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![Chemical proteomics reveals that IcsB targets multiple host membrane proteins for lysine Nε-fatty acylation
a, In-gel fluorescence visualization of the stearoylome profile of IcsB (WT or C306A)-transfected (left) and S. flexneri (WT, ΔicsB, ΔvirA or ΔvirAΔicsB)-infected (right) HeLa cells. Coomassie blue staining confirms equal protein loading. b, Scatter plots of SILAC proteomic data. Hits with an abundance increase of more than threefold (log2[H/L > 1.58] in the forward SILAC and log2[H/L < −1.58] in the reverse SILAC) in the IcsB group relative to the control group are shown in red. H/L represents the ratio between heavy and light label partners in the indicated (forward or reverse) experiment. c, Validation of IcsB modification of the proteomic hits. A total of 11 selected candidate substrates were individually transfected into 293T cells. The cells were infected with S. flexneri WT or ΔicsB in the presence of Alk-16, and subjected to in-gel fluorescence assay. Anti-Flag immunoblotting confirms equal loading of the indicated samples. d, Effects of lysine mutation in the PBR of selected substrates on their modification by IcsB. 293T cells were co-transfected with IcsB and Flag-tagged RhoA, VAMP8 or Rab13, or the indicated lysine mutant. The cells were metabolized with Alk-16 and subjected to in-gel fluorescence assay. Data are representative of two (a and b) or three (c and d) independent experiments.](publication/326690738/figure/fig4/AS:731581509541900@1551434032247/Chemical-proteomics-reveals-that-IcsB-targets-multiple-host-membrane-proteins-for-lysine.png)
Chemical proteomics reveals that IcsB targets multiple host membrane proteins for lysine Nε-fatty acylation a, In-gel fluorescence visualization of the stearoylome profile of IcsB (WT or C306A)-transfected (left) and S. flexneri (WT, ΔicsB, ΔvirA or ΔvirAΔicsB)-infected (right) HeLa cells. Coomassie blue staining confirms equal protein loading. b, Scatter plots of SILAC proteomic data. Hits with an abundance increase of more than threefold (log2[H/L > 1.58] in the forward SILAC and log2[H/L < −1.58] in the reverse SILAC) in the IcsB group relative to the control group are shown in red. H/L represents the ratio between heavy and light label partners in the indicated (forward or reverse) experiment. c, Validation of IcsB modification of the proteomic hits. A total of 11 selected candidate substrates were individually transfected into 293T cells. The cells were infected with S. flexneri WT or ΔicsB in the presence of Alk-16, and subjected to in-gel fluorescence assay. Anti-Flag immunoblotting confirms equal loading of the indicated samples. d, Effects of lysine mutation in the PBR of selected substrates on their modification by IcsB. 293T cells were co-transfected with IcsB and Flag-tagged RhoA, VAMP8 or Rab13, or the indicated lysine mutant. The cells were metabolized with Alk-16 and subjected to in-gel fluorescence assay. Data are representative of two (a and b) or three (c and d) independent experiments.
Source publication
Shigella flexneri, an intracellular Gram-negative bacterium causative for shigellosis, employs a type III secretion system to deliver virulence effectors into host cells. One such effector, IcsB, is critical for S. flexneri intracellular survival and pathogenesis, but its mechanism of action is unknown. Here, we discover that IcsB is an 18-carbon f...
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The type 3 secretion system (T3SS) is a nanomachine required for virulence of many bacterial pathogens that infect humans. The system delivers bacterial virulence proteins into the cytosol of human cells, where the virulence proteins promote bacterial infection. The T3SS forms a translocon pore in the membranes of target cells. This pore is the por...
Citations
... Besides that, Yang et al [16] suggested that Salmonella escapes host immune responses by inhibiting autophagy degradation. Previously, a large number of bacteria have been shown to evade NOD pathway-mediated intestinal immune surveillance by inhibiting autophagy [17,18]. Molecule evidence has been found that autophagy is involved in the secretion of membrane vesicles from Listeria monocytogenes in vitro [19]. ...
Autophagy is a cellular catabolic process characterized by the formation of double-membrane autophagosomes. Transmission electron microscopy is the most rigorous method to clearly visualize autophagic engulfment and degradation. A large number of studies have shown that autophagy is closely related to the digestion, secretion, and regeneration of gastrointestinal (GI) cells. However, the role of autophagy in GI diseases remains controversial. This article focuses on the morphological and biochemical characteristics of autophagy in GI diseases, in order to provide new ideas for their diagnosis and treatment.
