The Type II Secretion System Delivers Matrix Proteins for Biofilm Formation by Vibrio cholerae

Gram-negative bacteria have evolved several highly dedicated pathways for extracellular protein secretion, including the type II secretion (T2S) system. Since substrates secreted via the T2S system include both virulence factors and degradative enzymes, this secretion system is considered a major survival mechanism for pathogenic and environmental species. Previous analyses revealed that the T2S system mediates the export of ≥20 proteins in Vibrio cholerae, a human pathogen that is indigenous to the marine environment. Here we demonstrate a new role in biofilm formation for the V. cholerae T2S system, since wild-type V. cholerae was found to secrete the biofilm matrix proteins RbmC, RbmA, and Bap1 into the culture supernatant, while an isogenic T2S mutant could not. In agreement with this finding, the level of biofilm formation in a static microtiter assay was diminished in T2S mutants. Moreover, inactivation of the T2S system in a rugose V. cholerae strain prevented the development of colony corrugation and pellicle formation at the air-liquid interface. In contrast, extracellular secretion of the exopolysaccharide VPS, an essential component of the biofilm matrix, remained unaffected in the T2S mutants. Our results indicate that the T2S system provides a mechanism for the delivery of extracellular matrix proteins known to be important for biofilm formation by V. cholerae. Because the T2S system contributes to the pathogenicity of V. cholerae by secreting proteins such as cholera toxin and biofilm matrix proteins, elucidation of the molecular mechanism of T2S has the potential to lead to the development of novel preventions and therapies.     

Cell Envelope Perturbation Induces Oxidative Stress and Changes in Iron Homeostasis in Vibrio cholerae

