![YacC is a novel member of the PulS-OutS family of proteins.
(A) The conserved domain architecture tool (CDART) was used to map the regions of PulS from Klebsiella oxytoca 10-5250 (EHT07154.1), OutS from Dickeya dadantii 3937 (YP_003883937.1), EtpO from Shiga toxin-producing E. coli (STEC) O157:H7 (CAA70966.1) and YacC (CAS07673.1) from EPEC. Numbers refer to the amino acids of each protein sequence, and the broken blue bar denotes that the N-terminal and C-terminal residues of YacC diverge from the consensus features of the PulS-OutS family. Pairwise sequence alignment over 80 residues showing similarity of the previously characterized EtpO (CAA70966.1) and YacC (CAS07673.1). Identical residues are highlighted between the two sequences, and conserved substitutions are shown (+). (B) CLANS analysis graphically depicts homology in large datasets of proteins, utilizing all-against-all pairwise BLAST to cluster representations (colored dots) of individual protein sequences in three-dimensional space. Lines are shown between the most similar sequences, with an E-value cut-off of 1e−5. The analysis shows that proteins from diverse species cluster into two groups: the PulS/OutS group and YacC-related proteins (blue), and the AspS-related proteins (red). There are numerous relationships between the PulS/OutS proteins and YacC proteins, but no relationship links these to the AspS group of proteins. (C) Wild-type EPEC (WT), and the indicated mutants of EPEC were grown in culture and post-cell supernatants containing secreted proteins (200 µg of protein) were analyzed by SDS-PAGE and Coomassie blue staining. Mass spectrometry was used to identify SslE, FliC and EspC, consistent with a previous study [72].](https://www.researchgate.net/profile/Konstantin-Korotkov-2/publication/234158768/figure/fig10/AS:340764782546958@1458256063726/YacC-is-a-novel-member-of-the-PulS-OutS-family-of-proteins-A-The-conserved-domain_Q320.jpg)
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Assembly of the Type II Secretion System such as Found in Vibrio cholerae Depends on the Novel Pilotin AspS
PLOS Pathogens
Abstract and Figures
Author Summary
The type 2 secretion system (T2SS) is a sophisticated, multi-component molecular machine that drives the secretion of fully-folded protein substrates across the bacterial outer membrane. In Vibrio cholerae, for example, the T2SS mediates the secretion of cholera toxin. We find that there are three distinct forms of T2SS, based on the sequence characteristics of the secretin. A targeting paradigm, developed for the Klebsiella-type secretin PulD, could not previously be applied to the T2SS in Vibrio cholerae and many other bacterial species whose genomes encode no homolog of the crucial targeting factor PulS (also called OutS, EtpO or GspS). Using bioinformatics we find, remarkably, that these bacteria have instead evolved a structurally distinct protein to serve in place of PulS. We crystallized and solved the structure of this distinct factor, AspS, measured its activity in novel assays for T2SS assembly, and show that the protein is essential for the function of the Vibrio-type T2SS. A structural homolog of AspS found here in Pseudomonas suggests widespread use of the pilotin-secretin targeting paradigm for T2SS assembly.
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Supplementary resources (9)
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... The translocation of T2SS secretins from the inner membrane to the outer membrane is an essential step required for T2SS assembly, however, it is still unclear how this translocation is regulated in the bacterial cell envelope. Based on biochemistry experiments, there exist two possible pathways: (1) the scaffolding proteins GspA and GspB bind and increase the pore size of peptidoglycan, and translocate the secretin to the outer membrane, as found in Klebsiella-type secretins ExeD and OutD 12-14 ; (2) a small pilotin GspS could bind to the S domain of secretin, and the pilotin itself can translocate to the outer membrane through the Lol sorting pathway, as found in the Vibriotype secretins 4,15,16 . Besides these two pathways, since some T2SS exist without GspA and GspB or the pilotin 11,17,18 , other pathways also possibly exist. ...
... Also, GspD β has a different outer membrane targeting mechanism. Instead of using scaffolding proteins or enlarging the peptidoglycan pore size, a small protein GspS, named pilotin, could bind to the S domain of GspD β and translocate GspD β to the outer membrane 4,15 . Therefore, to visualize GspD β particles on the outer membrane and verify whether GspS forms a complex with GspD β there, we performed two experiments. ...
... As biochemistry results have shown that GspD β could form multimers on the inner membrane when gspS is knocked out 4,15 , to visualize GspD β on the inner membrane, we induced expression of GspD β within ΔgspS E. coli cells (Fig. 3h). Within the reconstructed tomograms, particles are all visualized on the inner membrane, with the nontransmembrane domains located in the periplasm (Fig. 3f, i). ...