... Future studies will involve the identification of the acetylation of the eIF3-K subunit and other potential substrates of Lpg0103 from infected amoebae and differentiated U937 cells. Furthermore, the modification of multiple lysine residues within the vicinity of each other on the host target has been adopted by other bacterial effectors, such as IcsB from Shigella flexneri that fatty acylates multiple lysine residues on the C-terminal polybasic tail of multiple members of the Ras, Rho, and Ras small GTPase families (37). The biological significance of this modification for S. flexneri infection has not been elucidated. ...
The survival of Legionella spp. as intracellular pathogens relies on the combined action of protein effectors delivered inside their eukaryotic hosts by the Dot/Icm (defective in organelle trafficking/intracellular multiplication) type IVb secretion system. The specific repertoire of effector arsenals varies dramatically across over 60 known species of this genera with Legionella pneumophila responsible for most cases of Legionnaires’ disease in humans encoding over 360 Dot/Icm effectors. However, a small subset of “core” effectors appears to be conserved across all Legionella species raising an intriguing question of their role in these bacteria’s pathogenic strategy, which for most of these effectors remains unknown. L. pneumophila Lpg0103 effector, also known as VipF, represents one of the core effector families that features a tandem of Gcn5-related N-acetyltransferase (GNAT) domains. Here, we present the crystal structure of the Lha0223, the VipF representative from Legionella hackeliae in complex with acetyl-coenzyme A determined to 1.75 Å resolution. Our structural analysis suggested that this effector family shares a common fold with the two GNAT domains forming a deep groove occupied by residues conserved across VipF homologs. Further analysis suggested that only the C-terminal GNAT domain of VipF effectors retains the active site composition compatible with catalysis, whereas the N-terminal GNAT domain binds the ligand in a non-catalytical mode. We confirmed this by in vitro enzymatic assays which revealed VipF activity not only against generic small molecule substrates, such as chloramphenicol, but also against poly-L-lysine and histone-derived peptides. We identified the human eukaryotic translation initiation factor 3 (eIF3) complex co-precipitating with Lpg0103 and demonstrated the direct interaction between the several representatives of the VipF family, including Lpg0103 and Lha0223 with the K subunit of eIF3. According to our data, these interactions involve primarily the C-terminal tail of eIF3-K containing two lysine residues that are acetylated by VipF. VipF catalytic activity results in the suppression of eukaryotic protein translation in vitro, revealing the potential function of VipF “core” effectors in Legionella’s pathogenic strategy.
IMPORTANCE
By translocating effectors inside the eukaryotic host cell, bacteria can modulate host cellular processes in their favor. Legionella species, which includes the pneumonia-causing Legionella pneumophila, encode a widely diverse set of effectors with only a small subset that is conserved across this genus. Here, we demonstrate that one of these conserved effector families, represented by L. pneumophila VipF (Lpg0103), is a tandem Gcn5-related N-acetyltransferase interacting with the K subunit of human eukaryotic initiation factor 3 complex. VipF catalyzes the acetylation of lysine residues on the C-terminal tail of the K subunit, resulting in the suppression of eukaryotic translation initiation factor 3-mediated protein translation in vitro. These new data provide the first insight into the molecular function of this pathogenic factor family common across Legionellae.
... However, the Shigella ipgD mutant showed no effect on Rab13 residence on the BCV (20.4 ± 2.6 min) despite its interaction with Rab8, a Rab GTPase that is recognized as the same family as Rab13 (Fig. 5A). Another report on the Shigella effector IcsB had suggested that it modifies a cohort of host proteins, including Rab13, by its acyltransferase activity to possibly enhance membrane anchor of the modified proteins 43 . We thus also tested the Rab13 recruitment in the presence of the icsB mutant and found that Rab13 stayed for a shorter time at the BCV compared to the infection of the WT strain (14.3 ± 2.2 min) (Fig. 5A). ...
Intracellular bacterial pathogens gain entry to mammalian cells inside a vacuole derived from the host membrane. Some of them escape the bacteria-containing vacuole (BCV) and colonize the cytosol. Bacteria replicating within BCVs coopt the microtubule network to position it within infected cells, whereas the role of microtubules for cyto-invasive pathogens remains obscure. Here, we show that the microtubule motor cytoplasmic dynein-1 and specific activating adaptors are hijacked by the enterobacterium Shigella flexneri. These host proteins were found on infection-associated macropinosomes (IAMs) formed during Shigella internalization. We identified Rab8 and Rab13 as mediators of dynein recruitment and discovered that the Shigella effector protein IpaH7.8 promotes Rab13 retention on moving BCV membrane remnants, thereby facilitating membrane uncoating of the Shigella-containing vacuole. Moreover, the efficient unpeeling of BCV remnants contributes to a successful intercellular spread. Taken together, our work demonstrates how a bacterial pathogen subverts the intracellular transport machinery to secure a cytosolic niche.
