Fig 1 - uploaded by Ivaylo Gentschev
Content may be subject to copyright.
![Topological model of hemolysin secretion. In response to HlyA engagement, a HlyD trimer [33], which bends at the gap between the coiled-coil regions, interacts with the trimeric TolC protein to form a continuous trans-periplasmic export channel. Supported by the observation that the carboxyterminal sequence of HlyD is in contact with periplasmic loop(s) of HlyB, Schlör et al. proposed HlyD has a hairpin conformation, which is essential for these interactions [63]. The HlyD coiled-coil regions are indicated and the asterisk marks the carboxy-terminal domain of HlyD. Only two HlyD molecules are shown for clarity. The circles labeled ATP denote the ATP-binding sites of HlyB. ATP hydrolysis by HlyB provides the energy for the transport process and leads to direct secretion of HlyA into the extracellular medium without formation of periplasmic intermediates. Modified from [26,30].](profile/Ivaylo-Gentschev/publication/11602605/figure/fig1/AS:905163200004099@1592819132465/Topological-model-of-hemolysin-secretion-In-response-to-HlyA-engagement-a-HlyD-trimer.png)
Topological model of hemolysin secretion. In response to HlyA engagement, a HlyD trimer [33], which bends at the gap between the coiled-coil regions, interacts with the trimeric TolC protein to form a continuous trans-periplasmic export channel. Supported by the observation that the carboxyterminal sequence of HlyD is in contact with periplasmic loop(s) of HlyB, Schlör et al. proposed HlyD has a hairpin conformation, which is essential for these interactions [63]. The HlyD coiled-coil regions are indicated and the asterisk marks the carboxy-terminal domain of HlyD. Only two HlyD molecules are shown for clarity. The circles labeled ATP denote the ATP-binding sites of HlyB. ATP hydrolysis by HlyB provides the energy for the transport process and leads to direct secretion of HlyA into the extracellular medium without formation of periplasmic intermediates. Modified from [26,30].
Source publication
Many Gram-negative bacteria use a type I secretion system to translocate proteins, including pore-forming toxins, proteases, lipases and S-layer proteins, across both the inner and outer membranes into the extracellular surroundings. The Escherichia coli alpha-hemolysin (HlyA) secretion system is the prototypical and best characterized type I secre...
Context in source publication
Context 1
... secretion of HlyA has been analyzed in great detail by several groups. HlyA seems to interact with the cytoplasmic region of the pre-formed HlyB-D complex (Fig. 1). After the binding of the HlyA secretion signal by the HlyB-D complex, HlyD induces the interaction with TolC. Koronakis et al. [29] have produced crystal structure data for TolC that suggest a model for a possible TolC-HlyD interaction. A HlyD trimer has a mass very similar to the TolC trimer and could provide a cylinder of almost ...
Citations
... The hlyA secretion system of Escherichia coli has already been used successfully for secretion of antigens [6,7]. The hly operon consists of four essential components, the transcription enhancer sequence hlyR, the HlyA acylating enzyme HlyC and the inner membrane complex HlyB-HlyD [8]. HlyB transports antigens which have been fused to the hlyA-signal across the inner membrane, while HlyD interacts with TolC in the outer membrane for secretion into the lumen [8]. ...
... The hly operon consists of four essential components, the transcription enhancer sequence hlyR, the HlyA acylating enzyme HlyC and the inner membrane complex HlyB-HlyD [8]. HlyB transports antigens which have been fused to the hlyA-signal across the inner membrane, while HlyD interacts with TolC in the outer membrane for secretion into the lumen [8]. Actively exported antigens become accessible to the host immune system without the bacteria being degraded [8][9][10]. ...
... HlyB transports antigens which have been fused to the hlyA-signal across the inner membrane, while HlyD interacts with TolC in the outer membrane for secretion into the lumen [8]. Actively exported antigens become accessible to the host immune system without the bacteria being degraded [8][9][10]. ...
