholesterol-depleting agent, resulted in a diminished injection of toxin. Moreover, the PopB translocator was found in the membrane fraction obtained from sucrose-gradient purifications and containing lipid-raft marker flotillin. Through a pharmacological approach, we brought evidence that, in addition to membrane composition, some general signalling pathways involved in actin polymerization may be critical for the formation of a functional pore.
... Les protéines constitutives de l'appareil de translocation peuvent être toxiques pour les cellules bactériennes. Les protéines chaperonnes de type II se fixent préférentiellement à ces protéines permettant de neutraliser leur toxicité (Neyt et Cornelis, 1999 Broms et al., 2003 ;Faudry et al., 2007 ;Job et al., 2010 PopD + + PscE-PscG ≈ PscF III + + Quinaud et al., 2005Quinaud et al., et 2007Plé et al., 2010 SpcS (Orf1) ExoS nd + + Shen et al., 2008 ;Yahr et al., 1995 ExoT + Bogdanove et al., 1998 ;van Dijk et al., 2002 ShcS1 § HopS1 Ia ∀ + nd Guo et al., 2005 ;Kabisch et al., 2005 HopO1-1 + -HopO1-2 nd nd ShcS2 § HopS1 Ia ∀ nd nd Guo et al., 2005 HopS2 nd nd Schoehn et al., 2003). La chaperonne de type II, PcrH de P. aeruginosa, est responsable de la reconnaissance et de la stabilisation de ces deux protéines constitutives (Faudry et al., 2007 ;Job et al., 2010). ...
... Les protéines chaperonnes de type II se fixent préférentiellement à ces protéines permettant de neutraliser leur toxicité (Neyt et Cornelis, 1999 Broms et al., 2003 ;Faudry et al., 2007 ;Job et al., 2010 PopD + + PscE-PscG ≈ PscF III + + Quinaud et al., 2005Quinaud et al., et 2007Plé et al., 2010 SpcS (Orf1) ExoS nd + + Shen et al., 2008 ;Yahr et al., 1995 ExoT + Bogdanove et al., 1998 ;van Dijk et al., 2002 ShcS1 § HopS1 Ia ∀ + nd Guo et al., 2005 ;Kabisch et al., 2005 HopO1-1 + -HopO1-2 nd nd ShcS2 § HopS1 Ia ∀ nd nd Guo et al., 2005 HopS2 nd nd Schoehn et al., 2003). La chaperonne de type II, PcrH de P. aeruginosa, est responsable de la reconnaissance et de la stabilisation de ces deux protéines constitutives (Faudry et al., 2007 ;Job et al., 2010). PcrH forme un complexe avec ces protéines partenaires et la stabilité de cette interaction doit être strictement maintenue pour garantir la capacité d'infection de la bactérie . ...
L'objectif de cette thèse est de contribuer à faire progresser les connaissances sur les interactions bénéfiques entre les plantes et les microorganismes en évaluant la contribution des systèmes de sécrétion de type III (SST3). Une synthèse des connaissances disponibles relatives aux SST3 chez les Pseudomonas non pathogènes, saprotrophes ou mutualistes, présentée chapitre I, montre que les SST3 ne sont pas cantonnés aux interactions parasites ou pathogènes avec les plantes. Dans l'étude expérimentale présentée chapitre II, nous avons utilisé différents génotypes de Medicago truncatula Gaertn. cv. Jemalong capables (Myc+) ou non (Myc-) d'établir une symbiose mycorhizienne. Ce travail nous a permis de montrer que les Pseudomonas spp. fluorescents possédant un SST3 (SST3+) sont préférentiellement associés aux racines mycorhizées des génotypes Myc+ de M. truncatula (J5 et TRV48) plutôt qu'aux racines du mutant Myc- (TRV25) et au sol nu. Ainsi, la plante seule n'est pas à l'origine de la présence accrue des Pseudomonas SST3+. La colonisation de la racine par les champignons mycorhizogènes à arbuscules (CMA), le développement du mycélium intraradiculaire et/ou la formation associée d'arbuscules, sont également déterminants. Dans l'étude présentée chapitre III, nous avons comparé les effets de la souche modèle promotrice de mycorhization (MHB) P. fluorescens C7R12 (SST3+) et de son mutant C7SM7 (SST3-), sur la mycorhization et la croissance de M. truncatula dans un sol non stérile. Ce travail a permis de montrer que le SST3 de C7R12 contribue à l'effet MHB de la bactérie. La promotion de la colonisation de la racine de M. truncatula par les CMA indigènes induite par le SST3 de C7R12 s'est traduite par une amélioration de la croissance de la plante. En revanche, l'inactivation du SST3 chez C7SM7 a eu un impact délétère sur la colonisation de la racine de M. truncatula par les CMA du sol étudié et sur la croissance de la plante. L'observation d'effets quantitatifs opposés entre C7R12 et C7SM7, nous a conduits à nous interroger sur l'existence d'un effet différentiel de l'inoculation de ces bactéries sur la structure et la diversité des communautés des microorganismes associés. Dans une étude présentée chapitre IV, le suivi dynamique en parallèle de la structure des communautés totales bactériennes (B-RISA) et fongiques (F-RISA) et de la colonisation de la racine par les CMA a été réalisée. Aucun effet de l'inoculation n'a été observé sur la structure des communautés fongiques de la rhizosphère ou des racines. En revanche, la structure des communautés bactériennes a varié selon que les plantes aient été inoculées ou non et selon la souche inoculée. Néanmoins, ces différences ont été observées plusieurs semaines après les effets de l'inoculation de C7R12 ou de C7SM7 sur la colonisation de la racine par les CMA. Ce décalage dans le temps, suggère que les différences observées dans la structure des communautés bactériennes pourraient être une conséquence plutôt qu'une cause des variations observées sur la mycorhization de M. truncatula. Nos résultats n'ont pas permis de mettre en évidence d'effets de l'inoculation sur la diversité des populations des bactéries fixatrices d'azote présentes dans les nodosités de M. truncatula. L'analyse des séquences de la grande sous-unité de l'ADN ribosomique (LSU rDNA) amplifiées à partir d'ADN extrait des racines, a montré pour les plantes inoculées et non inoculées, que les populations de CMA étaient majoritairement apparentées à Glomus intraradices. Un groupe d'isolats spécifiquement associé aux racines inoculées avec C7R12 et apparenté à G. claroideum a été décrit. Le groupe spécifique pourrait être associé à l'amélioration de la mycorhization observée dans les racines inoculées avec C7R12. Néanmoins, compte tenu de sa faible représentation numérique (8%), il semble probable que l'inoculation de C7R12 ait aussi un effet quantitatif sur la colonisation de la racine de M. truncatula par les CMA. etc
... PopD contains 295 residues and has a putative transmembrane region and an amphipathic helix (15). Neither crystallographic structure has been solved to date, most likely because these proteins are intrinsically flexible (17,18). Purified PopB and PopD are membrane-active, but PopB shows significantly greater pore-forming activity in liposome leakage assays as compared to PopD (14). ...
... To remove PcrH from the translocators, the complexes were dialyzed into a buffer containing 6 M guanidine. The use of chaotropic agents to dissociate the translocators from their chaperone was shown to preserve the structural and functional properties of the translocators while preventing aggregation during longterm storage (17,19). This strategy ensures reproducible results by providing a maximum population of translocators active toward membranes. ...
Pseudomonas aeruginosa is a major agent of hospital-acquired infections, and a pathogen of immunocompromised, cystic fibrosis and burn patients. It uses a type III secretion system for the injection of toxins directly into host cells, through a translocon assembled in the host cell membrane. The hydrophobic translocator subunits of this system, PopB and PopD, have membrane permeabilizing activity based on previous dye leakage experiments, but little is known about the mechanism of assembly and the pore properties of this translocon. Using electrophysiology, we have observed that an equimolar mixture of PopB and PopD induces current fluctuations in planar lipid bilayers, with a unitary conductance of 57 pS in 1 M KCl and numerous larger conductance levels. The activity depends on voltage magnitude and polarity, and increases with protein concentration and the duration of the voltage step. PopB alone is sufficient for producing current fluctuations. PopD rarely displays any transitions, but accelerates PopB onset of activity. The effects of pH, ionic strength, and lipid composition have also been explored. Our data provide new, to our knowledge, insights into the behavior of PopB and PopD by highlighting similarities with secreted pore-forming peptides, and by suggesting that PopB/PopD may form channels via the toroidal pore model. We believe that the events we report here represent the initial steps of insertion and assembly of these translocators in the membrane.
... For other group of translocators, conservation is observed throughout the length of the protein without any preference for a particular region (Supplementary Figure 1). However, the C-terminus of YopD possesses a putative coiled coil domain; and an a-helical amphipathic domain, which is responsible for interaction of YopD with LcrV and formation of YopD oligomers [3,12,14,35,36,38]. ...
... During the course of purification of recombinant YspC, we noticed that solubility, stability and yield of YspC are very high. Contrary to this, other minor translocators like PopD localizes in the inclusion bodies, AopD can be solubilised, only in the presence of 2 % TritonX-100 and shows non-specific heterogeneous aggregation during SEC [14,35,36]. IpaC also localizes in the inclusion bodies at 37°C, when expressed recombinantly in E. coli [32]. ...
YspC is an annotated translocator of Yersinia secretion apparatus-Yersinia secretion protein type three secretion system of Yersinia enterocolitica, it forms an 1:1 complex with its cognate chaperone SycB. Unlike other translocators, YspC is highly soluble inspite of having a transmembrane region. Size exclusion chromatography shows that YspC exists predominantly in a monomeric form. Multiple sequence alignment and ConSurf (a web based bioinformatic tool) analysis confirm its significant deviation from the closest class of minor translocators. YspC also possesses a tertiary structure signal seen from near UV CD, further confirming its unique nature amongst the groups of translocators. Far UV CD depicts that YspC is predominantly an α-helical protein; however, its secondary structure alters in the YspC-SycB complex. Thermal denaturation curve predicts a cooperative melting behaviour for YspC which is altered in the YspC-SycB complex. Furthermore, trypsinolysis data confirms a different digestion pattern for YspC in isolation, when compared to the complex form with SycB. From the Forsters resonance energy transfer analysis, it can be predicted that the two tetratricopeptide repeat regions of SycB are masked while it forms a complex with YspC and this is further confirmed by the interaction studies of YspC with two truncated forms of SycB. YspC interacted with ∆SycB₁₋₁₁₄ and ∆SycB₃₆₋₁₁₄ (possessing only the two TPR regions). However, the complexes formed between YspC and truncated forms of SycB have altered physiological states.
... The first proteins proposed to be exported through the needle are PcrV, PopB and PopD, playing an essential role in toxin translocation across the plasma membrane of target cell [8,9]. As a majority of T3S substrates, the three translocators are kept in bacterial cytoplasm in complexes with their cognate chaperons, PcrG for PcrV, and PcrH shared by PopB and PopD [10,11,12,13]. PcrV belongs to a family of surface-exposed, soluble antigens, some of them shown to oligomerize on the secretion needle extremity [14,15,16,17]. ...
... There is still little structural information on PopB/D proteins [9], probably due to their hydrophobic characteristics, molten globule conformation and/or oligomerization capacity [12,19]. PopB shares secondary fold with its Salmonella orthologue SipB, for which exhaustive proteolysis experiments showed that it is deeply inserted into lipid bilayer, with the N-terminal region being exposed and cleaved off [46]. ...