... Labelled proteins were enriched and identified by mass spectrometry. 123 IcsB was shown to acylate a great number of small GTPases of the Ras superfamily to disturb bacterial autophagy and facilitate its survival. 123 These examples show how pathogens can modify the lipidation of host proteins. ...
... 123 IcsB was shown to acylate a great number of small GTPases of the Ras superfamily to disturb bacterial autophagy and facilitate its survival. 123 These examples show how pathogens can modify the lipidation of host proteins. On the other hand, bacteria may exploit host lipidation enzymes to acylate their effector proteins for functional activation, stabilisation, and sub-cellular localisation. ...
With the rapid emergence and the dissemination of microbial resistance to conventional chemotherapy, the shortage of novel antimicrobial drugs has raised a global health threat. As molecular interactions between microbial pathogens and their mammalian hosts are crucial to establish virulence, pathogenicity, and infectivity, a detailed understanding of these interactions has the potential to reveal novel therapeutic targets and treatment strategies. Bidirectional molecular communication between microbes and eukaryotes is essential for both pathogenic and commensal organisms to colonise their host. In particular, several devastating pathogens exploit host signalling to adjust the expression of energetically costly virulent behaviours. Chemical proteomics has emerged as a powerful tool to interrogate the protein interaction partners of small molecules and has been successfully applied to advance host–pathogen communication studies. Here, we present recent significant progress made by this approach and provide a perspective for future studies.
... Xenophagy is partially avoided by ABM (Yoshikawa et al., 2009), but additional mechanisms appear required to efficiently avoid autophagosomal degradation. These include the T3SS effector protein IcsB of S. flexneri (Ogawa et al., 2005) with an acyltransferase activity affecting small GTPases and membrane fusion machinery (Liu et al., 2018), or secreted Listeriolysin O and phospholipases of L. monocytogenes targeting autophagosomal membranes (Birmingham et al., 2007). For SPA, the high speed of (Birmingham et al., 2006). ...
Salmonella enterica is a common foodborne, facultative intracellular enteropathogen. Typhoidal serovars like Paratyphi A (SPA) are human restricted and cause severe systemic diseases, while many serovars like Typhimurium (STM) have a broad host range, and usually lead to self‐limiting gastroenteritis. There are key differences between typhoidal and non‐typhoidal Salmonella in pathogenesis, but underlying mechanisms remain largely unknown. Transcriptomes and phenotypes in epithelial cells revealed induction of motility, flagella and chemotaxis genes for SPA but not STM. SPA exhibited cytosolic motility mediated by flagella. In this study, we applied single‐cell microscopy to analyze triggers and cellular consequences of cytosolic motility. Live‐cell imaging (LCI) revealed that SPA invades host cells in a highly cooperative manner. Extensive membrane ruffling at invasion sites led to increased membrane damage in nascent Salmonella‐containing vacuole, and subsequent cytosolic release. After release into the cytosol, motile bacteria showed the same velocity as under culture conditions in media. Reduced capture of SPA by autophagosomal membranes was observed by LCI and electron microscopy. Prior work showed that SPA does not use flagella‐mediated motility for cell exit via the intercellular spread. However, cytosolic motile SPA was invasion‐primed if released from host cells. Our results reveal flagella‐mediated cytosolic motility as a possible xenophagy evasion mechanism that could drive disease progression and contributes to the dissemination of systemic infection.
... Previous studies have shown that the protein expression [19,20], as well as the protein PTMs, such as phosphorylation and crotonylation [21][22][23], in host cells can be mediated by microbes, including viruses and bacteria. Some bacterial toxin effectors influence the host through PTM regulations, such as ubiquitination [24], glycosylation [26,124], and fatty acylation [28,125]. Gut microbiota-derived metabolites, such as inositol phosphate and short-chain fatty acids, play critical regulatory roles in host PTMs, such as crotonylation and acetylation [21,126]. ...
Background
Gut fungi are increasingly recognized as important contributors to host physiology, although most studies have focused on gut bacteria. Post-translational modifications (PTMs) of proteins play vital roles in cell metabolism. However, the contribution of gut fungi to host protein PTMs remains unclear. Mining gut fungi that mediate host protein PTMs and dissecting their mechanism are urgently needed.