The Vibrio cholerae type II secretion (T2S) machinery is a multiprotein complex that spans the cell envelope. When the T2S system is inactivated, cholera toxin and other exoproteins accumulate in the periplasmic compartment. Additionally, loss of secretion via the T2S system leads to a reduced growth rate, compromised outer membrane integrity, and induction of the extracytoplasmic stress factor RpoE (A. E. Sikora, S. R. Lybarger, and M. Sandkvist, J. Bacteriol. 189:8484-8495, 2007). In this study, gene expression profiling reveals that inactivation of the T2S system alters the expression of genes encoding cell envelope components and proteins involved in central metabolism, chemotaxis, motility, oxidative stress, and iron storage and acquisition. Consistent with the gene expression data, molecular and biochemical analyses indicate that the T2S mutants suffer from internal oxidative stress and increased levels of intracellular ferrous iron. By using a tolA mutant of V. cholerae that shares a similar compromised membrane phenotype but maintains a functional T2S machinery, we show that the formation of radical oxygen species, induction of oxidative stress, and changes in iron physiology are likely general responses to cell envelope damage and are not unique to T2S mutants. Finally, we demonstrate that disruption of the V. cholerae cell envelope by chemical treatment with polymyxin B similarly results in induction of the RpoE-mediated stress response, increased sensitivity to oxidants, and a change in iron metabolism. We propose that many types of extracytoplasmic stresses, caused either by genetic alterations of outer membrane constituents or by chemical or physical damage to the cell envelope, induce common signaling pathways that ultimately lead to internal oxidative stress and misregulation of iron homeostasis.Vibrio cholerae, a rod-shaped, highly motile, gram-negative bacterium, is the causative agent of the life threatening diarrheal disease cholera (59). The type II secretion (T2S) system plays an important role in the pathogenesis of V. cholerae by secreting cholera toxin (63), which is largely responsible for the symptoms of the disease (33). The T2S system is widespread and well conserved in gram-negative bacteria inhabiting a variety of ecological niches and likely contributes to environmental survival as well as to virulence (11, 21). In V. cholerae, secretion via the T2S machinery is supported by a transenvelope complex of 12 Eps proteins (EpsC to EpsN) and the type 4 prepilin peptidase PilD (VcpD) (25, 44, 63). Transport of exoproteins by the T2S system occurs via a two-step process. The first step, which is either Sec or Tat dependent, requires recognition of the N-terminal signal peptide of the exoproteins and translocation through the inner membrane to the periplasm. Then the folded proteins engage the T2S machinery and are subsequently exported across the outer membrane to the extracellular milieu (23, 29).Besides periplasmic accumulation of exoproteins, additional phenotypes of T2S mutants are reported for an increasing number of species, possibly indicating involvement of the T2S system in other important cellular processes. For example, alterations in outer membrane protein composition have been described for T2S mutants of V. cholerae, Aeromonas hydrophila, marine Vibrio sp. strain 60, and Shewanella oneidensis (30, 32, 63, 64). The levels of outer membrane porins OmpU, OmpT, and OmpS are decreased in T2S mutants of V. cholerae (63, 65), and likewise, disruption of T2S genes in A. hydrophila leads to diminished quantities of OmpF and OmpS (30). Similarly, the amounts of the c-type cytochromes MtrC and OmcA in the outer membranes of S. oneidensis T2S mutants are reduced (64). Furthermore, we have shown that inactivation of the T2S system in V. cholerae results in a reduced growth rate, compromised outer membrane integrity, and, as a consequence, induction of RpoE activity. In addition, our studies showed that V. cholerae T2S mutants are unable to survive the passage through the infant mouse gastrointestinal tract (65). Growth defects at low temperatures under laboratory conditions as well as in tap water and amoebae were also observed for T2S mutants of Legionella pneumophila (68).Interestingly, differential abundance of proteins involved in phosphate metabolism and iron uptake has been revealed by proteomic analysis of culture supernatants isolated from wild-type and T2S mutant strains of Pseudoaltermonas tunicata (22). Based on these results, it has been suggested that the T2S system might be involved in iron acquisition. Similarly, certain T2S mutants of Erwinia chrysanthemi exhibit defects indicative of changes in iron homeostasis (17). It has also been noted that the level of aconitate hydratase, an iron-sulfur cluster-containing enzyme, is reduced in L. pneumophila T2S mutants (16).In this study, in an attempt to explain the phenotypes associated with loss of T2S, we performed microarray gene expression profiling of wild-type and T2S-deficient strains. Our data revealed that inactivation of the T2S machinery results in a metabolic feedback loop leading to oxidative stress and changes in iron metabolism. By analyzing another V. cholerae mutant that shares a similar cell envelope phenotype while remaining competent for T2S, we show that the changes in iron homeostasis and oxidative stress are linked to cell envelope damage and extracytoplasmic stress.     

Vibrio cholerae VciB promotes iron uptake via ferrous iron transporters

Vibrio cholerae uses a variety of strategies for obtaining iron in its diverse environments. In this study we report the identification of a novel iron utilization protein in V. cholerae, VciB. The vciB gene and its linked gene, vciA, were isolated in a screen for V. cholerae genes that permitted growth of an Escherichia coli siderophore mutant in low-iron medium. The vciAB operon encodes a predicted TonB-dependent outer membrane receptor, VciA, and a putative inner membrane protein, VciB. VciB, but not VciA, was required for growth stimulation of E. coli and Shigella flexneri strains in low-iron medium. Consistent with these findings, TonB was not needed for VciB-mediated growth. No growth enhancement was seen when vciB was expressed in an E. coli or S. flexneri strain defective for the ferrous iron transporter Feo. Supplying the E. coli feo mutant with a plasmid encoding either E. coli or V. cholerae Feo, or the S. flexneri ferrous iron transport system Sit, restored VciB-mediated growth; however, no stimulation was seen when either of the ferric uptake systems V. cholerae Fbp and Haemophilus influenzae Hit was expressed. These data indicate that VciB functions by promoting iron uptake via a ferrous, but not ferric, iron transport system. VciB-dependent iron accumulation via Feo was demonstrated directly in iron transport assays using radiolabeled iron. A V. cholerae vciB mutant did not exhibit any growth defects in either in vitro or in vivo assays, possibly due to the presence of other systems with overlapping functions in this pathogen.     