Salmonella enterica Serovar Typhi Ty21a (Ty21a) is the only licensed oral vaccine against typhoid fever. Due to its excellent safety profile, it has been used as a promising vector strain for the expression of heterologous antigens for mucosal immunization. As the efficacy of any bacterial live vector vaccine correlates with its ability to express and present sufficient antigen, the genes for antigen expression are traditionally located on plasmids with antibiotic resistance genes for stabilization. However, for use in humans, antibiotic selection of plasmids is not applicable, leading to segregational loss of the antigen-producing plasmid. Therefore, we developed an oral Ty21a-based vaccine platform technology, the JMU-SalVac-system (Julius-Maximilians-Universität Würzburg) in which the antigen delivery plasmids (pSalVac-plasmid-series) are stabilized by a ΔtyrS/tyrS⁺-based balanced-lethal system (BLS). The system is made up of the chromosomal knockout of the essential tyrosyl-tRNA-synthetase gene (tyrS) and the in trans complementation of tyrS on the pSalVac-plasmid. Further novel functional features of the pSalVac-plasmids are the presence of two different expression cassettes for the expression of protein antigens. In this study, we present the construction of vaccine strains with BLS plasmids for antigen expression. The expression of cytosolic and secreted mRFP and cholera toxin subunit B (CTB) proteins as model antigens is used to demonstrate the versatility of the approach. As proof of concept, we show the induction of previously described in vivo inducible promoters cloned into pSalVac-plasmids during infection of primary macrophages and demonstrate the expression of model vaccine antigens in these relevant human target cells. Therefore, antigen delivery strains developed with the JMU-SalVac technology are promising, safe and stable vaccine strains to be used against mucosal infections in humans.
... E. coli can secrete hemolysin and induce erythrocyte lysis. [14] However, the PDMC-PEG nanocoating significantly reduced red blood cell hemolysis from 8.27% to 1.64% at a concentration of 5 × 10 8 cfu mL −1 (Figure 2I). Considering that bacterial proliferation was inhibited at such a high concentration of E. coli@PDMC-PEG, the decreased hemolytic ratio was mainly due to limited hemolysin secretion rather than the proliferative capacity of E. coli. ...
Stimulating the cyclic guanosine monophophate(GMP)‐adenosine monophosphate (AMP) synthase (cGAS)‐stimulator of interferon genes (STING) pathway is a crucial strategy by which bacteria activate the tumor immune system. However, the limited stimulation capability poses significant challenges in advancing bacterial immunotherapy. Here, an adenosine 5′‐triphosphate (ATP)‐responsive manganese (Mn)‐based bacterial material (E. coli@PDMC‐PEG (polyethylene glycol)) is engineered successfully, which exhibits an exceptional ability to synergistically activate the cGAS‐STING pathway. In the tumor microenvironment, which is characterized by elevated ATP levels, this biohybrid material degrades, resulting in the release of divalent manganese ions (Mn²⁺) and subsequent bacteria exposure. This combination synergistically activates the cGAS‐STING pathway, as Mn²⁺ enhances the sensitivity of cGAS to the extracellular DNA (eDNA) secreted by the bacteria. The results of the in vivo experiments demonstrate that the biohybrid materials E. coli@PDMC‐PEG and VNP20009@PDMC‐PEG effectively inhibit the growth of subcutaneous melanoma in mice and in situ liver cancer in rabbits. Valuable insights for the development of bacteria‐based tumor immunotherapy are provided here.
... As highlighted by investigations of other secretion systems (62)(63)(64)(65), extracellular secretion via the MccV system could provide opportunities for in vivo small-protein delivery. Oral delivery of proteins is challenging because they are quickly degraded during passage through the stomach (66). ...