Results
We studied the gut fungal communities of 56 weaned piglets and 56 finishing pigs from seven pig breeds using internal transcribed spacer (ITS) gene amplicon sequencing and metagenomics. The results showed that Kazachstania slooffiae was the most abundant gut fungal species in the seven breeds of weaned piglets. K. slooffiae decreased intestinal epithelial lysine succinylation levels, and these proteins were especially enriched in the glycolysis pathway. We demonstrated that K. slooffiae promoted intestinal epithelial glycolysis by decreasing lysine succinylation by activating sirtuin 5 (SIRT5). Furthermore, K. slooffiae-derived 5′-methylthioadenosine metabolite promoted the SIRT5 activity.
Conclusions
These findings provide a landscape of gut fungal communities of pigs and suggest that K. slooffiae plays a crucial role in intestinal glycolysis metabolism through lysine desuccinylation. Our data also suggest a potential protective strategy for pigs with an insufficient intestinal energy supply.
6wx82pHd_v1w2-kqvetnhKVideo Abstract
... This defence mechanism is mediated by autophagy related proteins (ARP). The IcsB effector can be recognised as ARP, masks the region that is unmasked by ARP and inhibits the autophagy mechanism of the host cell [69,70]. Non-Dysenteriae Shigella species inside the host cells secrete different toxins and toxic compounds. ...
Shigella species are the main cause of bacillary diarrhoea or shigellosis in humans. These organisms are the inhabitants of the human intestinal tract; however, they are one of the main concerns in public health in both developed and developing countries. In this study, we reviewed and summarised the previous studies and recent advances in molecular mechanisms of pathogenesis of Shigella Dysenteriae and non-Dysenteriae species. Regarding the molecular mechanisms of pathogenesis and the presence of virulence factor encoding genes in Shigella strains, species of this bacteria are categorised into Dysenteriae and non-Dysenteriae clinical groups. Shigella species uses attachment, invasion, intracellular motility, toxin secretion and host cell interruption mechanisms, causing mild diarrhoea, haemorrhagic colitis and haemolytic uremic syndrome diseases in humans through the expression of effector delivery systems, protein effectors, toxins, host cell immune system evasion and iron uptake genes. The investigation of these genes and molecular mechanisms can help us to develop and design new methods to detect and differentiate these organisms in food and clinical samples and determine appropriate strategies to prevent and treat the intestinal and extraintestinal infections caused by these enteric pathogens.
... Upon completion of the bacterial invasion process, a thick, so-called "cocoon" forms de novo around the BCV (Kühn et al., 2020) (Figure 1) This actin cocoon represents a unique structure implicating various cytoskeletal proteins and actin nucleation by the Arp2/3 complex. It is possible to speculate that through its Ne-fatty acetylase activity the T3SS effector IcsB is required for persistent activity of the Arp2/3 complex at the actin cocoon, while the initial recruitment of this actin nucleating complex depends on the activation of the Cdc42 small GTPase, at the onset of bacterial invasion (Liu et al., 2018). The role of the actin cocoon needs to be further investigated, however evidence shows that it is required for the efficient removal of damaged vacuolar remnants following rupture, that would otherwise impair bacterial spreading. ...
Shigella, the causative agent of bacillary dysentery, subvert cytoskeletal and trafficking processes to invade and replicate in epithelial cells using an arsenal of bacterial effectors translocated through a type III secretion system. Here, we review the various roles of the type III effector IpgD, initially characterized as phosphatidylinositol 4,5 bisphosphate (PI4,5P2) 4-phosphatase. By decreasing PI4,5P2 levels, IpgD triggers the disassembly of cortical actin filaments required for bacterial invasion and cell migration. PI5P produced by IpgD further stimulates signaling pathways regulating cell survival, macropinosome formation, endosomal trafficking and dampening of immune responses. Recently, IpgD was also found to exhibit phosphotransferase activity leading to PI3,4P2 synthesis adding a new flavor to this multipotent bacterial enzyme. The substrate of IpgD, PI4,5P2 is also the main substrate hydrolyzed by endogenous phospholipases C to produce inositoltriphosphate (InsP3), a major Ca²⁺ second messenger. Hence, beyond the repertoire of effects associated with the direct diversion of phoshoinositides, IpgD indirectly down-regulates InsP3-mediated Ca²⁺ release by limiting InsP3 production. Furthermore, IpgD controls the intracellular lifestyle of Shigella promoting Rab8/11 -dependent recruitment of the exocyst at macropinosomes to remove damaged vacuolar membrane remnants and promote bacterial cytosolic escape. IpgD thus emerges as a key bacterial effector for the remodeling of host cell membranes.