SciN is an outer membrane lipoprotein required for type VI secretion in enteroaggregative Escherichia coli

Enteroaggregative Escherichia coli (EAEC) is a pathogen implicated in several infant diarrhea or diarrheal outbreaks in areas of endemicity. Although multiple genes involved in EAEC pathogenesis have been identified, the overall mechanism of virulence is not well understood. Recently, a novel secretion system, called type VI secretion (T6S) system (T6SS), has been identified in EAEC and most animal or plant gram-negative pathogens. T6SSs are multicomponent cell envelope machines responsible for the secretion of at least two putative substrates, Hcp and VgrG. In EAEC, two copies of T6S gene clusters, called sci-1 and sci-2, are present on the pheU pathogenicity island. In this study, we focused our work on the sci-1 gene cluster. The Sci-1 apparatus is probably composed of all, or a subset of, the 21 gene products encoded on the cluster. Among these subunits, some are shared by all T6SSs identified to date, including a ClpV-type AAA⁺ ATPase (SciG) and an IcmF (SciS) and an IcmH (SciP) homologue, as well as a putative lipoprotein (SciN). In this study, we demonstrate that sciN is a critical gene necessary for T6S-dependent secretion of the Hcp-like SciD protein and for biofilm formation. We further show that SciN is a lipoprotein, as shown by the inhibition of its processing by globomycin and in vivo labeling with [³H]palmitic acid. SciN is tethered to the outer membrane and exposed in the periplasm. Sequestration of SciN at the inner membrane by targeting the +2 residue responsible for lipoprotein localization (Gly2Asp) fails to complement an sciN mutant for SciD secretion and biofilm formation. Together, these results support a model in which SciN is an outer membrane lipoprotein exposed in the periplasm and essential for the Sci-1 apparatus function.     

Secretion of TcpF by the Vibrio cholerae Toxin-Coregulated Pilus Biogenesis Apparatus Requires an N-Terminal Determinant

Type IV pili are important for microcolony formation, biofilm formation, twitching motility, and attachment. We and others have shown that type IV pili are important for protein secretion across the outer membrane, similar to type II secretion systems. This study explored the relationship between protein secretion and pilus formation in Vibrio cholerae. The toxin-coregulated pilus (TCP), a type IV pilus required for V. cholerae pathogenesis, is necessary for the secretion of the colonization factor TcpF (T. J. Kirn, N. Bose, and R. K. Taylor, Mol. Microbiol. 49:81–92, 2003). This phenomenon is not unique to V. cholerae; secreted virulence factors that are dependent on the presence of components of the type IV pilus biogenesis apparatus for secretion have been reported with Dichelobacter nodosus (R. M. Kennan, O. P. Dhungyel, R. J. Whittington, J. R. Egerton, and J. I. Rood, J. Bacteriol. 183:4451–4458, 2001) and Francisella tularensis (A. J. Hager et al., Mol. Microbiol. 62:227–237, 2006). Using site-directed mutagenesis, we demonstrated that the secretion of TcpF is dependent on the presence of selected amino acid R groups at position five. We were unable to find other secretion determinants, suggesting that Y5 is the major secretion determinant within TcpF. We also report that proteins secreted in a type IV pilus biogenesis apparatus-dependent manner have a YXS motif within the first 15 amino acids following the Sec cleavage site. The YXS motif is not present in proteins secreted by type II secretion systems, indicating that this is unique to type IV pilus-mediated secretion. Moreover, we show that TcpF interacts with the pilin TcpA, suggesting that these proteins are secreted by the type IV pilus biogenesis system. These data provide a starting point for understanding how type IV pili can mediate secretion of virulence factors important for bacterial pathogenesis.     