The GspD secretin is the outer membrane channel of the bacterial type II secretion system (T2SS) which secrets diverse toxins that cause severe diseases such as diarrhea and cholera. GspD needs to translocate from the inner to the outer membrane to exert its function, and this process is an essential step for T2SS to assemble. Here, we investigate two types of secretins discovered so far in Escherichia coli, GspDα, and GspDβ. By electron cryotomography subtomogram averaging, we determine in situ structures of key intermediate states of GspDα and GspDβ in the translocation process, with resolution ranging from 9 Å to 19 Å. In our results, GspDα and GspDβ present entirely different membrane interaction patterns and ways of transitioning the peptidoglycan layer. From this, we hypothesize two distinct models for the membrane translocation of GspDα and GspDβ, providing a comprehensive perspective on the inner to outer membrane biogenesis of T2SS secretins.
... In the assembled secretin channel, the bound pilotins stabilize the external b-barrel by stapling tandemly arranged secretin subunits (15,43). The T2SS pilotins of the AspS/ExeS family that were identified in Vibrio, Aeromonas, and enterotoxigenic and enteropathogenic E. coli seem to fulfil exactly the same functions as OutS/PulS while adopting a profoundly different fold (15,(43)(44)(45). Targeting and assembly of secretins from the T3SS and T4P systems are also facilitated by specific piloting lipoproteins with different structures and modes of action. ...
... Specifically, at a 1% false discovery rate (FDR), several best spectra (Fig. 6A and Fig. S6D). The OutD residue, being cross-linked to pBPA, could be assigned from S 41 to M 45 with prevalence of D 43 and M 44 (Fig. S6D). ...
Gram-negative bacteria have two cell membranes sandwiching a peptidoglycan net that together form a robust protective cell envelope. To translocate effector proteins across this multilayer envelope, bacteria have evolved several specialized secretion systems.
... Pilotins are secretin-specific lipoprotein chaperones supporting the oligomerization, assembly, and correct insertion of GspD subunits into the outer membrane (Dunstan et al., 2013;Nickerson et al., 2011). They may be co-located with other T2SS genes in a single operon as for yghG in ETEC (Strozen et al., 2012) or separated and positioned elsewhere in the chromosome (Chernyatina and Low, 2019;Koo et al., 2012). ...
... The Klebsiella-type pilotins have a fold comprised of four α-helices Korotkov and Hol, 2013;Rehman et al., 2013;Tosi et al., 2011) (Figure 1a). In contrast, the Vibrio-type pilotins constitute five stranded β-sheets flanked by four α-helices (Dunstan et al., 2013;Yin et al., 2018) (Figure 1b). Each pilotin has a groove into which the C-terminal helix of the S-domain inserts. ...
The type II secretion system (T2SS) is a multi‐protein complex used by many bacteria to move substrates across their cell membrane. Substrates released into the environment serve as local and long‐range effectors that promote nutrient acquisition, biofilm formation and pathogenicity. In both animals and plants, the T2SS is increasingly recognised as a key driver of virulence. The T2SS spans the bacterial cell envelope and extrudes substrates through an outer membrane secretin channel using a pseudopilus. An inner membrane assembly platform and a cytoplasmic motor controls pseudopilus assembly. This microreview focusses on the structure and mechanism of the T2SS. Advances in cryo‐electron microscopy are enabling increasingly elaborate sub‐complexes to be resolved. However, key questions remain regarding mechanism of pseudopilus extension and retraction, and how this is coupled to the choreography of substrate moving through the secretion system. The T2SS is part of an ancient type IV filament superfamily that may have been present within the last universal common ancestor (LUCA). Overall, mechanistic principles that underlie T2SS function have implication for other closely related systems such as the type IV and tight adherence pilus systems.
... Thirty-nine DEGs were common among the genes upregulated, while 63 genes were significantly downregulated in S. plymuthica HRO-C48 due to the contact with the VOCs of all three fungi ( Figure 3B). Some of the upregulated genes were predicted to code for phage shock proteins, or proteins involved in stress response and virulence, such as for example, Type III secretion system lipoprotein chaperone (Tosi et al., 2011;Horstman and Darwin, 2012;Dunstan et al., 2013). Among the downregulated genes those coding for membrane proteins, multiple stress resistance proteins, murein hydrolase proteins and putative Ferritin-like protein were identified. ...