... Legionella pneumophila inhibits the formation of autophagosomes by cutting the lipid chain of LC3-PE, thereby achieving the goal of survival by regulating autophagy [17]. Shigella flexneri delivers virulence effectors IcsB into host cells, which alters the host membrane of the fatty acylation landscape to get rid of host autophagy [18]. Furthermore, inhibition of autophagy significantly reduces the over-replication of cytoplasmic S. Infantis [19]. ...
... S. Infantis infection in newly weaned piglets causes gut microbiota dysbiosis and results in enteritis, diarrhea, fever, and poor growth performance [1,[23][24][25]. Lactobacillus has been shown protective against Salmonella infection [3][4][5]18,23]. In the piglet model, L. johnsonii L531 could increase SCFAs levels and downregulate the NF-κB-SQSTM1 mitophagy signaling pathway, thus, alleviating intestinal inflammation and limiting S. Infantis dissemination [3,4]. ...
... Probiotics such as Bifidobacterium species have a protective effect on ER stress in Caco2 monolayers S. Infantis infection in newly weaned piglets causes gut microbiota dysbiosis and results in enteritis, diarrhea, fever, and poor growth performance [1,[23][24][25]. Lactobacillus has been shown protective against Salmonella infection [3][4][5]18,23]. In the piglet model, L. johnsonii L531 could increase SCFAs levels and downregulate the NF-κB-SQSTM1 mitophagy signaling pathway, thus, alleviating intestinal inflammation and limiting S. Infantis dissemination [3,4]. ...
Salmonella enterica serovar Infantis (S. Infantis) is an intracellular bacterial pathogen. It is prevalent but resistant to antibiotics. Therefore, the therapeutic effect of antibiotics on Salmonella infection is limited. In this study, we used the piglet diarrhea model and the Caco2 cell model to explore the mechanism of probiotic Lactobacillus johnsonii L531 (L. johnsonii L531) against S. Infantis infection. L. johnsonii L531 attenuated S. Infantis-induced intestinal structural and cellular ultrastructural damage. The expression of NOD pathway-related proteins (NOD1/2, RIP2), autophagy-related key proteins (ATG16L1, IRGM), and endoplasmic reticulum (ER) stress markers (GRP78, IRE1) were increased after S. Infantis infection. Notably, L. johnsonii L531 pretreatment not only inhibited the activation of the above signaling pathways but also played an anti-S. Infantis infection role in accelerating autophagic degradation. However, RIP2 knockdown did not interfere with ER stress and the activation of autophagy induced by S. Infantis in Caco2 cells. Our data suggest that L. johnsonii L531 pretreatment alleviates the intestinal damage caused by S. Infantis by inhibiting NOD activation and regulating ER stress, as well as promoting autophagic degradation.
... SIRT2 can hydrolyze long-chain fatty acyl groups on protein lysine residues. Interestingly, another effector from Shigella, IcsB, is able to fatty acylate host proteins 15 . Therefore, we hypothesized that, upon Shigella infection, SIRT2 is upregulated to remove the PTM installed by IcsB as a host defense mechanism. ...
... Host cells use autophagy to target intracellular bacteria and to restrict bacterial growth 33 . IcsB is important for Shigella to escape host autophagy by modifying CHMP5 15,34 . We hypothesized that SIRT2, working against IcsB, would promote the innate immunity and suppress Shigella escaping from autophagosomes. ...
Enzymes from pathogens often modulate host protein post-translational modifications (PTMs), facilitating survival and proliferation of pathogens. Shigella virulence factors IpaJ and IcsB induce proteolytic cleavage and lysine fatty acylation on host proteins, which cause Golgi stress and suppress innate immunity, respectively. However, it is unknown whether host enzymes could reverse such modifications introduced by pathogens’ virulence factors to suppress pathogenesis. Herein, we report that SIRT2, a potent lysine defatty-acylase, is upregulated by the transcription factor CREB3 under Golgi stress induced by Shigella infection. SIRT2 in turn removes the lysine fatty acylation introduced by Shigella virulence factor IcsB to enhance host innate immunity. SIRT2 knockout mice are more susceptible to Shigella infection than wildtype mice, demonstrating the importance of SIRT2 to counteract Shigella infection.