Constitutive Type VI Secretion System Expression Gives Vibrio cholerae Intra- and Interspecific Competitive Advantages

The type VI secretion system (T6SS) mediates protein translocation across the cell membrane of Gram-negative bacteria, including Vibrio cholerae – the causative agent of cholera. All V. cholerae strains examined to date harbor gene clusters encoding a T6SS. Structural similarity and sequence homology between components of the T6SS and the T4 bacteriophage cell-puncturing device suggest that the T6SS functions as a contractile molecular syringe to inject effector molecules into prokaryotic and eukaryotic target cells. Regulation of the T6SS is critical. A subset of V. cholerae strains, including the clinical O37 serogroup strain V52, express T6SS constitutively. In contrast, pandemic strains impose tight control that can be genetically disrupted: mutations in the quorum sensing gene luxO and the newly described regulator gene tsrA lead to constitutive T6SS expression in the El Tor strain C6706. In this report, we examined environmental V. cholerae isolates from the Rio Grande with regard to T6SS regulation. Rough V. cholerae lacking O-antigen carried a nonsense mutation in the gene encoding the global T6SS regulator VasH and did not display virulent behavior towards Escherichia coli and other environmental bacteria. In contrast, smooth V. cholerae strains engaged constitutively in type VI-mediated secretion and displayed virulence towards prokaryotes (E. coli and other environmental bacteria) and a eukaryote (the social amoeba Dictyostelium discoideum). Furthermore, smooth V. cholerae strains were able to outcompete each other in a T6SS-dependent manner. The work presented here suggests that constitutive T6SS expression provides V. cholerae with an advantage in intraspecific and interspecific competition.     

YscO of Yersinia pestis Is a Mobile Core Component of the Yop Secretion System

The Yersinia pestis low-Ca²⁺ response stimulon is responsible for the temperature- and Ca²⁺-regulated expression and secretion of plasmid pCD1-encoded antihost proteins (V antigen and Yops). We have previously shown that lcrD, yscC, yscD, yscG, and yscR encode proteins that are essential for high-level expression and secretion of V antigen and Yops at 37°C in the absence of Ca²⁺. In this study, we characterized yscO of the Yop secretion (ysc) operon that contains yscN through yscU by determining the localization of its gene product and the phenotype of an in-frame deletion. The yscO mutant grew and expressed the same levels of Yops as the parent at 37°C in the presence of Ca²⁺. In the absence of Ca²⁺, the mutant grew independently of Ca²⁺, expressed only basal levels of V antigen and Yops, and failed to secrete these. These defects could be partially complemented by providing yscO in trans in the yscO mutant. Overexpression of YopM and V antigen in the mutant failed to restore the export of either protein, showing that the mutation had a direct effect on secretion. These results indicated that the yscO gene product is required for high-level expression and secretion of V antigen and Yops. YscO was found by immunoblot analysis in the soluble and membrane fractions of bacteria growing at 37°C irrespective of the presence of Ca²⁺ and in the culture medium in the absence of Ca²⁺. YscO is the only mobile protein identified so far in the Yersinia species that is required for secretion of V antigen and Yops.     

Structure of the minor pseudopilin EpsH from the Type 2 secretion system of Vibrio cholerae

Many Gram-negative bacteria use the multi-protein type II secretion system (T2SS) to selectively translocate virulence factors from the periplasmic space into the extracellular environment. In Vibrio cholerae the T2SS is called the extracellular protein secretion (Eps) system,which translocates cholera toxin and several enzymes in their folded state across the outer membrane. Five proteins of the T2SS, the pseudopilins, are thought to assemble into a pseudopilus, which may control the outer membrane pore EpsD, and participate in the active export of proteins in a “piston-like” manner. We report here the 2.0 Å resolution crystal structure of an N-terminally truncated variant of EpsH, a minor pseudopilin from Vibrio cholerae. While EpsH maintains an N-terminal α-helix and C-terminal β-sheet consistent with the type 4a pilin fold, structural comparisons reveal major differences between the minor pseudopilin EpsH and the major pseudopilin GspG from Klebsiella oxytoca: EpsH contains a large β-sheet in the variable domain, where GspG contains an α-helix. Most importantly, EpsH contains at its surface a hydrophobic crevice between its variable and conserved β-sheets, wherein a majority of the conserved residues within the EpsH family are clustered. In a tentative model of a T2SS pseudopilus with EpsH at its tip, the conserved crevice faces away from the helix axis. This conserved surface region may be critical for interacting with other proteins from the T2SS machinery.     