Pseudomonas aeruginosa type III secretion apparatus exports and translocates four exotoxins into the cytoplasm of the host cell. The translocation requires two hydrophobic bacterial proteins, PopB and PopD, that are found associated with host cell membranes following infection. In this work we examined the influence of host cell elements on exotoxin translocation efficiency. We developed a quantitative flow cytometry based assay of translocation that used protein fusions between either ExoS or ExoY and the ß-lactamase reporter enzyme. In parallel, association of translocon proteins with host plasma membranes was evaluated by immunodetection of PopB/D following sucrose gradient fractionation of membranes. A pro-myelocytic cell line (HL-60) and a pro-monocytic cell line (U937) were found resistant to toxin injection even though PopB/D associated with host cell plasma membranes. Differentiation of these cells to either macrophage- or neutrophil-like cell lines resulted in injection-sensitive phenotype without significantly changing the level of membrane-inserted translocon proteins. As previous in vitro studies have indicated that the lysis of liposomes by PopB and PopD requires both cholesterol and phosphatidyl-serine, we first examined the role of cholesterol in translocation efficiency. Treatment of sensitive HL-60 cells with methyl-ß-cyclodextrine, a cholesterol-depleting agent, resulted in a diminished injection of ExoS-Bla. Moreover, the PopB translocator was found in the membrane fraction, obtained from sucrose-gradient purifications, containing the lipid-raft marker flotillin. Examination of components of signalling pathways influencing the toxin injection was further assayed through a pharmacological approach. A systematic detection of translocon proteins within host membranes showed that, in addition to membrane composition, some general signalling pathways involved in actin polymerization may be critical for the formation of a functional pore. In conclusion, we provide new insights in regulation of translocation process and suggest possible cross-talks between eukaryotic cell and the pathogen at the level of exotoxin translocation.
... A soluble internal 150 -287-residue YopD polypeptide has been found to exist stably in an unfolded state (31). This is comparable with the behavior of the PopD N-terminal region, which exists in a loosely folded, highly flexible, molten, globule state (32). Evidence also indicates that EspB from enteropathogenic Escherichia coli and IpaC from Shigella flexneri both assume a partially folded state with a significant degree of disordered structure (33,34). ...
... Evidence also indicates that EspB from enteropathogenic Escherichia coli and IpaC from Shigella flexneri both assume a partially folded state with a significant degree of disordered structure (33,34). All of these structural characteristics may confer several benefits to this protein family; limiting the need for unfolding may promote their efficient secretion and/or pore formation into biological membranes or increase the potential surface area to facilitate physical contacts with multiple protein interaction partners (31)(32)(33)(34). ...
YopD-like translocator proteins encoded by several Gram-negative bacteria are important for type III secretion-dependent delivery of anti-host effectors into eukaryotic cells. This probably depends on their ability to form pores in the infected cell plasma membrane, through which effectors may gain access to the cell interior. In addition, Yersinia YopD is a negative regulator essential for the control of effector synthesis and secretion. As a prerequisite for this functional duality, YopD may need to establish molecular interactions with other key T3S components. A putative coiled-coil domain and an alpha-helical amphipathic domain, both situated in the YopD C terminus, may represent key protein-protein interaction domains. Therefore, residues within the YopD C terminus were systematically mutagenized. All 68 mutant bacteria were first screened in a variety of assays designed to identify individual residues essential for YopD function, possibly by providing the interaction interface for the docking of other T3S proteins. Mirroring the effect of a full-length yopD gene deletion, five mutant bacteria were defective for both yop regulatory control and effector delivery. Interestingly, all mutations clustered to hydrophobic amino acids of the amphipathic domain. Also situated within this domain, two additional mutants rendered YopD primarily defective in the control of Yop synthesis and secretion. Significantly, protein-protein interaction studies revealed that functionally compromised YopD variants were also defective in self-oligomerization and in the ability to engage another translocator protein, LcrV. Thus, the YopD amphipathic domain facilitates the formation of YopD/YopD and YopD/LcrV interactions, two critical events in the type III secretion process.
... They act mainly as "bodyguards" preventing their substrates from generating premature or nonspecific interactions with other proteins but are also thought to play a role in secretion. The class II chaperones bind to hydrophobic translocators and keep them in a soluble state (13,31). SycD of Yersinia binds YopB and YopD translocators, while PcrH from Pseudomonas is responsible for recognition of PopB and PopD (4,9,13,21). ...
... The class II chaperones bind to hydrophobic translocators and keep them in a soluble state (13,31). SycD of Yersinia binds YopB and YopD translocators, while PcrH from Pseudomonas is responsible for recognition of PopB and PopD (4,9,13,21). These chaperones display all-helical structures with three tetratricopeptide repeat (TPR) motifs, with a single TPR module being composed of two antiparallel ␣ helices; the overall structure forms a concave substrate-binding groove (4,21,23). ...
Type III secretion (T3S) systems allow the export and translocation of bacterial effectors into the host cell cytoplasm. Secretion
is accomplished by an 80-nm-long needle-like structure composed, in Pseudomonas aeruginosa, of the polymerized form of a 7-kDa protein, PscF. Two proteins, PscG and PscE, stabilize PscF within the bacterial cell
before its export and polymerization. In this work we screened the 1,320-Å2 interface between the two chaperones, PscE and PscG, by site-directed mutagenesis and determined hot spot regions that are
important for T3S function in vivo and complex formation in vitro. Three amino acids in PscE and five amino acids in PscG, found to be relevant for complex formation, map to the central part
of the interacting surface. Stability assays on selected mutants performed both in vitro on purified PscE-PscG complexes and in vivo on P. aeruginosa revealed that PscE is a cochaperone that is essential for the stability of the main chaperone, PscG. Notably, when overexpressed
from a bicistronic construct, PscG and PscF compensate for the absence of PscE in cytotoxic P. aeruginosa. These results show that all of the information needed for needle protein stabilization and folding, its presentation to
the T3 secreton, and its export is present within the sequence of the PscG chaperone.
... 13,23 It has also been reported that the translocator has a molten globule conformation both in its free and chaperone bound forms. 35 To speculate that exposed regions may be present in the complexes formed between AcrH and AopB or AopD, we carried out limited protease digestions on these complexes. Intact chaperone binding regions mapped this way represent actual functional binding domains and are different from arbitrary boundaries determined by assessing loss of chaperone binding by use of deletion mutants. ...
... 32,37 In PopD, the putative transmembrane fragment was buried within the PcrH-PopD complex. 35 However, unlike in the cases of AopD and YopD, the C-terminal amphipathic helix of PopD was not bound to PcrH and was susceptible to trypsin digestion. The region (residues 20-49) at the N-terminus of DF1 is aligned to a region in IpaC (residues 50-80), a homolog of AopD from Shigella flexneri, which had been reported to be critical for its invasion function and also essential for chaperone binding. ...
In the type III secretion system (T3SS) of Aeromonas hydrophila, AcrH acts as a chaperone for translocators AopB and AopD. AcrH forms a stable 1:1 monomeric complex with AopD, whereas the 1:1 AcrH-AopB complex exists mainly as a metastable oligomeric form and only in minor amounts as a stable monomeric form. Limited protease digestion shows that these complexes contain highly exposed regions, thus allowing mapping of intact functional chaperone binding regions of AopB and AopD. AopD uses the transmembrane domain (DF1, residues 16-147) and the C-terminal amphipathic helical domain (DF2, residues 242-296) whereas AopB uses a discrete region containing the transmembrane domain and the putative N-terminal coiled coil domain (BF1, residues 33-264). Oligomerization of the AcrH-AopB complex is mainly through the C-terminal coiled coil domain of AopB, which is dispensable for chaperone binding. The three proteins, AcrH, AopB, and AopD, can be coexpressed to form an oligomeric and metastable complex. These three proteins are also oligomerized mainly through the C-terminal domain of AopB. Formation of such an oligomeric and metastable complex may be important for the proper formation of translocon of correct topology and stoichiometry on the host membrane.
... PopB is the larger of the translocators and possesses two nonpolar segments in its primary structure, while PopD possesses only one non-polar segment [22,38]. Both proteins adopt a molten globular conformation and are prone to aggregation in aqueous solution [23,39,40]. PopB has been shown to assist with the proper insertion of PopD by causing a conformational change, leaving the N-terminal region of PopD exposed to the host cytosol only when forming functional translocons [41]. ...
Multidrug resistant Pseudomonas aeruginosa poses a serious threat to hospitalized patients. This organism expresses an arsenal of virulence factors that enables it to readily establish infections and to disseminate in the host. The Type III secretion System (T3SS) and its associated effectors play a crucial role in the pathogenesis of P. aeruginosa, making them attractive targets for the development of novel therapeutic agents. The T3SS translocon, comprised of PopD and PopB, is an essential component of the T3SS secretion apparatus. In the properly assembled translocon, the N-terminus of PopD protrudes into the cytoplasm of the target mammalian cell, which can be exploited as a molecular indicator of functional translocon assembly. In this manuscript, we describe a novel whole-cell-based assay that employs the split NanoLuc luciferase detection system to provide a readout for translocon assembly.
The assay demonstrates a favorable signal/noise ratio (17.9) and robustness (z’=0.73), making it highly suitable for high-throughput screening of small molecule inhibitors targeting T3SS translocon assembly.
... PopD is the smallest of the two P. aeruginosa translocators with a molecular mass of 31.3 kDa. Like PopB, PopD adopts a molten globule state composed of mostly helical structure and the chaperone PcrH maintain PopD soluble in the cytosol before secretion (Faudry et al. 2007;Tan et al. 2009;Romano et al. 2011;Discola et al. 2014). Purified, recombinantly-expressed PopD aggregates in solution and does not significantly bind to membranes at neutral pH (Romano et al. 2016). ...
Type III secretion (T3S) systems are complex bacterial structures used by many pathogens to inject proteins directly into the cytosol of the host cell. These secretion machines evolved from the bacterial flagella and they have been grouped into families by phylogenetic analysis. The T3S system is composed of more than 20 proteins grouped into five complexes: the cytosolic platform, the export apparatus, the basal body, the needle, and the translocon complex. While the proteins located inside the bacterium are conserved, those exposed to the external media present high variability among families. This suggests that the T3S systems have adapted to interact with different cells or tissues in the host, and/or have been subjected to the evolutionary pressure of the host immune defenses. Such adaptation led to changes in the sequence of the T3S needle tip and translocon suggesting differences in the mechanism of assembly and structure of this complex.
Graphical abstract
... These proteins are targeted for T3S to the extracellular environment, where they should remain soluble until they approach the host membrane and adopt a transmembrane orientation [21--21-23]. This ability to transfer from soluble to membrane-embedded is likely supported by their dual structural fluctuation from molten globule conformation in aqueous solution to transmembrane embedded ringlike structures with a stoichiometry of 6-8 subunits of each protein [8,22,[24][25][26][27]. Before their secretion, the SctB and SctE proteins are associated with class II T3SS chaperones that prevent their premature folding and target them to the sorting platform of the T3S apparatus [28--28-33]. ...