Volatile organic compounds (VOCs) are involved in microbial interspecies communication and in the mode of action of various antagonistic interactions. They are important for balancing host-microbe interactions and provide the basis for developing biological control strategies to control plant pathogens. We studied the interactions between the bacterial antagonist Serratia plymuthica HRO-C48 and three fungal plant pathogens Rhizoctonia solani, Leptosphaeria maculans and Verticillium longisporum. Significant differences in fungal growth inhibition by the Serratia-emitted VOCs in pairwise dual culture assays and changes in the transcriptome of the bacterium and in the volatilomes of both interacting partners were observed. Even though the rate of fungal growth inhibition by Serratia was variable, the confrontation of the bacterium with the VOCs of all three fungi changed the levels of expression of the genes involved in stress response, biofilm formation, and the production of antimicrobial VOCs. Pairwise interacting microorganisms switched between defense (downregulation of gene expression) and attack (upregulation of gene expression and metabolism followed by growth inhibition of the interacting partner) modes, subject to the combinations of microorganisms that were interacting. In the attack mode HRO-C48 significantly inhibited the growth of R. solani while simultaneously boosting its own metabolism; by contrast, its metabolism was downregulated when HRO-C48 went into a defense mode that was induced by the L. maculans and V. longisporum VOCs. L. maculans growth was slightly reduced by the one bacterial VOC methyl acetate that induced a strong downregulation of expression of genes involved in almost all metabolic functions in S. plymuthica. Similarly, the interaction between S. plymuthica and V. longisporum resulted in an insignificant growth reduction of the fungus and repressed the rate of bacterial metabolism on the transcriptional level, accompanied by an intense volatile dialogue. Overall, our results indicate that VOCs substantially contribute to the highly break species-specific interactions between pathogens and their natural antagonists and thus deserving of increased consideration for pathogen control.
... Klebsiella oxytoca et Dickeya dadantii a été prouvée comme étant responsable de la localisation du SST2 dans la membrane interne [199]. [201]. ...
Pseudomonas aeruginosa est une bactérie responsable de sévères infections nosocomiales. Ces infections sont un réel problème pour la santé publique car P. aeruginosa fait partie des pathogènes ESKAPE qui ont développé des facteurs de résistance aux antibiotiques ce qui leur permet d’échapper aux traitements classiques. P. aeruginosa, en plus d’être résistante aux antibiotiques est capable d’exprimer de nombreux facteurs de virulences. Parmi ces facteurs on retrouve les systèmes de sécrétion de type V (SST5) sur lequel j’ai pu travailler durant ma thèse.Une nouvelle souche de P. aeruginosa a été découverte à Grenoble il y a quelques années. Cette souche n’exprime pas les mêmes facteurs de virulence que les souches communément étudiées mais en demeure bien plus toxique. La toxicité de cette souche est due à l’expression de deux protéines ExlB et ExlA faisant partie de la famille du SST5b et ayant un mécanisme d’action complètement nouveau car aucun homologue n’est connu. La toxine ExlA est capable de faire des pores dans les membranes des cellules eucaryotes entrainant leur mort. Le but de ma thèse était donc de comprendre à l’échelle moléculaire le fonctionnement de ExlA.Pour identifier le mécanisme d’action de cette toxine, nous l’avons divisée en deux domaines que nous avons étudiés séparément. En utilisant des techniques de RMN, de SEC-MALLS, de flottaison différentielle, de SAXS et d’AFM, nous avons pu définir que le domaine C-terminal de cette protéine était un « molten globule» en solution et était capable de faire des trous dans les membranes lipidiques reconstituées. Ce domaine semble être le domaine responsable de l’interaction d’ExlA avec les lipides. Ceci additionné à des études menées in vivo sur la toxine ExlA complète et comportant uniquement son domaine N-terminal nous a permis de définir un possible mécanisme d’action de la toxine ExlA. La somme de nos études fait l’objet d’un article en cours d’écriture.
Bacterial membrane proteins account for around one-third of the proteome in many species and can represent much more than half of the mass of the membranes. Classic techniques in cell biology can be applied to characterize bacterial membranes and their membrane protein constituents, and here we describe a protocol for the purification of outer membranes and inner membranes from Escherichia coli. This allows for compositional analysis of the membranes as well as functional analyses. The procedure can be applied with minor modifications to other bacterial species including those carrying capsular polysaccharide attached to the outer membrane.