Ecotin and LamB in Escherichia coli influence the susceptibility to Type VI secretion-mediated interbacterial competition and killing by Vibrio cholerae

BackgroundA prevailing action of the Type VI secretion system (T6SS) in several Gram-negative bacterial species is inter-bacterial competition. In the past several years, many effectors of T6SS were identified in different bacterial species and their involvement in inter-bacterial interactions were described. However, possible defence mechanisms against T6SS attack among prey bacteria were not well clarified yet.MethodsEscherichia coli was assessed for susceptibility to T6SS-mediated killing by Vibrio cholerae. TheT6SS-mediated bacterial killing assays were performed in absence or presence of different protease inhibitors and with different mutant E. coli strains. Expression levels of selected proteins were monitored using SDS-PAGE and immunoblot analyses.ResultsThe T6SS-mediated killing of E. coli by V. cholerae was partly blocked when the serine protease inhibitor Pefabloc was present. E. coli lacking the periplasmic protease inhibitor Ecotin showed enhanced susceptibility to killing by V. cholerae. Mutations affecting E. coli membrane stability also caused increased susceptibility to killing by V. cholerae. E. coli lacking the maltodextrin porin protein LamB showed reduced susceptibility to killing by V. cholerae whereas E. coli with induced high levels of LamB showed reduced survival in inter-bacterial competition.ConclusionsOur study identified two proteins in E. coli, the intrinsic protease inhibitor Ecotin and the outer membrane porin LamB, that influenced E. coli susceptibility to T6SS-mediated killing by V. cholerae.General significanceWe envision that it is feasible to explore these findings to target and modulate their expression to obtain desired changes in inter-bacterial competition in vivo, e.g. in the gastrointestinal microbiome.     

Specificity of the Type II Secretion Systems of Enterotoxigenic Escherichia coli and Vibrio cholerae for Heat-Labile Enterotoxin and Cholera Toxin