Many bacterial pathogens utilize a specialized secretion system, termed type III secretion system (T3SS), to translocate effector proteins into host cells and establish bacterial infection. The T3SS is anchored within the bacterial membranes and contains a long needle/filament that extends toward the host-cell and forms, at its distal end, a pore complex within the host membrane. The T3SS pore complex consists of two bacterial proteins, termed SctB and SctE, which have conflicting targeting indications; a signal sequence that targets to secretion to the extracellular environment via the T3SS, and transmembrane domains (TMDs) that target to membrane localization. In this study, we investigate whether the TMD sequences of SctB and SctE have special features that differentiate them from classical TMDs and allow them to escape bacterial membrane integration. For this purpose, we exchanged the SctB and SctE native TMDs for alternative hydrophobic sequences and found that the TMD sequences of SctB and SctE dictate membrane destination (bacterial versus host membrane). Moreover, we examined the role of the SctB TMD sequence in the activity of the full-length protein, post secretion, and found that the TMD does not serve only as a hydrophobic segment, but is also involved in the ability of the protein to translocate itself and other proteins into and across the host cell membrane.
... 94 The equivalent of YopD in P. aeruginosa, PopD, including the the major translocon protein (PopB) have been reported to exist as molten globules. 81,95 The major translocon proteins. Results of secondary predictions, structural studies, deletions and point mutations have contributed towards our current understanding of the various structural and functional domains of the major translocon protein [ Fig. 5(B)]. ...
Many Gram‐negative bacteria pathogenic to plants and animals deploy the type III secretion system (T3SS) to inject virulence factors into their hosts. All bacteria that rely on the T3SS to cause infectious diseases in humans have developed antibiotic resistance. The T3SS is an attractive target for developing new antibiotics because it is essential in virulence, and part of its structural component is exposed on the bacterial surface. The structural component of the T3SS is the needle apparatus, which is assembled from over 20 different proteins and consists of a base, an extracellular needle, a tip, and a translocon. This review summarizes the current knowledge on the structure and assembly of the needle, tip, and translocon.
... Variations in ε can partially denature well-structured proteins [73,74], increase the secondary structure content of many different IDPs [41,48,75,76] and induce, in some well-folded proteins, a transformation to a molten globule-like state in aqueous solution under mild denaturing conditions [69,77]. The latter phenomenon is hypothesized to be important for protein translocation across the membrane [78,79,80]. ...
Background: Golgi Reassembly and Stacking Proteins (GRASPs) are widely spread among eukaryotic cells (except plants) and are considered as key components in both the stacking of the Golgi cisternae and its lateral connection. Furthermore, GRASPs were also proved essential in the unconventional secretion pathway of several proteins, even though the mechanism remains obscure. It was previously observed that the GRASP homologue in Cryptococcus neoformans has a molten globule-like behavior in solution.
Methods: We used circular dichroism, synchrotron radiation circular dichroism and steady-state as well as time-resolved fluorescence.
Results: We report the disorder-to-order transition propensities for a native molten globule-like protein in the presence of different mimetics of cell conditions. Changes in the dielectric constant (such as those experienced close to the membrane surface) seem to be the major factor in inducing multiple disorder-to-order transitions in GRASP, which shows very distinct behavior when in conditions that mimic the vicinity of the membrane surface as compared to those found when free in solution. Other folding factors such as molecular crowding, counter ions, pH and phosphorylation exhibit lower or no effect on GRASP secondary structure and/or stability.
General Significance: To the best of our knowledge, this is the first study focusing on understanding the disorder-to-order transitions of a molten globule structure without the need of any mild denaturing condition. A model is also introduced aiming at describing how the cell could manipulate the GRASP sensitivity to changes in the dielectric constant during different cell-cycle periods.
Keywords: Golgi Reassembly and Stacking Protein; intrinsically disordered proteins; molten globule; disorder-to-order transition; spectroscopy
... Some membranes enriched in negative phospholipids may create an environment in which proteins in close proximity could change in conformation to form MG states. It is quite interesting that for some opportunistic pathogens, the injection of virulence factors into the host cell through its membrane requires specialized translocation systems and MG states for membrane insertion [50]. Accordingly, during virus fusion to host cells, H+ pumps in host-endocytic vesicles promote exposure of fusion peptides in virus proteins that facilitate invasion [51]. ...
The combination of biophysical and structural techniques has allowed the visualization of species classified as dry molten-globule states. Further destabilization causes these structures to follow through a wet-globule stage to reach an unfolded chain. We have recently combined small angle X-ray scattering and nuclear magnetic resonance to observe these species, and we introduce a push-and-pull hypothesis to explain the dissimilar actions of urea and high pressure on proteins. The implications of these molten-globule states are further discussed in light of their potential physiological and pathological roles, especially in protein misfolding diseases.
... SRB was selected as fluorescent probe because of its high quantum yield independently of the pH. Fluorescence was normalized as previously described (14). The initial rate (V 0 ) was deduced from the slope at the origin of the curves. ...
Diphtheria toxin (DT) is a bacterial toxin with intracellular target, secreted by Corynebacterium diphtheriae. During cell intoxication, the translocation domain of DT (T) inserts into the endosomal membrane under acidic conditions and assists the translocation of the catalytic (C) domain in the cytosol. T undergoes conformational changes and adopts a molten globule (MG) state, competent for the interaction with the membrane.
We first identified by mutagenesis the residues in the T domain whose protonation triggers the formation of the MG state in solution and the interaction with the membrane. The results showed that the concerted protonation of the six histidines in the T domain is implicated. We found that the pair His223-257 et His251 are more implicated in the formation of the MG state in solution and that the pair His322-323 (and His251) is more implicated in the interaction with the membrane.
We then studied the conformational changes of both C and T domains within a CT protein corresponding to the whole toxin truncated at the R domain to understand the effects of the covalent link between the two domains on the conformations of C and T as a function of pH. We produced two Trp mutants, CTW50/153F and CTW206/281F, that allow to follow up the Trp fluorescence of each domain in the CT protein. The results showed that the T domain leads the way in the first steps of translocation (MG formation, binding and membrane insertion), and that it may play the role of a membrane chaperone by stabilizing the MG state of C.
... Dans le cytoplasme bactérien, PopB et PopD sont maintenus à l'état monomérique par leur chaperonne commune, PcrH, à laquelle ils s'associent dans un ratio 1:1, comme expliqué dans la partie III-B-1) de ce chapitre (Faudry et al. 2006 ;Schoehn et al. 2003 ;Job et al. 2010). Le domaine transmembranaire de PopD est capturé au coeur de la chaperonne (Faudry et al. 2007). ...
Pseudomonas aeruginosa, a Gram negative bacterium responsible for nosocomial infections, exhibits numerous virulence factors to infect its hosts. In particular, the Type III Secretion System (T3SS) allows the injection of effectors directly into the host cell cytoplasm. This work focuses on the study of two proteins from the T3SS of P. aeruginosa: the ATPase PscN and the ExsB protein.
Several approaches were used to study the ATPase PscN, an enzyme essential for T3SS activity. Site-directed mutations, made on PscN, lead to non cytotoxic strains, and this effect is dominant negative. Another approach allowed the partial purification of active PscN, visualized as large complexes by electron microscopy. These partially purified samples also contain other T3SS proteins, which could interact with PscN.
The ExsB protein was characterized for the first time. After checking its expression in P. aeruginosa, its association with the outer membrane was shown. The phenotypic analysis of a strain lacking exsB gene gave insights into the role of this protein. We did not identified any function of ExsB in the T3SS regulation. After showing the involvement of ExsB in the bacterial virulence during acute animal infections, ExsB role in T3SS activity was established. Finally, we showed that ExsB has a pilotin activity as it participates in the assembly of the secretin, the outer membrane component of T3SS.
... Nevertheless, there are virtually no investigations employing multiple substitutions of amino acid residues for analyzing intermediate states of proteins, although intermediate states of some proteins are functionally important and in many respects determine the properties of proteins. For example, it has been shown that in the molten globule state, proteins are transported via the cell membrane and can catalyze reactions [33][34][35][36][37][38], and many proteins form aggregates and amyloidal structures in conditions when they pass to intermediate states [39][40][41][42]. ...
At present it is unclear which interactions in proteins reveal the presence of intermediate states, their stability and formation rate. In this study, we have investigated the effect of substitutions of hydrophobic amino acid residues in the hydrophobic core of protein and on its surface on a molten globule type intermediate state of apomyoglobin. It has been found that independent of their localization in protein, substitutions of hydrophobic amino acid residues do not affect the stability of the molten globule state of apomyoglobin. It has been shown also that introduction of a disulfide bond on the protein surface can stabilize the molten globule state. However in the case of apomyoglobin, stabilization of the intermediate state leads to relative destabilization of the native state of apomyoglobin. The result obtained allows us not only to conclude which mutations can have an effect on the intermediate state of the molten globule type, but also explains why the introduction of a disulfide bond (which seems to "strengthen" the protein) can result in destabilization of the protein native state of apomyoglobin.
... Stoichiometry and dimerization interfaces of class II chaperones vary in the structures observed to date. Structures of the full-length translocon/effector complex could help discern the ternary structure of the complex; however, the full-length translocon may interact with the chaperone in a molten globule conformation [62], impeding crystallization. ...
The Type III Secretion System (T3SS) is a multi-mega Dalton apparatus assembled from more than twenty components and is found in many species of animal and plant bacterial pathogens. The T3SS creates a contiguous channel through the bacterial and host membranes, allowing injection of specialized bacterial effector proteins directly to the host cell. In this review, we discuss our current understanding T3SS assembly and structure, as well as highlight structurally characterized Salmonella effectors. This article is part of a Special Issue entitled: Protein Trafficking & Secretion.
... Generally, class III needle protein T3SC are thought to prevent premature association or polymerization of monomeric needle proteins within the bacterial cytoplasm (5). Class II T3SC of translocon proteins bind their cargos within the bacterial cell and in many cases are required for translocon protein secretion (6). For effector binding class I T3SC, there are two subclasses: IA and IB (7). ...
The enteropathogenic Escherichia coli (EPEC) multicargo chaperone CesT interacts with at least 10 effector proteins and is central to pathogenesis. CesT has been implicated in coordinating effector hierarchy although the mechanisms behind this regulation are poorly understood. To address this question, we set out to functionally characterize CesT with respect to roles in i) effector binding, ii) effector recruitment to the T3SS, and iii) effector translocation into host cells. A CesT variant expression library was screened in EPEC using a newly developed semi high-throughput secretion assay. Among many deficient CesT variants, a predominant number were localized to a novel CesT C-terminal region. These CesT C-terminal variants exhibited normal effector binding yet reduced effector secretion levels. Structural correlation and thermal spectroscopy analyses of purified CesT variants implicated multiple surface exposed residues, a terminal helix region and a flexible C-terminal triple serine stretch in effector secretion. Site directed mutagenesis of the flexible CesT C-terminal triple serine sequence produced differential effector secretion, implicating this region in secretion events. Infection assays further indicated that the C-terminal region of CesT was important for NleA translocation into host cells, but was dispensable for Tir translocation. The findings implicate the CesT C-terminus in effector secretion and contribute to a model for multiple cargo chaperone function and effector translocation into host cells during infection.
... Molten globule conformations in proteins emerge as a particularly efficient mechanism to regulate binding/enzymatic activities and allosteric interactions (Demarest et al., 2002; Liu and Nussinov, 2008; Pervushin et al., 2007) and may even be a common feature in TTS (Dawson et al., 2009; Faudry et al., 2007). The CesAB chaperone provides a compelling example of a system that exploits structural instability for function as has been shown previously for a number of biological systems (Dyson and Wright, 2005). ...