Secretins are outer membrane (OM) channels found in various bacterial nanomachines that secrete or assemble large extracellular structures. High-resolution 3D structures of type 2 secretion system (T2SS) secretins revealed bimodular channels with a C-module, holding a conserved central gate and an optional top gate, followed by an N-module for which multiple structural organizations have been proposed. Here, we perform a structure-driven in vivo study of the XcpD secretin, which validates one of the organizations of the N-module whose flexibility enables alternative conformations. We also show the existence of the central gate in vivo and its required flexibility, which is key for substrate passage and watertightness control. Last, functional, genomic, and phylogenetic analyses indicate that the optional top gate provides a gain of watertightness. Our data illustrate how the gating properties of T2SS secretins allow these large channels to overcome the duality between the necessity of preserving the OM impermeability while simultaneously promoting the secretion of large, folded effectors.
The GspD secretin is the outer membrane channel of the bacterial type II secretion system (T2SS) which secrets diverse effector proteins or toxins that cause severe diseases such as diarrhea and cholera. GspD needs to translocate from the inner to the outer membrane to exert its function, and this process is an essential step for T2SS to assemble. Here, we investigate two types of secretins discovered so far in Escherichia coli , GspD α and GspD β , respectively. By electron cryotomography subtomogram averaging, we determine in situ structures of all the key intermediate states of GspD α and GspD β in the translocation process, with resolution ranging from 9 Å to 19 Å. In our results, GspD α and GspD β present entirely different membrane interaction patterns and ways of going across the peptidoglycan layer. We propose two distinct models for the membrane translocation of GspD α and GspD β , providing a comprehensive perspective on the inner to outer membrane biogenesis of T2SS secretins.
To exchange and communicate with their surroundings, bacteria have evolved multiple active and passive mechanisms for trans-envelope transport. Among the pore-forming complexes found in the outer membrane of Gram-negative bacteria, secretins are distinctive homo-oligomeric channels dedicated to the active translocation of voluminous structures such as folded proteins, assembled fibers, virus particles or DNA. Members of the bacterial secretin family share a common cylinder-shaped structure with a gated pore-forming part inserted in the outer membrane, and a periplasmic channel connected to the inner membrane components of the corresponding nanomachine. In this mini-review, we will present what recently determined 3D structures have told us about the mechanisms of translocation through secretins of large substrates to the bacterial surface or in the extracellular milieu.
Uptake of DNA from the environment into the bacterial cytoplasm is mediated by a macromolecular transport machinery that is similar in structure and function to type IV pili (T4P) and, indeed, DNA translocator and T4P share common components. One is the secretin PilQ which is assembled into homopolymeric complexes forming highly dynamic outer membrane (OM) channels mediating pilus extrusion and DNA uptake. How PilQ interacts with the motor is still enigmatic. Here, we have used biochemical and genetic techniques to study the interaction of PilQ with PilW, a unique protein which is essential for natural transformation and T4P extrusion of T. thermophilus. PilQ and PilW form high molecular mass complexes in the OM of T. thermophilus that were purified. When pilW was deleted, PilQ complexes were no longer observed but only PilQ monomers, accompanied by a loss of DNA uptake as well as a loss of T4P and twitching motility. Piliation of a ΔpilT2/ΔpilW double mutant suggests that PilW is important for stable assembly of PilQ complexes. To analyze the role of different regions of PilW, partial deletions (pilW∆2–40, pilW∆50–150, pilW∆163–216 and pilW∆216–292) were generated and the effect on DNA uptake, PilQ complex formation and T4P functions such as twitching motility, biofilm formation and cell-cell interaction was studied. These studies revealed that a central disordered region in PilW is required for pilus dynamics. We propose that PilW is part of a protein network that connects the transport ATPase to drive different functions of the DNA translocator and T4P.
The enterotoxigenic Escherichia coli (ETEC) pathotype, characterized by the prototypical strain H10407, is a leading cause of morbidity and mortality in the developing
world. A major virulence factor of ETEC is the type II secretion system (T2SS) responsible for secretion of the diarrheagenic
heat-labile enterotoxin (LT). In this study, we have characterized the two type II secretion systems, designated alpha (T2SSα) and beta (T2SSβ), encoded in the H10407 genome and describe the prevalence of both systems in other E. coli pathotypes. Under laboratory conditions, the T2SSβ is assembled and functional in the secretion of LT into culture supernatant, whereas the T2SSα is not. Insertional inactivation of the three genes located upstream of gspCβ (yghJ, pppA, and yghG) in the atypical T2SSβ operon revealed that YghJ is not required for assembly of the GspDβ secretin or secretion of LT, that PppA is likely the prepilin peptidase required for the function of T2SSβ, and that YghG is required for assembly of the GspDβ secretin and thus function of the T2SSβ. Mutational and physiological analysis further demonstrated that YghG (redesignated GspSβ) is a novel outer membrane pilotin protein that is integral for assembly of the T2SSβ by localizing GspDβ to the outer membrane, whereupon GspDβ forms the macromolecular secretin multimer through which T2SSβ substrates are translocated.