The Gram-negative type II secretion (T2S) system is a multiprotein complex mediating the release of virulence factors from a number of pathogens. While an understanding of the function of T2S components is emerging, little is known about what identifies substrates for export. To investigate T2S substrate recognition, we compared mutations affecting the secretion of two highly homologous substrates: heat-labile enterotoxin (LT) from enterotoxigenic Escherichia coli (ETEC) and cholera toxin (CT) from Vibrio cholerae. Each toxin consists of one enzymatic A subunit and a ring of five B subunits mediating the toxin''s secretion. Here, we report two mutations in LT''s B subunit (LTB) that reduce its secretion from ETEC without global effects on the toxin. The Q3K mutation reduced levels of secreted LT by half, and as with CT (T. D. Connell, D. J. Metzger, M. Wang, M. G. Jobling, and R. K. Holmes, Infect. Immun. 63:4091-4098, 1995), the E11K mutation impaired LT secretion. Results in vitro and in vivo show that these mutants are not degraded more readily than wild-type LT. The Q3K mutation did not significantly affect CT B subunit (CTB) secretion from V. cholerae, and the E11A mutation altered LT and CTB secretion to various extents, indicating that these toxins are identified as secretion substrates in different ways. The levels of mutant LTB expressed in V. cholerae were low or undetectable, but each CTB mutant expressed and secreted at wild-type levels in ETEC. Therefore, ETEC''s T2S system seems to accommodate mutations in CTB that impair the secretion of LTB. Our results highlight the exquisitely fine-tuned relationship between T2S substrates and their coordinate secretion machineries in different bacterial species.Gram-negative bacteria have evolved a number of methods to secrete proteins into the extracellular milieu, with at least six specific secretion systems currently described (14, 30). Type II secretion (T2S), or the main terminal branch of the general secretory pathway, is a feature of a number of proteobacteria and has been shown to be required for pathogenesis and maintenance of environmental niches in a large number of species (5). The T2S system is a multiprotein complex of 12 to 15 components that spans the inner and outer membranes, allowing for the controlled release of certain folded proteins that have been directed to the periplasm through the Sec or Tat machinery (21). Aside from providing a means of exporting freely released virulence factors from plant, animal, and human pathogens (5), the T2S system has been shown to export surface-associated virulence factors (18), fimbrial components (46), outer membrane cytochromes (36), and a surfactant required for sliding motility in Legionella pneumophila (39), among other substrates.While an increasing number of studies have focused on understanding the structure and function of the components of the T2S system itself, little is known about what identifies a periplasmic protein as a substrate for secretion (21, 32). Because proteins secreted from the same bacterial species need not share any obvious structural homology, it is not even clear how much of a T2S substrate interacts with the secretion machinery (32). Analysis of two similar substrates that can each be secreted by the T2S systems of two distinct species would provide information about species-specific identification of T2S substrates and, by extension, the nature of the “secretion motif” identifying those substrates. Heat-labile enterotoxin (LT) from enterotoxigenic Escherichia coli (ETEC) and cholera toxin (CT) from Vibrio cholerae represent one such pair of substrates.ETEC and V. cholerae are enteric pathogens causing significant morbidity and mortality worldwide (33). The causative agents of traveler''s diarrhea and cholera, respectively, these two pathogens share a number of similarities, including the nature of their disease symptoms (38). Each pathogen secretes an AB₅ toxin important for colonization and the induction of water and electrolyte efflux from intestinal epithelial cells (1, 29). These toxins, LT and CT, are both encoded by two-gene operons. After sec-dependent transport to the periplasm, holotoxin formation occurs spontaneously (13), with one catalytic A subunit (LTA or CTA) assembling with five B subunits (LTB or CTB), which are responsible for the binding properties of the toxins. Export of fully folded and assembled LT or CT is then accomplished by the T2S system (34, 40). In ETEC, this system is encoded by gspC to -M (40), while in V. cholerae, these genes are found in the eps operon (34).LT and CT are very similar in structure, sharing approximately 80% sequence homology and 83% identity in the mature B subunit (16, 24). ETEC is thought to have acquired the genes for CT through horizontal transfer, with the toxins evolving over time to possess slight differences (45). As such, these toxins share the same primary host receptor, the monosialoganglioside G_M1, and catalyze the same ADP-ribosylation reaction within host cells (38). However, LT is able to bind other host sphingolipids in addition to G_M1 and to interact with sugar residues from the A-type blood antigen, which CT cannot bind (16, 41). Both LT and CT are able to associate with sugar residues in lipopolysaccharide (LPS) on the surface of E. coli cells (17). Binding to each of these substrates can be impaired by point mutation (26, 43).In this study, we report point mutations impairing the release of LT from ETEC and CT from V. cholerae. We analyzed the specificity of the defects in substrate recognition by comparing the effects of substituting charged and neutral residues in key regions of LTB and CTB. To confirm that the identified mutations resulted specifically in a secretion defect, we tested the effect of the mutations on (i) ligand binding by each toxin, (ii) toxin stability, and (iii) formation of secretion-competent B-subunit pentamers. By introducing comparable mutations into both toxins, including one previously reported to impair the secretion of CT (6), and exchanging toxin substrates between the two species, we have revealed species-dependent differences in T2S substrate recognition. Although wild-type LT and CT can be heterologously expressed and secreted from V. cholerae and ETEC, respectively, the substrate residues identified by the secretion machinery in each species are distinct. Together, our results demonstrate that highly homologous T2S substrates are recognized in different ways when secreted by two distinct systems.     

The binding of cholera toxin to the periplasmic vestibule of the type II secretion channel

The type II secretion system (T2SS) is a large macromolecular complex spanning the inner and outer membranes of many Gram-negative bacteria. The T2SS is responsible for the secretion of virulence factors such as cholera toxin (CT) and heat-labile enterotoxin (LT) from Vibrio cholerae and enterotoxigenic Escherichia coli, respectively. CT and LT are closely related AB₅ heterohexamers, composed of one A subunit and a B-pentamer. Both CT and LT are translocated, as folded protein complexes, from the periplasm across the outer membrane through the type II secretion channel, the secretin GspD. We recently published the 19 Å structure of the V. cholerae secretin (VcGspD) in its closed state and showed by SPR measurements that the periplasmic domain of GspD interacts with the B-pentamer complex. Here we extend these studies by characterizing the binding of the cholera toxin B-pentamer to VcGspD using electron microscopy of negatively stained preparations. Our studies indicate that the pentamer is captured within the large periplasmic vestibule of VcGspD. These new results agree well with our previously published studies and are in accord with a piston-driven type II secretion mechanism.Key words: secretin, GspD, electron cryomicroscopy, type II secretion system (T2SS), cholera toxin     