Protein-protein interactions mediate a vast number of cellular processes. Here, we present a regulatory mechanism in protein-protein interactions mediated by finely tuned structural instability and coupled with molecular mimicry. We show that a set of type III secretion (TTS) autoinhibited homodimeric chaperones adopt a molten globule-like state that transiently exposes the substrate binding site as a means to become rapidly poised for binding to their cognate protein substrates. Packing defects at the homodimeric interface stimulate binding, whereas correction of these defects results in less labile chaperones that give rise to nonfunctional biological systems. The protein substrates use structural mimicry to offset the weak spots in the chaperones and to counteract their autoinhibitory conformation. This regulatory mechanism of protein activity is evolutionarily conserved among several TSS systems and presents a lucid example of functional advantage conferred upon a biological system by finely tuned structural instability.
... SRB was selected as fluorescent probe because of its high quantum yield independently of the pH. Fluorescence was normalized as previously described [27]. The initial rate (V 0 ) was deduced from the slope at the origin of the curves. ...
During cell intoxication by diphtheria toxin, endosome acidification triggers the translocation of the catalytic (C) domain into the cytoplasm. This event is mediated by the translocation (T) domain of the toxin. Previous work suggested that the T domain acts as a chaperone for the C domain during membrane penetration of the toxin. Using partitioning experiments with lipid vesicles, fluorescence spectroscopy, and a lipid vesicle leakage assay, we characterized the dominant behavior of the T domain over the C domain during the successive steps by which these domains interact with a membrane upon acidification: partial unfolding in solution and during membrane binding, and then structural rearrangement during penetration into the membrane. To this end, we compared, for each domain, isolated or linked together in a CT protein (the toxin lacking the receptor-binding domain), each of these steps. The behavior of the T domain is marginally modified by the presence or absence of the C domain, whereas that of the C domain is greatly affected by the presence of the T domain . All of the steps leading to membrane penetration of the C domain are triggered at higher pH by the T domain , by 0.5-1.6 pH units. The T domain stabilizes the partially folded states of the C domain corresponding to each step of the process. The results unambiguously demonstrate that the T domain acts as a specialized pH-dependent chaperone for the C domain. Interestingly, this chaperone activity acts on very different states of the protein: in solution, membrane-bound, and membrane-inserted.
... For this reason, EspB protein is classified as 'natively partially folded', rather than 'natively unfolded' or 'intrinsically disordered' [10]. A similar structural property has been observed with PopD which is a homolog of EspB expressed by Pseudomonas aeruginosa [37]. It should be noted here that, according to the original definition by Ohgushi & Wada [35], the 'molten globule' is a partially folded intermediate state with a significant amount of secondary structure, similar to the tightly packed native state, but lacks tertiary contacts. ...
Enterohemorrhagic and enteropathogenic Escherichia coli produce various effector proteins that are directly injected into the host-cell cytosol through the type III secretion system. E. coli secreted protein (Esp)B is one such effector protein, and affects host-cell morphology by reorganizing actin networks. Unlike most globular proteins that have well-ordered, rigid structures, the structures of type III secretion system effectors from pathogenic Gram-negative bacteria, including EspB, are often less well-ordered. This minireview focuses on the functional relationship between the structural properties of these proteins and their roles in type III secretion system-associated pathogenesis.
... Des chaperonnes seraient aussi nécessaires aux protéines formant la seringue. Il a été montré que PcrH pouvait limiter la protéolyse de PopD et aider à l'insertion de PopD dans la membrane des cellules eucaryotes [343] tandis que Pcr2 pourrait être associée à PopN [344]. ...
Pseudomonas aeruginosa (PA) is an opportunistic Gram-negative bacillus involved in life-threatening infections including ventilator-acquired pneumonia and lung disease in cystic fibrosis. The treatment of these infections is often difficult due to its natural resistance to a large number of antibiotics and an increasing number of strains with acquired multi-resistance.
Virulence factors of PA induce an increase in apical fluid secretion of airways epithelial cells by activating transepithelial transport and by modifying calcium homeostasis in these cells. However, the consequence of these effects remains largely to be clarified.
We showed a protective effect of EGTA on TTSS-induced cell death in a co-culture model of epithelial cells with PA. EGTA protects the cells by a decreasing bacterial adhesion and reducing TTSS toxins translocation into the epithelial cells. In the same model, we have shown a disorganization of cytoskeleton by both EGTA and TTSS toxins. The second part of this work demonstrated the role of LPS from PA on intracellular calcium increase and activation of chloride secretion of human bronchial epithelial cells. LPS induced a calcium release from reticulum and a calcium entry from extracellular space. This increase is related to a chloride secretion by activation of CFTR and calcium-activated chloride channel.
Effects of virulence factors of PA associated to the important role for intracellular calcium and chloride secretion of epithelial cells described here may represent the first signals sensed by the host cells. The understanding of these mechanisms is a crucial step to considerate the pathophysiology and to develop alternative therapeutics to antibiotics.
Two-partner secretion (TPS) is widespread in the bacterial world. The pore-forming TPS toxin ExlA of Pseudomonas aeruginosa is conserved in pathogenic and environmental Pseudomonas. While P. chlororaphis and P. entomophila displayed ExlA-dependent killing, P. putida did not cause damage to eukaryotic cells. ExlA proteins interacted with epithelial cell membranes, however only ExlAPch induced the cleavage of the adhesive molecule E-cadherin. ExlA proteins participated in insecticidal activity towards the larvae of Galleria mellonella and the fly Drosophila melanogaster. Evolutionary analyses demonstrated that the differences in the C-terminal domains are partly due to horizontal movements of the operon within the genus Pseudomonas. Reconstruction of the evolutionary history revealed the complex horizontal acquisitions. Together, our results provide evidence that conserved TPS toxins in environmental Pseudomonas play a role in bacteria-insect interactions and discrete differences in CTDs may determine their specificity and mode of action towards eukaryotic cells.
The characteristic features of cancer cells are aberrant (acidic) intracellular pH and elevated levels of phosphatidylserine. The primary focus of cancer research is concentrated on the discovery of biomarkers directed towards early diagnosis and therapy. It has been observed that azoxymethane-treated mice demonstrate an increased expression of calnuc (a multi-domain, Ca2+- and DNA-binding protein) in their colon, suggesting it to be a good biomarker of carcinogenesis. We show that culture supernatants from tumor cells have significantly higher amounts of secreted calnuc compared to non-tumor cells, selectively packaged into exosomes. Exosomal calnuc is causal for epithelial-mesenchymal transition and atypical migration in non-tumor cells, which are key events in tumorigenesis and metastasis. In vitro studies reveal a significant affinity for calnuc towards phosphatidylserine, specifically to its C-terminal region, leading to the formation of “molten globule” conformation. Similar structural changes are observed at acidic pH (pH 4), which demonstrates the role of the acidic microenvironment in causing the molten globule conformation and membrane interaction. On a precise note, we propose that the molten globule structure of calnuc caused by aberrant conditions in cancer cells to be the causative mechanism underlying its exosome-mediated secretion, thereby driving metastasis.
Pseudomonas aeruginosa is an opportunistic bacterium of which the main virulence factor is the Type III Secretion System. The ATPase of this machinery, PscN (SctN), is thought to be localized at the base of the secretion apparatus and to participate in the recognition, chaperone dissociation and unfolding of exported T3SS proteins. In this work, a protein-protein interaction ELISA revealed the interaction of PscN with a wide range of exported T3SS proteins including the needle, translocator, gate-keeper and effector. These interactions were further confirmed by Microscale Thermophoresis that also indicated a preferential interaction of PscN with secreted proteins or protein-chaperone complex rather than with chaperones alone, in line with the release of the chaperones in the bacterial cytoplasm after the dissociation from their exported proteins. Moreover, we suggest a new role of the gate-keeper complex and the ATPase in the regulation of early substrates recognition by the T3SS. This finding sheds a new light on the mechanism of secretion switching from early to middle substrates in P. aeruginosa.
Protein-translocating channels, or translocons, are ubiquitous membrane proteins found in all cell types as well as intracellular organelles. In bacteria, they often constitute one of the components of molecular machines that participate in membrane biogenesis, assembly of surface exposed appendages, and the secretion of protein substrates across the cell envelope. Their pore-forming properties make them directly amenable to electrophysiology, even though ionic movement is not their natural function. Several bacterial translocons have been investigated by patch-clamp or planar lipid bilayer electrophysiology. These studies have revealed that bacterial translocons are often dynamic channels, capable of sampling a large conformational landscape, and thus displaying multiplicity of conductance states. They typically form large pores, but are often gated by internal plug elements that limit the flow of ions and molecules in the resting state. Combined with structural data, electrophysiological studies have allowed the discovery of structure-function relationships that shed light on functional mechanisms. In some cases, the channel properties have been investigated in the presence of natural substrates to understand the molecular steps involved in translocation itself. This review highlights the electrophysiological investigations of SecYEG, BamA, TolC, secretins, ushers, type III secretion translocons, autotransporters and two-partner secretion systems.
Human α-lactalbumin made lethal to tumor cells (HAMLET), a complex formed by human α-lactalbumin and oleic acid, has a unique apoptotic activity for the selective killing of tumor cells. It has been hypothesized that HAMLET expresses its antitumor activity in the stomach of breast-fed infants, thereby protecting the infants from tumor development, and in this case the protein portion of HAMLET is in a flexible molten globule state. On the other hand, the primary biological function of α-lactalbumin in its rigid native structure is to modify the specificity of galactosyltransferase to produce lactose in mammary glands. α-Lactalbumin thus provides a unique example, in which a single globular protein has two independent biological functions in quite different locations. In this article, we summarize the historical background and recent progress of the studies on HAMLET and related protein-fatty acid complexes. It is shown that oleic acid forms an antitumor complex not only with α-lactalbumin but also with various globular proteins in the molten globule state by nonspecific hydrophobic interactions, although the strength of the activity varies somewhat depending on the protein species. Similarly, not only oleic acid but also various cytotoxic fatty acids (mono-and polyunsaturated cis fatty acids) are bound to α-lactalbumin in the molten globule state and exhibit the antitumor activities. It is thus concluded that the protein portion of these complexes is not the origin of their cytotoxicity but plays a role as the delivery carrier of cytotoxic fatty acid molecules into tumor cells across the cell membrane.
Gram-negative bacteria like Yersinia, Pseudomonas, and Aeromonas need type III secretion system (T3SS) for their pathogenicity. V-antigen and its regulator are essential for functioning of T3SS. There is significant functional conservation amongst V-antigen and its regulator belonging to the Ysc family. In this study, we have structurally characterized the inter-genus complexes of V-antigen and its regulator. ConSurf analysis demonstrates that V-antigens belonging to the Ysc family show high structural identity predominantly confined to the two long helical regions. The regulator of V-antigen shows high conservation in its first intramolecular coiled-coil domain, responsible for interaction with V-antigen. ∆LcrG(1-70) localizes within the groove formed by long helices of LcrV, as observed in PcrV-∆PcrG(13-72) interaction. Inter-genus complexes of LcrV-PcrG and PcrV-LcrG exhibited elongated conformation and 1:1 heterodimeric state like the native complex of PcrV-PcrG and LcrV-LcrG. Both native and inter-genus complexes showed rigid tertiary structure, solvent-exposed hydrophobic patches, and cooperative melting behavior with high melting temperature. LcrV-PcrG and PcrV-LcrG showed nanomolar affinity of interaction, identical to PcrV-PcrG interaction, but stronger than LcrV-LcrG interaction. Calcium (a secretion blocker of T3SS) propels all the complexes towards a highly monodisperse form. Calcium and magnesium increase the helicity of the native and inter-genus complexes, and causes helix-helix stabilization. Stabilization of helices leads to a slight increase in the melting temperature by 1.5-2.0 °C. However, calcium does not alter the affinity of interaction of V-antigen and its regulator, emphasizing the effect of divalent of cations at the structural level without any regulatory implications. Therefore, the structural conservation of these inter-genus complexes could be the basis for their functional complementation.