The TLSMD web server extracts information about dynamic properties of a protein based on information derived from a single-crystal structure. It does so by analyzing the spatial distribution of individual atomic thermal parameters present in an input structural model. The server partitions the protein structure into multiple, contiguous chain segments, each segment corresponding to one group in a multi-group description of the protein's overall dynamic motion. For each polypeptide chain of the input protein, the analysis generates the optimal partition into two segments, three segments, . . . up to 20 segments. Each such partition is optimal in the sense that it is the best approximation of the overall spatial distribution of input thermal parameters in terms of N chain segments, each acting as a rigid group undergoing TLS (translation/libration/screw) motion. This multi-group TLS model may be used as a starting point for further crystallographic refinement, or as the basis for analyzing inter-domain and other large-scale motions implied by the crystal structure.
The energy-dependent secretion of aerolysin by Aeromonas hydrophila requires the ExeA and ExeB proteins. An 85 kDa complex containing the two proteins was identified in wild-type cells but not in cells producing either protein alone. Radiolabelling followed by cross-linking, immunoprecipitation and then reduction of the cross-links confirmed the presence of the two proteins in the same complex. The complex could also be extracted intact from cell membranes with non-ionic detergents. A G229D substitution in the kinase-3a motif of ExeA strongly reduced the level of aerolysin secretion, as did the replacement of the invariant Lys of the kinase-1a motif (K56) with Arg. The G229D mutant contained very little of the ExeA–ExeB complex, but overexpression of the mutant complex until wild-type levels were achieved allowed normal secretion. In contrast, the K56R mutation had no effect on complex formation, but normal secretion levels occurred only when there was a far greater amount of the complex present. These results are consistent with a model in which binding of ATP by ExeA is required for ExeA–ExeB complex formation, while hydrolysis is required for its function in secretion once established.
Analysis of 14.162 kb of DNA derived from plasmid pO157 of enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain EDL933, extending in the 5′ direction of the recently described EHEC-hly operon, revealed 13 open reading frames (ORF) which showed great similarities to genes of members of the type II pathway secretion systems of Gram-negative bacteria. We named the ORFs etpC to etpO for HEC ype II secretion athway. In addition, an IS911-like insertion element was found to separate the etp genes from the EHEC-hlyC gene. Hybridization experiments with a specific etp probe and various categories of enteric E. coli pathotypes revealed that the etp gene cluster occurred in all 30 EHEC strains of serogroup O157 (100%) tested and is distributed sporadically among other EHEC serogroups (60%). In addition, the etp genes were rarely detected in STEC isolated from bovine feces (10%). Moreover, it was found not to occur in enteropathogenic E. coli, enteroaggregative E. coli, enterotoxigenic E. coli and enteroinvasive E. coli. The results obtained with the etp probe were confirmed by a PCR approach to specifically detect an internal fragment of the etpD gene.
The β-barrel assembly machinery (BAM) complex drives the assembly of β-barrel proteins into the outer membrane of gram-negative bacteria. It is composed of five subunits: BamA, BamB, BamC, BamD, and BamE. We find that the BAM complex isolated from the outer membrane of Escherichia coli consists of a core complex of BamA:B:C:D:E and, in addition, a BamA:B module and a BamC:D module. In the absence of BamC, these modules are destabilized, resulting in increased protease susceptibility of BamD and BamB. While the N-terminus of BamC carries a highly conserved region crucial for stable interaction with BamD, immunofluorescence, immunoprecipitation, and protease-sensitivity assays show that the C-terminal domain of BamC, composed of two helix-grip motifs, is exposed on the surface of E. coli. This unexpected topology of a bacterial lipoprotein is reminiscent of the analogous protein subunits from the mitochondrial β-barrel insertion machinery, the SAM complex. The modular arrangement and topological features provide new insight into the architecture of the BAM complex, towards a better understanding of the mechanism driving β-barrel membrane protein assembly.
Many gram-negative bacteria use the sophisticated type II secretion system (T2SS) to translocate a wide range of proteins from the periplasm across the outer membrane. The inner-membrane platform of the T2SS is the nexus of the system and orchestrates the secretion process through its interactions with the periplasmic filamentous pseudopilus, the dodecameric outer-membrane complex and a cytoplasmic secretion ATPase. Here, recent structural and biochemical information is reviewed to describe our current knowledge of the biogenesis and architecture of the T2SS and its mechanism of action.