Small proteins perform a diverse array of functions, from microbial competition, to endocrine signaling, to building biomaterials. Microbial systems that can produce recombinant small proteins enable discovery of new effectors, exploration of sequence activity relationships, and have the potential for in vivo delivery. However, we lack simple systems for controlling small-protein secretion from Gram-negative bacteria. Microcins are small-protein antibiotics secreted by Gram-negative bacteria that inhibit the growth of neighboring microbes. They are exported from the cytosol to the environment in a one-step process through a specific class of type I secretion systems (T1SSs). However, relatively little is known about substrate requirements for small proteins exported through microcin T1SSs. Here, we investigate the prototypic microcin V T1SS from Escherichia coli and show that it can export a remarkably wide range of natural and synthetic small proteins. We demonstrate that secretion is largely independent of the cargo protein's chemical properties and appears to be constrained only by protein length. We show that a varied range of bioactive sequences, including an antibacterial protein, a microbial signaling factor, a protease inhibitor, and a human hormone, can all be secreted and elicit their intended biological effect. Secretion through this system is not limited to E. coli, and we demonstrate its function in additional Gram-negative species that can inhabit the gastrointestinal tract. Our findings uncover the highly promiscuous nature of small-protein export through the microcin V T1SS, which has implications for native-cargo capacity and the use of this system in Gram-negative bacteria for small-protein research and delivery. IMPORTANCE Type I secretion systems for microcin export in Gram-negative bacteria transport small antibacterial proteins from the cytoplasm to the extracellular environment in a single step. In nature, each secretion system is generally paired with a specific small protein. We know little about the export capacity of these transporters and how cargo sequence influences secretion. Here, we investigate the microcin V type I system. Remarkably, our studies show that this system can export small proteins of diverse sequence composition and is only limited by protein length. Furthermore, we demonstrate that a wide range of bioactive small proteins can be secreted and that this system can be used in Gram-negative species that colonize the gastrointestinal tract. These findings expand our understanding of secretion through type I systems and their potential uses in a variety of small-protein applications.
... E. coli α-hemolysin is the best-studied repeat-in-toxin protein (repeat-in-toxin proteins are widespread among Gram-negative bacteria) released by the type I secretion system. It is an important virulence factor due to its cytolytic and cytotoxic activity against a diverse range of mammalian cell types (e.g., erythrocytes, monocytes, granulocytes, and endothelial cells) [52]. The α-hemolysin in meningitic E. coli K1 strain has been shown to reduce the TGFβ1 receptor TGFBRII and the critical transcription factor Gli2 of hedgehog signaling in BMECs, eventually leading to the BBB breakdown [53]. ...
Despite advances in supportive care and antimicrobial treatment, bacterial meningitis remains the most serious infection of the central nervous system (CNS) that poses a serious risk to life. This clinical dilemma is largely due to our insufficient knowledge of the pathology behind this disease. By controlling the entry of molecules into the CNS microenvironment, the blood–brain barrier (BBB), a highly selective cellular monolayer that is specific to the CNS’s microvasculature, regulates communication between the CNS and the rest of the body. A defining feature of the pathogenesis of bacterial meningitis is the increase in BBB permeability. So far, several contributing factors for BBB disruption have been reported, including direct cellular damage brought on by bacterial virulence factors, as well as host-specific proteins or inflammatory pathways being activated. Recent studies have demonstrated that targeting pathological factors contributing to enhanced BBB permeability is an effective therapeutic complement to antimicrobial therapy for treating bacterial meningitis. Hence, understanding how these meningitis-causing pathogens affect the BBB permeability will provide novel perspectives for investigating bacterial meningitis’s pathogenesis, prevention, and therapies. Here, we summarized the recent research progress on meningitis-causing pathogens disrupting the barrier function of BBB. This review provides handy information on BBB disruption by meningitis-causing pathogens, and helps design future research as well as develop potential combination therapies.
... It was not pathogenic to banana (NPB), but exhibited pathogenicity to cucurbits (Florent et al., 2016). Hemolysin secretion protein HlyB was the only unique VF detected in this strain, which functioned as an adenosine triphosphate (ATP)-binding cassette (ABC) transporter to enable the bacteria to secrete toxins at the expense of ATP hydrolysis (Gentschev et al., 2002;Zhou et al., 2018). In addition, 20 strains had single unique VFs, and 30 strains had fewer than 10 unique VFs, which may be related to their pathogenicity and host specificity. ...