Les héparanes sulfate (HS) sont des polysaccharides complexes appartenant à la famille des glycosaminoglycanes (GAGs), présents en abondance à la surface cellulaire et dans les matrices interstitielles. De par leur positionnement stratégique à l'interface entre la cellule et son micro-environnement et leur capacité à fixer et moduler l'activité d'un très grand nombre de protéines, les HS sont impliqués dans de nombreux processus physiologiques et pathologiques. Les travaux présentés viseront à illustrer l'importance biologique de ces polysaccharides et de leurs interactions avec les protéines, principalement dans les domaines des relations hôtes-pathogènes et des processus inflammatoires. Ils s'attacheront également à montrer les difficultés que présente l'étude de ces molécules, ainsi que les méthodologies développées afin de faciliter leur caractérisation structurale et fonctionnelle. Enfin, des perspectives de recherche seront proposées, visant à intégrer les études en cours dans un contexte physiologique, par l'analyse des HS exprimés par les cellules. La structure des HS, et donc leurs propriétés biologiques, varie en effet de manière considérable selon le type cellulaire étudié, son niveau d'activation ou de différenciation. La réponse cellulaire à des stimuli externes (protéines de signalisation, cytokines pro-inflammatoires, molécules intervenant dans la reconnaissance hôte/pathogènes...) est donc intimement liée au " paysage glycanique" présent à sa surface et à sa capacité à moduler ce paysage. La compréhension de ces mécanismes représente donc un enjeu évident pour l'étude de nombreuses fonctions cellulaires.
Pseudomonas aeruginosa, a gram-negative pathogen utilizes a specialized set of T3SS translocator proteins to establish virulence in the host cell. An understanding of the factors that govern translocation by the translocator protein-chaperone complex is thus, of immense importance. In the present work, experimental and computational techniques were employed to probe into the structure of the major translocator protein PopB from P. aeruginosa and to identify the important regions involved in functioning of the translocator protein. This study reveals that the binding sites of the common chaperone PcrH, needed for maintenance of the translocator PopB within the bacterial cytoplasm, which are primarily localized within the N-terminal domain. However, disordered and flexible residues located both at the N- and C-terminal domains are also observed to be involved in association with the chaperone. This intrinsic disorderliness of the terminal domains is conserved for all the major T3SS translocator proteins and is functionally important to maintain the intrinsically disordered state of the translocators. Our experimental and computational analyses suggest that a 'disorder-to-order' transition of PopB protein might take place upon PcrH binding. The long helical coiled-coil part of PopB protein perhaps helps in pore formation while the flexible apical region is involved in chaperone interaction. Thus, our computational model of translocator protein PopB and its binding analyses provide crucial functional insights into the T3SS translocation mechanism. © Proteins 2014;. © 2014 Wiley Periodicals, Inc.
Although human α-lactalbumin made lethal to tumor cells (HAMLET), a complex formed by human α-lactalbumin and oleic acid, has a unique apoptotic activity for the selective killing of tumor cells, the molecular mechanisms of expression of the HAMLET activity are not well understood. Therefore, we studied the molecular properties of HAMLET and its goat counterpart, goat α-lactalbumin made lethal to tumor cells (GAMLET), by pulse field-gradient NMR and 920-MHz two-dimensional NMR techniques. We also examined the expression of HAMLET-like Molecular mechanisms of the cytotoxicity of HAMLET activities of complexes between oleic acid and other proteins that form a stable molten globule state. We observed that both HAMLET and GAMLET at pH 7.5 were heterogeneous, composed of the native protein, the monomeric molten globule-like state, and the oligomeric species. At pH 2.0 and 50°C, HAMLET and GAMLET appeared in the monomeric state, and we identified the oleic acid-binding site in the complexes by two-dimensional NMR. Rather surprisingly, the binding site thus identified was markedly different between HAMLET and GAMLET. Furthermore, canine milk lysozyme, apo-myoglobin and β2-microglobulin all formed the HAMLET-like complex with the anti-tumor activity, when the protein was treated with oleic acid under conditions in which their molten globule states were stable. From these results, we conclude that the protein portion of HAMLET, GAMLET and the other HAMLET-like protein-oleic acid complexes is not the origin of their cytotoxicity to tumor cells and that the protein portion of these complexes plays a role in the delivery of cytotoxic oleic acid molecules into tumor cells across the cell membrane.
Gram-negative bacteria utilize a dedicated membrane-embedded apparatus, the type III secretion system (T3SS), to inject proteins into host cells. The passage of the proteins across the target membrane is accomplished by a proteinaceous pore-the translocon-formed within the host-cell cytoplasmic membrane. Translocators bound to their chaperones can be expressed in Escherichia coli and subsequently dissociated from the chaperone by guanidine treatment. The pore formation properties of the translocators can then be studied by an in-vitro liposome leakage assay. Sulforhodamine-B is encapsulated within lipid vesicles during liposome preparation. At high concentration, this fluorochrome exhibits self-quenching limiting fluorescence emission. Upon pore formation, liposome leakage leads to the dilution of Sulforhodamine-B in the medium and fluorescence emission increases. Alternatively, fluorochromes coupled to large dextran molecules can be encapsulated in order to estimate pore dimensions. Here we describe protein expression and purification, dye-liposome preparation, and leakage assay conditions.
The TTSS encoding "translocator operon" of Pseudomonas aeruginosa consists of a major translocator protein PopB, minor translocator protein PopD and their cognate chaperone PcrH. Far-UV CD spectra and secondary structure prediction servers predict an α-helical model for PopB, PcrH and PopB-PcrH complex. PopB itself forms a single species of higher order oligomer (15 mer) as seen from AUC, but in complex with PcrH, both monomeric (1:1) and oligomeric form exist. PopB has large solvent-exposed hydrophobic patches and exists as an unordered molten globule in its native state, but on forming complex with PcrH it gets transformed into an ordered molten globule. Tryptophan fluorescence spectrum indicates that PopB interacts with the first TPR region of dimeric PcrH to form a stable PopB-PcrH complex that has a partial rigid structure with a large hydrodynamic radius and few tertiary contacts. The pH-dependent studies of PopB, PcrH and complex by ANS fluorescence, urea induced unfolding and thermal denaturation experiments prove that PcrH not only provides structural support to the ordered molten globule PopB in complex but also undergoes conformational change to assist PopB to pass through the needle complex of TTSS and form pores in the host cell membrane. ITC experiments show a strong affinity (K(d) ~ 0.37 μM) of PopB for PcrH at pH 7.8, which reduces to ~0.68 μM at pH 5.8. PcrH also loses its rigid tertiary structure at pH 5 and attains a molten globule conformation. This indicates that the decrease in pH releases PopB molecules and thus triggers the TTSS activation mechanism for the formation of a functional translocon.
Oligopeptidase B, a processing enzyme of the prolyl oligopeptidase family, is considered as an important virulence factor in trypanosomiasis. Trypanosoma cruzi oligopeptidase B (OPBTc) is involved in host cell invasion by generating a Ca(2+)-agonist necessary for recruitment and fusion of host lysosomes at the site of parasite attachment. The underlying mechanism remains unknown and further structural and functional characterization of OPBTc may help clarify its physiological function and lead to the development of new therapeutic molecules to treat Chagas disease. In the present work, size exclusion chromatography and analytical ultracentrifugation experiments demonstrate that OPBTc is a dimer in solution, an association salt and pH-resistant and independent of intermolecular disulfide bonds. The enzyme retains its dimeric structure and is fully active up to 42°C. OPBTc is inactivated and its tertiary, but not secondary, structure is disrupted at higher temperatures, as monitored by circular dichroism and fluorescence spectroscopy. It has a highly stable secondary structure over a broad range of pH, undergoes subtle tertiary structure changes at low pH and is less stable under moderate ionic strength conditions. These results bring new insights into the structural properties of OPBTc, contributing to future studies on the rational design of OPBTc inhibitors as a promising strategy for Chagas disease chemotherapy.
Translocation of bacterial toxins or effectors into host cells using the type III secretion (T3S) system is a conserved mechanism shared by many Gram-negative pathogens. Pseudomonas aeruginosa injects different proteins across the plasma membrane of target cells, altering the normal metabolism of the host. Protein translocation presumably occurs through a proteinaceous transmembrane pore formed by two T3S secreted protein translocators, PopB and PopD. Unfolded translocators are secreted through the T3S needle prior to insertion into the target membrane. Purified PopB and PopD form pores in model membranes. However, their tendency to form heterogeneous aggregates in solution had hampered the analysis of how these proteins undergo the transition from a denatured state to a membrane-inserted state. Translocators were purified as stable complexes with the cognate chaperone PcrH and isolated from the chaperone using 6 M urea. We report here the assembly of stable transmembrane pores by dilution of urea-denatured translocators in the presence of membranes. PopB and PopD spontaneously bound liposomes containing anionic phospholipids and cholesterol in a pH-dependent manner as observed by two independent assays, time-resolved Förster resonance energy transfer and sucrose-step gradient ultracentrifugation. Using Bodipy-labeled proteins, we found that PopB interacts with PopD on the membrane surface as determined by excitation energy migration and fluorescence quenching. Stable transmembrane pores are more efficiently assembled at pH <5.0, suggesting that acidic residues might be involved in the initial membrane binding and/or insertion. Altogether, the experimental setup described here represents an efficient method for the reconstitution and analysis of membrane-inserted translocators.
The type III secretion system (T3SS) is a complex macromolecular machinery employed by a number of Gram-negative species to initiate infection. Toxins secreted through the system are synthesized in the bacterial cytoplasm and utilize the T3SS to pass through both bacterial membranes and the periplasm, thus being introduced directly into the eukaryotic cytoplasm. A key element of the T3SS of all bacterial pathogens is the translocon, which comprises a pore that is inserted into the membrane of the target cell, allowing toxin injection. Three macromolecular partners associate to form the translocon: two are hydrophobic and one is hydrophilic, and the latter also associates with the T3SS needle. In this review, we discuss recent advances on the biochemical and structural characterization of the proteins involved in translocon formation, as well as their participation in the modification of intracellular signalling pathways upon infection. Models of translocon assembly and regulation are also discussed.
Thermal stability of the pore-forming domain of colicin A was studied by high sensitivity differential scanning calorimetry and circular dichroism spectroscopy. In the pH range between 8 and 5, the thermal denaturation of the protein in solution occurs at 66-69 degrees C and is characterized by the calorimetric enthalpy of approximately 90 kcal/M. At pH below 5, there is a rapid pH-dependent destabilization of the pore-forming domain resulting in the lowering of the midpoint denaturation temperature and a decrease in the calorimetric enthalpy of denaturation. Circular dichroism spectra in the near and far ultraviolet show that the thermotropic transition is associated with collapse of the native tertiary structure of the pore-forming domain, although a large proportion of the helical secondary structure remains preserved. The present data indicate some similarity also between acid-induced and temperature-induced denaturation of the pore-forming domain of colicin A. Association of the pore-forming domain with phospholipid vesicles of dioleoylphosphatidylglycerol results in total disappearance of the calorimetric transition, even at pH values as high as 7. Since lipid binding also induces collapse of the near ultraviolet circular dichroism spectrum, these data indicate that interaction with the membrane facilitates a conformational change within the pore-forming domain to a looser (denaturated-like) state. These findings are discussed in relation to the recent model (van der Goot, F. G., Gonzalez-Manas, J. M., Lakey, J. H., Pattus, F. (1991) Nature 354, 408-410) which postulates that a flexible "molten globule" state is an intermediate on the pathway to membrane insertion of colicin A.