Assembly of the Type II Secretion System such as Found in Vibrio cholerae Depends on the Novel Pilotin AspS

The Type II Secretion System (T2SS) is a molecular machine that drives the secretion of fully-folded protein substrates across the bacterial outer membrane. A key element in the machinery is the secretin: an integral, multimeric outer membrane protein that forms the secretion pore. We show that three distinct forms of T2SSs can be distinguished based on the sequence characteristics of their secretin pores. Detailed comparative analysis of two of these, the Klebsiella-type and Vibrio-type, showed them to be further distinguished by the pilotin that mediates their transport and assembly into the outer membrane. We have determined the crystal structure of the novel pilotin AspS from Vibrio cholerae, demonstrating convergent evolution wherein AspS is functionally equivalent and yet structurally unrelated to the pilotins found in Klebsiella and other bacteria. AspS binds to a specific targeting sequence in the Vibrio-type secretins, enhances the kinetics of secretin assembly, and homologs of AspS are found in all species of Vibrio as well those few strains of Escherichia and Shigella that have acquired a Vibrio-type T2SS.     

Amyloids of Alpha-Synuclein Affect the Structure and Dynamics of Supported Lipid Bilayers

Interactions of monomeric alpha-synuclein (αS) with lipid membranes have been suggested to play an important role in initiating aggregation of αS. We have systematically analyzed the distribution and self-assembly of monomeric αS on supported lipid bilayers. We observe that at protein/lipid ratios higher than 1:10, αS forms micrometer-sized clusters, leading to observable membrane defects and decrease in lateral diffusion of both lipids and proteins. An αS deletion mutant lacking amino-acid residues 71–82 binds to membranes, but does not observably affect membrane integrity. Although this deletion mutant cannot form amyloid, significant amyloid formation is observed in the wild-type αS clusters. These results suggest that the process of amyloid formation, rather than binding of αS on membranes, is crucial in compromising membrane integrity.     

Chimeric adaptor proteins translocate diverse type VI secretion system effectors in Vibrio cholerae

Vibrio cholerae is a diverse species of Gram-negative bacteria, commonly found in the aquatic environment and the causative agent of the potentially deadly disease cholera. These bacteria employ a type VI secretion system (T6SS) when they encounter prokaryotic and eukaryotic competitors. This contractile puncturing device translocates a set of effector proteins into neighboring cells. Translocated effectors are toxic unless the targeted cell produces immunity proteins that bind and deactivate incoming effectors. Comparison of multiple V. cholerae strains indicates that effectors are encoded in T6SS effector modules on mobile genetic elements. We identified a diverse group of chimeric T6SS adaptor proteins required for the translocation of diverse effectors encoded in modules. An example for a T6SS effector that requires T6SS adaptor protein 1 (Tap-1) is TseL found in pandemic V. cholerae O1 serogroup strains and other clinical isolates. We propose a model in which Tap-1 is required for loading TseL onto the secretion apparatus. After T6SS-mediated TseL export is completed, Tap-1 is retained in the bacterial cell to load other T6SS machines.     