Ralstonia solanacearum species complex (RSSC) is a diverse group of plant pathogens that attack a wide range of hosts and cause devastating losses worldwide. In this study, we conducted a comprehensive analysis of 131 RSSC strains to detect their genetic diversity, pathogenicity, and evolution dynamics. Average nucleotide identity analysis was performed to explore the genomic relatedness among these strains, and finally obtained an open pangenome with 32,961 gene families. To better understand the diverse evolution and pathogenicity, we also conducted a series of analyses of virulence factors (VFs) and horizontal gene transfer (HGT) in the pangenome and at the single genome level. The distribution of VFs and mobile genetic elements (MGEs) showed significant differences among different groups and strains, which were consistent with the new nomenclatures of the RSSC with three distinct species. Further functional analysis showed that most HGT events conferred from Burkholderiales and played a great role in shaping the genomic plasticity and genetic diversity of RSSC genomes. Our work provides insights into the genetic polymorphism, evolution dynamics, and pathogenetic variety of RSSC and provides strong supports for the new taxonomic classification, as well as abundant resources for studying host specificity and pathogen emergence.
... Moreover, the comparative analysis of non-core genes revealed that W. coagulans exploits other less widespread secretion systems (Table 2). Indeed, 36D1 and CSL1 bear the hylD gene that is a component of the Escherichia coli α-hemolysin secretion system (type I secretion system) which is otherwise made up also of hlyA, hlyB, and tolC [45]. Most, but not all, W. coagulans strains are featured by genes of the type VII secretion system (cT7SS) which exports proteins lacking a canonical cleavable signal peptide but bearing a WXG motif at the N-terminus [46]. ...
The production of biochemicals requires the use of microbial strains with efficient substrate
conversion and excellent environmental robustness, such as Weizmannia coagulans species. So far, the
genomes of 47 strains have been sequenced. Herein, we report a comparative genomic analysis of
nine strains on the full repertoire of Carbohydrate-Active enZymes (CAZymes), secretion systems,
and resistance mechanisms to environmental challenges. Moreover, Clustered Regularly Interspaced
Short Palindromic Repeats (CRISPR) immune system along with CRISPR-associated (Cas) genes, was
also analyzed. Overall, this study expands our understanding of the strain’s genomic diversity of W.
coagulans to fully exploit its potential in biotechnological applications.
... Discrete mutations in these translocation proteins do not usually hamper the pore formation efficiency, rather the docking or translocation efficiency of those effector molecules are restricted (Adams et al., 2015). Even few T3SSs carrying bacteria can induce hemolysis in red blood cells, where the hemolytic activity is due to the assembly of International Journal of Bio-resource and Stress Management 2022, 13(2):162-171 the T3SS translocon in a host cell membrane (Gentschev et al., 2002). ...
Bacterial type III secretions system (T3SS) is a membrane embedded needle like macromolecular complex structure present in Gram-negative bacteria and mostly found in Yersinia, Salmonella, Shigella, Entero Pathogenic E. coli (EPEC), Entero Haemorrhagic E. coli (EHEC) and Pseudomonas aeruginosa. The infection dynamic involves several subcellular components for easy delivery of the effector molecules from bacteria to the host cells to survive the host immune mechanism. Mainly three categories of proteins are involved- structural, translocator and effector proteins. T3SSs can be classified into seven phylogenetic families, based on the genetic analysis of their components. Multiple T3SSs are found in the same bacteria with the purpose of causing infection in multiple steps.Their contribution to virulence mechanism is mainly through modification of the host cytoskeleton system or interfering with the signaling Pathways in the host cellular events related to the defensive mechanism. The infection requires multi-step regulatory strategies which include spatiotemporal regulation of a different set of effector
proteins encoded genes. Despite their contribution in virulence mechanism, they can be utilized by re-engineering them to deliver either various therapeutic protein agents or could be used as an alternative novel approach for antigen delivery into the host. Apart from its use as the delivery platform they can be targeted using broad-spectrum inhibitors against diverse sets of T3SS mediated diseases. Therefore, this review summarizes the basic structure, its regulatory mechanism in different bacteria and the future perspective.