The pore-forming α-toxin from Staphylococcus aureus is secreted as a soluble monomeric protein. In order to form a transmembrane channel, the protein has to undergo oligomerization
and membrane insertion. Previous studies have shown that channel formation is favored by acidic pH. We have analyzed the effect
of pH on the kinetics of channel formation as well as on the conformation of the toxin. Using a variety of spectroscopic probes
for protein structure, we have shown that α-toxin unfolded upon acidification and that the unfolding process occurred in at
least three steps. The various steps could be selectively affected by modifying the salt concentration or the temperature.
This unfolding was, however, only partial as the secondary structure remained native-like as witnessed by far UV CD measurements.
The first unfolding step, corresponding to a region of the C-terminal half of the toxin, is of particular importance as it
coincided with the exposure of hydrophobic patches on the surface of the protein as well as with the onset of channel formation.
Our observations strongly suggest that transition of the C-terminal half of α-toxin to a molten globule-like state is required
for channel formation.
Protein folding can be described in terms of the development of specific contacts between residues as a highly disordered polypeptide chain converts into the native state. Here we describe an NMR based strategy designed to detect such contacts by observation of nuclear Overhauser effects (NOEs). Experiments with alpha-lactalbumin reveal the existence of extensive NOEs between aromatic and aliphatic protons in the archetypal molten globule formed by this protein at low pH. Analysis of their time development provides direct evidence for near-native compactness of this state. Through a rapid refolding procedure the NOE intensity can be transferred efficiently into the resolved and assigned spectrum of the native state. This demonstrates the viability of using this approach to map out time-averaged interactions between residues in a partially folded protein.
Several Gram-negative bacterial pathogens have evolved a type III secretion system to deliver virulence effector proteins directly into eukaryotic cells, a process essential for disease. This specialized secretion process requires customized chaperones specific for particular effector proteins. The crystal structures of the enterohemorrhagic Escherichia coli O157:H7 Tir-specific chaperone CesT and the Salmonella enterica SigD-specific chaperone SigE reveal a common overall fold and formation of homodimers. Site-directed mutagenesis suggests that variable, delocalized hydrophobic surfaces observed on the chaperone homodimers are responsible for specific binding to a particular effector protein. Isothermal titration calorimetry studies of Tir-CesT and enzymatic activity profiles of SigD-SigE indicate that the effector proteins are not globally unfolded in the presence of their cognate chaperones.
Efficient folding of many newly synthesized proteins depends on assistance from molecular chaperones, which serve to prevent
protein misfolding and aggregation in the crowded environment of the cell. Nascent chain–binding chaperones, including trigger
factor, Hsp70, and prefoldin, stabilize elongating chains on ribosomes in a nonaggregated state. Folding in the cytosol is
achieved either on controlled chain release from these factors or after transfer of newly synthesized proteins to downstream
chaperones, such as the chaperonins. These are large, cylindrical complexes that provide a central compartment for a single
protein chain to fold unimpaired by aggregation. Understanding how the thousands of different proteins synthesized in a cell
use this chaperone machinery has profound implications for biotechnology and medicine.
The study of the membrane insertion of the translocation domain of diphtheria toxin deepens our insight into the interactions
between proteins and membranes. During cell intoxication, this domain undergoes a change from a soluble and folded state at
alkaline pH to a functional membrane-inserted state at acid pH. We found that hydrophobic and electrostatic interactions occur
in a sequential manner between the domain and the membrane during the insertion. The first step involves hydrophobic interactions
by the C-terminal region. This is because of the pH-induced formation of a molten globule specialized for binding to the membrane.
Accumulation of this molten globule follows a precise molecular mechanism adapted to the toxin function. The second step,
as the pH decreases, leads to the functional inserted state. It arises from the changes in the balance of electrostatic attractions
and repulsions between the N-terminal part and the membrane. Our study shows how the structural changes and the interaction
with membranes of the translocation domain are finely tuned by pH changes to take advantage of the cellular uptake system.
Gram-negative bacteria commonly interact with animal and plant hosts using type III secretion systems (TTSSs) for translocation of proteins into eukaryotic cells during infection. 10 of the 25 TTSS-encoding genes are homologous to components of the bacterial flagellar basal body, which the TTSS needle complex morphologically resembles. This indicates a common ancestry, although no TTSS sequence homologues for the genes encoding the flagellum are found. We here present an approximately 16-A structure of the central component, the needle, of the TTSS. Although the needle subunit is significantly smaller and shares no sequence homology with the flagellar hook and filament, it shares a common helical architecture ( approximately 5.6 subunits/turn, 24-A helical pitch). This common architecture implies that there will be further mechanistic analogies in the functioning of these two bacterial systems.
The primary effector for Shigella invasion of epithelial cells is IpaC, which is secreted via a type III secretion system. We recently reported that the IpaC N terminus is required for type III secretion and possibly other functions. In this study, mutagenesis was used to identify an N-terminal secretion signal and to determine the functional importance of the rest of the IpaC N terminus. The 15 N-terminal amino acids target IpaC for secretion by Shigella flexneri, and placing additional amino acids at the N terminus does not interfere with IpaC secretion. Furthermore, amino acid sequences with no relationship to the native IpaC secretion signal can also direct its secretion. Deletions introduced beyond amino acid 20 have no effect on secretion and do not adversely affect IpaC function in vivo until they extend beyond residue 50, at which point invasion function is completely eliminated. Deletions introduced at amino acid 100 and extending toward the N terminus reduce IpaC's invasion function but do not eliminate it until they extend to the N-terminal side of residue 80, indicating that a region from amino acid 50 to 80 is critical for IpaC invasion function. To explore this further, the ability of an IpaC N-terminal peptide to associate in vitro with its translocon partner IpaB and its chaperone IpgC was studied. The N-terminal peptide binds tightly to IpaB, but the IpaC central hydrophobic region also appears to participate in this binding. The N-terminal peptide also associates with the chaperone IpgC and IpaB is competitive for this interaction. Based on additional biophysical data, we propose that a region between amino acids 50 and 80 is required for chaperone binding, and that the IpaB binding domain is located downstream from, and possibly overlapping, this region. From these data, we propose that the secretion signal, chaperone binding region, and IpaB binding domain are located at the IpaC N terminus and are essential for presentation of IpaC to host cells during bacterial entry; however, IpaC effector activity may be located elsewhere.
Pseudomonas aeruginosa is the agent of opportunistic infections in immunocompromised individuals and chronic respiratory illnesses in cystic fibrosis patients. Pseudomonas aeruginosa utilizes a type III secretion system for injection of toxins into the host cell cytoplasm through a channel on the target membrane (the 'translocon'). Here, we have functionally and structurally characterized PopB and PopD, membrane proteins implicated in the formation of the P.aeruginosa translocon. PopB and PopD form soluble complexes with their common chaperone, PcrH, either as stable heterodimers or as metastable heterooligomers. Only oligomeric forms are able to bind to and disrupt cholesterol-rich membranes, which occurs within a pH range of 5-7 in the case of PopB/PcrH, and only at acidic pH for PcrH-free PopD. Electron microscopy reveals that upon membrane association PopB and PopD form 80 A wide rings which encircle 40 A wide cavities. Thus, formation of metastable oligomers precedes membrane association and ring generation in the formation of the Pseudomonas translocon, a mechanism which may be similar for other pathogens that employ type III secretion systems.
Pseudomonas aeruginosa efficiently intoxicates eukaryotic cells through the activity of the type III secretion-translocation system (TTSS). Gene
deletions within the translocation operon pcrGVH-popBD abolish pore-forming activity of P. aeruginosa strains with macrophages and TTSS-dependent hemolysis. Here we investigated the requirements for PcrV, PopB, and PopD in
pore formation by analyzing specific mutants using red blood cells (RBCs) and fibroblasts expressing green fluorescent protein
fused to actin. Simultaneous secretion of three proteins, PopB, PopD, and PcrV, was required to achieve wild-type hemolysis
and effector translocation. Deletion of pcrV in a cytotoxic strain did not affect secretion of PopB and PopD but abolished hemolytic activity and translocation of effectors
into fibroblasts. Notably, the PcrV-deficient mutant was not capable of inserting PopD into host cell membranes, whereas PopB
and PopD, but not PcrV, were readily found within membranes of wild-type-infected RBCs. Immunoprecipitation experiments performed
by using a liposome model of pore assembly revealed a direct interaction between PopD and PopB but not between PopD and PcrV.
Consequently, PcrV is necessary for the functional assembly of the PopB/D translocon complex but does not interact directly
with pore-forming Pop proteins.
Bacterial injectisomes deliver effector proteins straight into the cytosol of eukaryotic cells (type III secretion, T3S). Many effectors are associated with a specific chaperone that remains inside the bacterium when the effector is delivered. The structure of such chaperones and the way they interact with their substrate is well characterized but their main function remains elusive. Here, we describe and characterize SycO, a new chaperone for the Yersinia effector kinase YopO. The chaperone-binding domain (CBD) within YopO coincides with the membrane localization domain (MLD) targeting YopO to the host cell membrane. The CBD/MLD causes intrabacterial YopO insolubility and the binding of SycO prevents this insolubility but not folding and activity of the kinase. Similarly, SycE masks the MLD of YopE and SycT covers an aggregation-prone domain of YopT, presumably corresponding to its MLD. Thus, SycO, SycE and most likely SycT mask, inside the bacterium, a domain needed for proper localization of their cognate effector in the host cell. We propose that covering an MLD might be an essential function of T3S effector chaperones.
Type III secretion systems are essential virulence determinants for many Gram-negative bacterial pathogens. The type III secretion system consists of cytoplasmic, transmembrane, and extracellular domains. The extracellular domain is a hollow needle protruding above the bacterial surface and is held within a basal body that traverses both bacterial membranes. Effector proteins are translocated, via this external needle, directly into host cells, where they subvert normal cell functions to aid infection. Physical contact with host cells initiates secretion and leads to formation of a pore, thought to be contiguous with the needle channel, in the host-cell membrane. Here, we report the crystal structure of the Shigella flexneri needle subunit MxiH and a complete model for the needle assembly built into our three-dimensional EM reconstruction. The model, combined with mutagenesis data, reveals that signaling of host-cell contact is relayed through the needle via intersubunit contacts and suggests a mode of binding for a tip complex.
• needle complex
• protein secretion
• Shigella
The type III secretion injectisome is a complex nanomachine that allows bacteria to deliver protein effectors across eukaryotic cellular membranes. In recent years, significant progress has been made in our understanding of its structure, assembly and mode of operation. The principal structural components of the injectisome, from the base located in the bacterial cytosol to the tip of the needle protruding from the cell surface, have been investigated in detail. The structures of several constituent proteins were solved at the atomic level and important insights into the assembly process have been gained. However, despite the ongoing concerted efforts of molecular and structural biologists, the role of many of the constituent components of this nanomachine remain unknown.
Efficient type-III secretion depends on cytosolic molecular chaperones, which bind specifically to the translocators and effectors. In the past there has been a tendency to shoe-horn all type-III-secretion chaperones into a single structural and functional class. However, we have shown that the LcrH/SycD-like chaperones consist of three central tetratricopeptide-like repeats that are predicted to fold into an all-alpha-helical array that is quite distinct from the known structure of the SycE class of chaperones. Furthermore, we predict that this array creates a peptide-binding groove that is utterly different from the helix-binding groove in SycE. We present a homology model of LcrH/SycD that is consistent with existing mutagenesis data. We also report the existence of tetratricopeptide-like repeats in regulators of type-III secretion, such as HilA from Salmonella enterica and HrpB from Ralstonia solanacearum. The discovery of tetratricopeptide-like repeats in type-III-secretion regulators and chaperones provides a new conceptual framework for structural and mutagenesis studies and signals a potential unification of prokaryotic and eukaryotic chaperone biology.
The pore-forming alpha-toxin from Staphylococcus aureus is secreted as a soluble monomeric protein. In order to form a transmembrane channel, the protein has to undergo oligomerization and membrane insertion, Previous studies have shown that channel formation is favored by acidic pH. We have analyzed the effect of pH on the kinetics of channel formation as well as on the conformation of the toxin, Using a variety of spectroscopic probes for protein structure, we have shown that alpha-toxin unfolded upon acidification and that the unfolding process occurred in at least three steps, The various steps could be selectively affected by modifying the salt concentration or the temperature. This unfolding was, however, only partial as the secondary structure remained native-like as witnessed by far UV CD measurements. The first unfolding step, corresponding to a region of the C-terminal half of the toxin, is of particular importance as it coincided with the exposure of hydrophobic patches on the surface of the protein as well as with the onset of channel formation, Our observations strongly suggest that transition of the C-terminal half of alpha-toxin to a molten globule-like state is required for channel formation.
The 'molten' globular conformation of a protein is compact with a native secondary structure but a poorly defined tertiary structure. Molten globular states are intermediates in protein folding and unfolding and they may be involved in the translocation or insertion of proteins into membranes. Here we investigate the membrane insertion of the pore-forming domain of colicin A, a bacteriocin that depolarizes the cytoplasmic membrane of sensitive cells. We find that this pore- forming domain, the insertion of which depends on pH, undergoes a native to molten globule transition at acidic pH. The variation of the kinetic constant of membrane insertion of the protein into negatively charged lipid vesicles as a function of the interfacial pH correlates with the appearance of the acidic molten globular state, indicating that this state could be an intermediate formed during the insertion of colicin A into membranes.
Delivery of Yop effector proteins by pathogenic Yersinia across the eukaryotic cell membrane requires LcrV, YopB and YopD. These proteins were also required for channel formation in infected erythrocytes and, using different osmolytes, the contact-dependent haemolysis assay was used to study channel size. Channels associated with LcrV were around 3 nm, whereas the homologous PcrV protein of Pseudomonas aeruginosa induced channels of around 2 nm in diameter. In lipid bilayer membranes, purified LcrV and PcrV induced a stepwise conductance increase of 3 nS and 1 nS, respectively, in 1 M KCl. The regions important for channel size were localized to amino acids 127–195 of LcrV and to amino acids 106–173 of PcrV. The size of the channel correlated with the ability to translocate Yop effectors into host cells. We suggest that LcrV is a size-determining structural component of the Yop translocon.
The 'molten' globular conformation of a protein is compact with a native secondary structure but a poorly defined tertiary structure. Molten globular states are intermediates in protein folding and unfolding and they may be involved in the translocation or insertion of proteins into membranes. Here we investigate the membrane insertion of the pore-forming domain of colicin A, a bacteriocin that depolarizes the cytoplasmic membrane of sensitive cells. We find that this pore-forming domain, the insertion of which depends on pH, undergoes a native to molten globule transition at acidic pH. The variation of the kinetic constant of membrane insertion of the protein into negatively charged lipid vesicles as a function of the interfacial pH correlates with the appearance of the acidic molten globular state, indicating that this state could be an intermediate formed during the insertion of colicin A into membranes.
Binding of the hydrophobia fluorescent probe, 1-anilino-naphthalene-8-sulfonate (ANS), to synthetic polypeptides and proteins with a different structural organization has been studied. It has been shown that ANS has a much stronger affinity to the protein “molten globule” state, with a pronounced secondary structure and compactness, but without a tightly packed tertiary structure as compared with its affinity to the native and coil-like proteins, or to coil-like, α-helical, or β-structural hydrophilic homopolypeptides.
The possibility of using ANS for the study of equilibrium and kinetic molten globule intermediates is demonstrated, with carbonic anhydrase, β-lactamase, and α-lactalbumin as examples.
The refolding of apo bovine alpha-lactalbumin has been monitored in real time by NMR spectroscopy following rapid in situ dilution of a chemically denatured state. By examining individual resonances in the time-resolved NMR spectra, the native state has been shown to emerge in a cooperative manner from an intermediate formed in the dead-time of the experiments. The kinetics of folding to the native state are closely similar to those observed by stopped-flow fluorescence and near-UV circular dichroism. The NMR spectrum of the transient intermediate resembles closely that of the well characterized stable molten globule state formed at low pH. The results suggest that NMR can play a key role in describing at an atomic level the structural transitions occurring during protein folding.
Shigella species cause bacillary dysentery in humans by invading colonic epithelial cells. IpaB and IpaC, two major invasins of these pathogens, are secreted into the extracellular milieu. We show here that IpaB and IpaC form a complex in the extracellular medium and that each binds independently to a 17 kDa polypeptide, IpgC, in the bacterial cytoplasm. The IpgC polypeptide was found to be necessary for bacterial entry into epithelial cells, to stabilize the otherwise unstable IpaB protein, and to prevent the proteolytic degradation of IpaC that occurs through its association with unprotected IpaB. We propose that IpgC, which is not secreted and thus acts as a molecular chaperone, serves as a receptor that prevents premature oligomerization of IpaB and IpaC within the cytoplasm of Shigella cells.
Extracellular Yersinia adhering at the surface of a eukaryotic cell translocate effector Yops across the plasma membrane of the cell by a mechanism requiring YopD and YopB, the latter probably mediating pore formation. We studied the role of SycD, the intrabacterial chaperone of YopD. By producing GST-YopB hybrid proteins and SycD in Escherichia coli, we observed that SycD also binds specifically to YopB and that this binding reduces the toxicity of GST-YopB in E. coli. By analysis of a series of truncated GST-YopB proteins, we observed that SycD does not bind to a discrete segment of YopB. Using the same approach, we observed that YopD can also bind to YopB. Binding between YopB and YopD occurred even in the presence of SycD, and a complex composed of these three proteins could be immunoprecipitated from the cytoplasm of Yersinia. In a sycD mutant, the intracellular pool of YopB and YopD was greatly reduced unless the lcrV gene was also deleted. As LcrV is known to interact with YopB and YopD and to promote their secretion, we speculate that SycD prevents a premature association between YopB-YopD and LcrV.
Polypeptides emerging from the ribosome must fold into stable three-dimensional structures and maintain that structure throughout
their functional lifetimes. Maintaining quality control over protein structure and function depends on molecular chaperones
and proteases, both of which can recognize hydrophobic regions exposed on unfolded polypeptides. Molecular chaperones promote
proper protein folding and prevent aggregation, and energy-dependent proteases eliminate irreversibly damaged proteins. The
kinetics of partitioning between chaperones and proteases determines whether a protein will be destroyed before it folds properly.
When both quality control options fail, damaged proteins accumulate as aggregates, a process associated with amyloid diseases.
This chapter deals with the structure of the molten globules of various globular proteins revealed by the recent experimental studies. Recent advances in experimental techniques, including hydrogen-exchange NMR, solution X-ray scattering, and protein engineering, have provided detailed pictures of the molten globules for these proteins. The molten globule state has heterogeneous structures, in which one portion of a molecule is more organized and native-like with the other portions being less organized, although the overall structure satisfies the criteria of the molten globule state (compactness, the presence of secondary structure, and the lack of rigid tertiary structure). The chapter describes how the molten globule state has been identified as the intermediate of kinetic refolding and discusses the kinetic roles of the molten globule state in protein folding. The chapter also discusses thermodynamic stability and cooperativity of the molten globule state from the viewpoint of the hierarchy of protein folding, in which the molten globule state plays a role as a junction of two levels of the hierarchy.
The enteropathogen Yersinia pseudotuberculosis is a model system used to study the molecular mechanisms by which Gram-negative pathogens translocate effector proteins into target eukaryotic cells by a common type III secretion machine. Of the numerous proteins produced by Y. pseudotuberculosis that act in concert to establish an infection, YopD (Yersinia outer protein D) is a crucial component essential for yop regulation and Yop effector translocation. In this study, we describe the mechanisms by which YopD functions to control these processes. With the aid of the yeast two-hybrid system, we investigated the interaction between YopD and the cognate chaperone LcrH. We confirmed that non-secreted LcrH is necessary for YopD stabilization before secretion, presumably by forming a complex with YopD in the bacterial cytoplasm. At least in yeast, this complex depends upon the N-terminal domain and a C-terminal amphipathic alpha-helical domain of YopD. Introduction of amino acid substitutions within the hydrophobic side of the amphipathic alpha-helix abolished the YopD-LcrH interaction, indicating that hydrophobic, as opposed to electrostatic, forces of attraction are important for this process. Suppressor mutations isolated within LcrH could compensate for defects in the amphipathic domain of YopD to restore binding. Isolation of LcrH mutants unable to interact with wild-type YopD revealed no single domain responsible for YopD binding. The YopD and LcrH mutants generated in this study will be relevant tools for understanding YopD function during a Yersinia infection.
The type III secretion (TTS) system of Gram-negative pathogenic bacteria is composed of proteins that assemble into the TTS machinery, proteins that are secreted by this machinery and specific chaperones that are required for storage and sometimes secretion of these proteins. Many sequential protein interactions are involved in the TTS pathway to deliver effector proteins to host cells. We used the yeast two-hybrid system to investigate the interaction partners of the Shigella flexneri effectors and chaperones. Libraries of preys containing random fusions with fragments of the TTS proteins were screened using effectors and chaperones as baits. Interactions between the effectors IpaB and IpaC and their chaperone IpgC were detected by this method, and interaction domains were identified. Using a His-tagged IpgC protein to co-purify truncated IpaB and IpaC proteins, we showed that the chaperone-binding domain was unique and located in the N-terminus of these proteins. This domain was not required for the secretion of recombinant proteins but was involved in the stability of IpaC and instability of IpaB. Homotypic interactions were identified with the baits IpaA, IpaB and IpaC. Interactions between effectors and components of the TTS machinery were also selected that might give insights into regulation of the TTS process.
Many Gram-negative plant and animal pathogenic bacteria use a specialized type III secretion system (TTSS) as a molecular syringe to inject effector proteins directly into the host cell. Protein translocation across the eukaryotic host cell membrane is presumably mediated by a bacterial translocon. The structure of this predicted transmembrane complex and the mechanism of transport are far from being understood. In bacterial pathogens of animals, several putative type III secretion translocon proteins (TTPs) have been identified. Interestingly, TTP sequences are not conserved among different bacterial species, however, there are structural similarities such as transmembrane segments and coiled-coil regions. Accumulating evidence suggests that TTPs are components of oligomeric protein channels that are inserted into the host cell membrane by the TTSS.