... Despite a long list of successfully secreted heterologous proteins, HlyA1 is still far away from being an universal secretion platform, due to a narrow range of possible substrates as well as low yields of secretion. [14][15][16][17] It should be noted that an engineered T1SS has recently been developed to allow efficient production of peptides and small proteins. [18] The HlyA secretion system forms a tripartite doublemembrane-spanning channel trough association of an inner membrane ATP-binding cassette transporter (HlyB), a membrane fusion protein (HlyD), and an outer membrane protein (TolC). ...
Type 1 secretion systems (T1SS) have a relatively simple architecture compared to other classes of secretion systems and therefore, are attractive to be optimized by protein engineering. Here, we report a KnowVolution campaign for the hemolysin (Hly) enhancer fragment, an untranslated region upstream of the hlyA gene, of the hemolysin T1SS of Escherichia coli to enhance its secretion efficiency. The best performing variant of the Hly enhancer fragment contained five nucleotide mutations at five positions (A30U, A36U, A54G, A81U, and A116U) resulted in a 2‐fold increase in the secretion level of a model lipase fused to the secretion carrier HlyA1. Computational analysis suggested that altered affinity to the generated enhancer fragment towards the S1 ribosomal protein contributes to the enhanced secretion levels. Furthermore, we demonstrate that involving a native terminator region along with the generated Hly enhancer fragment increased the secretion levels of the Hly system up to 5‐fold.
... Moreover, the comparative analysis of non-core genes revealed that B. coagulans exploits other less widespread secretion systems (Table 2). Indeed, 36D1 and CSL1 bear the hylD gene that is a component of the Escherichia coli αhemolysin secretion system (type I secretion system) which is otherwise made up also of hlyA, hlyB, and tolC [41]. ...
The production of bio-chemicals requires the use of microbial strains with efficient substrate conversion and excellent environmental robustness, such as Bacillus coagulans spp. So far the genomes of about 50 strains have been sequenced. Herein, we report a comparative genomic analysis of nine strains on the full repertoire of CAZymes, secretion systems, and resistance mechanisms to environmental challenges. Moreover, B. coagulans Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) immune system along with CRISPR-associated Cas) genes, was also analysed. Overall, this study expands our understanding of the strains genomic diversity of B. coagulans to fully exploit its potential in biotechnological applications.
... 240 The secretory pathway of hly is well known, as it is both the prototypical and best characterized type I secretion system. 241 HlyA reaches the extracellular space in a soluble form without the formation of periplasmic intermediates. 242 Interestingly, a fraction of hlyA appears to be very tightly associated with outer membrane vesicles. ...
Growing evidence suggests that imbalances in resident microbes (dysbiosis) can promote chronic inflammation, immune-subversion, and production of carcinogenic metabolites, thus leading to neoplasia. Yet, evidence to support a direct link of individual bacteria species to human sporadic cancer is still limited. This chapter focuses on several emerging bacterial toxins that have recently been characterized for their potential oncogenic properties toward human orodigestive cancer and the presence of which in human tissue samples has been documented. These include cytolethal distending toxins produced by various members of gamma and epsilon Proteobacteria, Dentilisin from mammalian oral Treponema, Pasteurella multocida toxin, two Fusobacterial toxins, FadA and Fap2, Bacteroides fragilis toxin, colibactin, cytotoxic necrotizing factors and α-hemolysin from Escherichia coli, and Salmonella enterica AvrA. It was clear that these bacterial toxins have biological activities to induce several hallmarks of cancer. Some toxins directly interact with DNA or chromosomes leading to their breakdowns, causing mutations and genome instability, and others modulate cell proliferation, replication and death and facilitate immune evasion and tumor invasion, prying specific oncogene and tumor suppressor pathways, such as p53 and β-catenin/Wnt. In addition, most bacterial toxins control tumor-promoting inflammation in complex and diverse mechanisms. Despite growing laboratory evidence to support oncogenic potential of selected bacterial toxins, we need more direct evidence from human studies and mechanistic data from physiologically relevant experimental animal models, which can reflect chronic infection in vivo, as well as take bacterial-bacterial interactions among microbiome into consideration.