To establish an infection, Yersinia pseudotuberculosis utilizes a plasmid-encoded type III secretion machine that permits the translocation of several anti-host factors into the cytosol of target eukaryotic cells. Secreted YopD is essential for this process. Pre-secretory stabilization of YopD is mediated by an interaction with its cognate chaperone, LcrH. YopD possesses LcrH binding domains located in the N-terminus and in a predicted amphipathic domain located near the C-terminus. This latter domain is also critical for Yersinia virulence. In this study, we designed synthetic peptides encompassing the C-terminal amphipathic domain of YopD. A solution structure of YopD278-300, a peptide that strongly interacted with LcrH, was obtained by NMR methods. The structure is composed of a well-defined amphipathic alpha helix ranging from Phe280 to Tyr291, followed by a type I beta turn between residues Val292 and His295. The C-terminal truncated peptides, YopD278-292 and YopD271-292, lacked helical structure, implicating the beta turn in helix stability. An interaction between YopD278-300 and its cognate chaperone, LcrH, was observed by NMR through line-broadening effects and chemical shift differences between the free peptide and the peptide-LcrH complex. These effects were not observed for the unstructured peptide, YopD278-292, which confirms that the alpha helical structure of the YopD amphipathic domain is a critical binding region of LcrH.
Proteins are linear polymers synthesized by ribosomes from activated amino acids. The product of this biosynthetic process is a polypeptide chain, which has to adopt the unique three-dimensional structure required for its function in the cell. In 1972, Christian Anfinsen was awarded the Nobel Prize for Chemistry for showing that this folding process is autonomous in that it does not require any additional factors or input of energy. Based on in vitro experiments with purified proteins, it was suggested that the correct three-dimensional structure can form spontaneously in vivo once the newly synthesized protein leaves the ribosome. Furthermore, proteins were assumed to maintain their native conformation until they were degraded by specific enzymes. In the last decade this view of cellular protein folding has changed considerably. It has become clear that a complicated and sophisticated machinery of proteins exists which assists protein folding and allows the functional state of proteins to be maintained under conditions in which they would normally unfold and aggregate. These proteins are collectively called molecular chaperones, because, like their human counterparts, they prevent unwanted interactions between their immature clients. In this review, we discuss the principal features of this peculiar class of proteins, their structure-function relationships, and the underlying molecular mechanisms.
The tetratricopeptide repeat (TPR) is a 34-amino acid alpha-helical motif that occurs in over 300 different proteins. In the different proteins, three to sixteen or more TPR motifs occur in tandem arrays and function to mediate protein-protein interactions. The binding specificity of each TPR protein is different, although the underlying structural motif is the same. Here we describe a statistical approach to the design of an idealized TPR motif. We present the high-resolution X-ray crystal structures (to 1.55 and 1.6 A) of designed TPR proteins and describe their solution properties and stability. A detailed analysis of these structures provides an understanding of the TPR motif, how it is repeated to give helical arrays with different superhelical twists, and how a very stable framework may be constructed for future functional designs.
Several medically important Gram-negative bacterial pathogens inject virulence factors into host cells through a type III secretion system and specialized bacterial chaperones are required for their effective delivery. Recent structural work shows that these chaperones maintain virulence factors in a partially non-globular conformation that is primed for unfolding and translocation through the 'injectisome'.
Mass spectrometry has grown in recent years to a well-accepted and increasingly important complementary technique in structural biology. Especially electrospray ionization mass spectrometry is well suited for the detection of non-covalent protein complexes and their interactions with DNA, RNA, ligands, and cofactors. Over the last decade, significant advances have been made in the ionization and mass analysis techniques, which makes the investigation of even larger and more heterogeneous intact assemblies feasible. These technological developments have paved the way to study intact non-covalent protein-protein interactions, assembly and disassembly in real time, subunit exchange, cooperativity effects, and effects of cofactors, allowing us a better understanding of proteins in cellular processes. In this review, we describe some of the latest developments and several highlights.
The structural properties of EspB, a virulence factor of the Escherichia coli O157 type III secretion system, were characterized. Far-UV and near-UV CD spectra, recorded between pH 1.0 and pH 7.0, show that the protein assumes alpha-helical structures and that some tyrosine tertiary contacts may exist. All tyrosine side-chains are exposed to water, as determined by acrylamide fluorescence quenching spectroscopy. An increase in the fluorescence intensity of 8-anilinonaphthalene-1-sulfonate was observed at pH 2.0 in the presence of EspB, whereas no such increase in fluorescence was observed at pH 7.0. These data suggest the formation of a molten globule state at pH 2.0. Destabilization of EspB at low pH was shown by urea-unfolding transitions, monitored by far-UV CD spectroscopy. The result from a sedimentation equilibrium study indicated that EspB assumes a monomeric form at pH 7.0, although its Stokes radius (estimated by multiangle laser light scattering) was twice as large as expected for a monomeric globular structure of EspB. These data suggest that EspB, at pH 7.0, assumes a relatively expanded conformation. The chemical shift patterns of EspB 15N-1H heteronuclear single quantum correlation spectra at pH 2.0 and 7.0 are qualitatively similar to that of urea-unfolded EspB. Taken together, the properties of EspB reported here provide evidence that EspB is a natively partially folded protein, but with less exposed hydrophobic surface than traditional molten globules. This structural feature of EspB may be advantageous when EspB interacts with various biomolecules during the bacterial infection of host cells.
Despite the fact that type III chaperones were discovered approximately 10 years ago, the precise role of most of them is still mysterious. A panoply of functions has been proposed for the members of this family of proteins. Type III chaperones have been suggested to act as anti-aggregation and stabilizing factors. They have also been proposed to keep their substrates in unfolded or partially folded structures, set a hierarchy on secretion, and participate in the regulation of the transcription of the type III substrates. Here, we review this enigmatic family of proteins, and discuss the experimental data supporting the roles proposed for type III chaperones.
Bacterial type III secretion systems are thought to translocate virulence proteins directly from the bacterial cytoplasm into host cells through a continuous molecular channel. Little is known about how the apparatus itself interacts with membranes and whether insertion of this structure into the host membrane has consequences for the bacteria apart from its beneficial role in delivering virulence proteins. New evidence suggests that membrane insertion of the bacterial type III apparatus might turn on a calcium-dependent signaling pathway resulting in phagolysosomal fusion.
Type III protein secretion systems are essential virulence factors of many bacteria pathogenic to humans, animals and plants. These systems mediate the transfer of bacterial virulence proteins directly into the host cell cytoplasm. Proteins are thought to travel this pathway in a largely unfolded manner, and a family of customized cytoplasmic chaperones, which specifically bind cognate secreted proteins, are essential for secretion. Here we show that InvC, an ATPase associated with a Salmonella enterica type III secretion system, has a critical function in substrate recognition. Furthermore, InvC induces chaperone release from and unfolding of the cognate secreted protein in an ATP-dependent manner. Our results show a similarity between the mechanisms of substrate recognition by type III protein secretion systems and AAA + ATPase disassembly machines.
Non-flagellar type III secretion systems (T3SSs) transport proteins across the bacterial cell and into eukaryotic cells. Targeting of proteins into host cells requires a dedicated translocation apparatus. Efficient secretion of the translocator proteins that make up this apparatus depends on molecular chaperones. Chaperones of the translocators (also called class-II chaperones) are characterized by the possession of three tandem tetratricopeptide repeats (TPRs). We wished to dissect the relations between chaperone structure and function and to validate a structural model using site-directed mutagenesis. Drawing on a number of experimental approaches and focusing on LcrH, a class-II chaperone from the Yersinia Ysc-Yop T3SS, we examined the contributions of different residues, residue classes and regions of the protein to chaperone stability, chaperone-substrate binding, substrate stability and secretion and regulation of Yop protein synthesis. We confirmed the expected role of the conserved canonical residues from the TPRs to chaperone stability and function. Eleven mutations specifically abrogated YopB binding or secretion while three mutations led to a specific loss of YopD secretion. These are the first mutations described for any class-II chaperone that allow interactions with one translocator to be dissociated from interactions with the other. Strikingly, all mutations affecting the interaction with YopB mapped to residues with side chains projecting from the inner, concave surface of the modelled TPR structure, defining a YopB interaction site. Conversely, all mutations preventing YopD secretion affect residues that lie on the outer, convex surface of the triple-TPR cluster in our model, suggesting that this region of the molecule represents a distinct interaction site for YopD. Intriguingly, one of the LcrH double mutants, Y40A/F44A, was able to maintain stable substrates inside bacteria, but unable to secrete them, suggesting that these two residues might influence delivery of substrates to the secretion apparatus.
The type III secretion system (T3SS) is a specialized apparatus evolved by Gram-negative bacteria to deliver effector proteins into host cells, thus facilitating the establishment of an infection. Effector translocation across the target cell plasma membrane is believed to occur via pores formed by at least two secreted translocator proteins, the functions of which are dependent upon customized class II T3SS chaperones. Recently, three internal tetratricopeptide repeats (TPRs) were identified in this class of chaperones. Here, defined mutagenesis of the class II chaperone PcrH of Pseudomonas aeruginosa revealed these TPRs to be essential for chaperone activity towards the translocator proteins PopB and PopD and subsequently for the translocation of exoenzymes into host cells.
Salmonella invasion protein A (SipA) is translocated into host cells by a type III secretion system (T3SS) and comprises two regions: one domain binds its cognate type III secretion chaperone, InvB, in the bacterium to facilitate translocation, while a second domain functions in the host cell, contributing to bacterial uptake by polymerizing actin. We present here the crystal structures of the SipA chaperone binding domain (CBD) alone and in complex with InvB. The SipA CBD is found to consist of a nonglobular polypeptide as well as a large globular domain, both of which are necessary for binding to InvB. We also identify a structural motif that may direct virulence factors to their cognate chaperones in a diverse range of pathogenic bacteria. Disruption of this structural motif leads to a destabilization of several chaperone-substrate complexes from different species, as well as an impairment of secretion in Salmonella.
Efficient type-III secretion depends on cytosolic molecular chaperones, which bind specifically to the translocators and effectors. In the past there has been a tendency to shoe-horn all type-III-secretion chaperones into a single structural and functional class. However, we have shown that the LcrH/SycD-like chaperones consist of three central tetratricopeptide-like repeats that are predicted to fold into an all-alpha-helical array that is quite distinct from the known structure of the SycE class of chaperones. Furthermore, we predict that this array creates a peptide-binding groove that is utterly different from the helix-binding groove in SycE. We present a homology model of LcrH/SycD that is consistent with existing mutagenesis data. We also report the existence of tetratricopeptide-like repeats in regulators of type-III secretion, such as HilA from Salmonella enterica and HrpB from Ralstonia solanacearum. The discovery of tetratricopeptide-like repeats in type-III-secretion regulators and chaperones provides a new conceptual framework for structural and mutagenesis studies and signals a potential unification of prokaryotic and eukaryotic chaperone biology.
Type III secretion/translocation systems are essential actors in the pathogenicity of Gram-negative bacteria. The injection of bacterial toxins across the host cell plasma membranes is presumably accomplished by a proteinaceous structure, the translocon. In vitro, Pseudomonas aeruginosa translocators PopB and PopD form ringlike structures observed by electron microscopy. We demonstrate here that PopB and PopD are functionally active and sufficient to form pores in lipid vesicles. Furthermore, the two translocators act in synergy to promote membrane permeabilization. The size-based selectivity observed for the passage of solutes indicates that the membrane permeabilization is due to the formation of size-defined pores. Our results provide also new insights into the mechanism of translocon pore formation that may occur during the passage of toxins from the bacterium into the cell. While proteins bind to lipid vesicles equally at any pH, the kinetics of membrane permeabilization accelerate progressively with decreasing pH values. Electrostatic interactions and the presence of anionic lipids were found to be crucial for pore formation whereas cholesterol did not appear to play a significant role in functional translocon formation.
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