PKA Regulates Vacuolar H+-ATPase Localization and Activity via Direct Phosphorylation of the A Subunit in Kidney Cells

The vacuolar H⁺-ATPase (V-ATPase) is a major contributor to luminal acidification in epithelia of Wolffian duct origin. In both kidney-intercalated cells and epididymal clear cells, cAMP induces V-ATPase apical membrane accumulation, which is linked to proton secretion. We have shown previously that the A subunit in the cytoplasmic V₁ sector of the V-ATPase is phosphorylated by protein kinase A (PKA). Here we have identified by mass spectrometry and mutagenesis that Ser-175 is the major PKA phosphorylation site in the A subunit. Overexpression in HEK-293T cells of either a wild-type (WT) or phosphomimic Ser-175 to Asp (S175D) A subunit mutant caused increased acidification of HCO₃⁻-containing culture medium compared with cells expressing vector alone or a PKA phosphorylation-deficient Ser-175 to Ala (S175A) mutant. Moreover, localization of the S175A A subunit mutant expressed in HEK-293T cells was more diffusely cytosolic than that of WT or S175D A subunit. Acute V-ATPase-mediated, bafilomycin-sensitive H⁺ secretion was up-regulated by a specific PKA activator in HEK-293T cells expressing WT A subunit in HCO₃⁻-free buffer. In cells expressing the S175D mutant, V-ATPase activity at the membrane was constitutively up-regulated and unresponsive to PKA activators, whereas cells expressing the S175A mutant had decreased V-ATPase activity that was unresponsive to PKA activation. Finally, Ser-175 was necessary for PKA-stimulated apical accumulation of the V-ATPase in a polarized rabbit cell line of collecting duct A-type intercalated cell characteristics (Clone C). In summary, these results indicate a novel mechanism for the regulation of V-ATPase localization and activity in kidney cells via direct PKA-dependent phosphorylation of the A subunit at Ser-175.     

Endochitinase Is Transported to the Extracellular Milieu by the eps-Encoded General Secretory Pathway of Vibrio cholerae

The chiA gene of Vibrio cholerae encodes a polypeptide which degrades chitin, a homopolymer of N-acetylglucosamine (GlcNAc) found in cell walls of fungi and in the integuments of insects and crustaceans. chiA has a coding capacity corresponding to a polypeptide of 846 amino acids having a predicted molecular mass of 88.7 kDa. A 52-bp region with promoter activity was found immediately upstream of the chiA open reading frame. Insertional inactivation of the chromosomal copy of the gene confirmed that expression of chitinase activity by V. cholerae required chiA. Fluorescent analogues were used to demonstrate that the enzymatic activity of ChiA was specific for β,1-4 glycosidic bonds located between GlcNAc monomers in chitin. Antibodies against ChiA were obtained by immunization of a rabbit with a MalE-ChiA hybrid protein. Polypeptides with antigenic similarity to ChiA were expressed by classical and El Tor biotypes of V. cholerae and by the closely related bacterium Aeromonas hydrophila. Immunoblotting experiments using the wild-type strain 569B and the secretion mutant M14 confirmed that ChiA is an extracellular protein which is secreted by the eps system. The eps system is also responsible for secreting cholera toxin, an oligomeric protein with no amino acid homology to ChiA. These results indicate that ChiA and cholera toxin have functionally similar extracellular transport signals that are essential for eps-dependent secretion.Chitin, a homopolymer of N-acetylglucosamine (GlcNAc), is a major component of the cell walls of fungi and the integuments of crustaceans and insects (38). The molecule is one of the most abundant biopolymers in nature and is used by many microorganisms as a source of carbon. Utilization of chitin as a nutrient usually requires degradation of the molecule to GlcNAc monomers. Complete degradation of chitin in both prokaryotes and eukaryotes is a two-step process which involves successive hydrolysis of the β,1-4 glycosidic bonds linking the GlcNAc subunits. In the first stage, endochitinase binds and degrades tetrameric and longer polymeric forms of GlcNAc to produce the disaccharide chitobiose. In the second step, chitobiase hydrolyzes chitobiose to GlcNAc monomers. The enzymes for chitin degradation are usually coordinately regulated and in several organisms are induced by chitosan, chitobiose, GlcNAc, or glucosamine (2, 7, 44).Members of the family Vibrionaceae thrive in marine environments where chitin is abundant. It is not surprising that many Vibrionaceae evolved systems for utilizing chitin as a nutrient source. Chitinases have been identified in Vibrio vulnificus (56, 61), V. harveyi (57), and V. parahemolyticus (29, 30). Nucleotide sequence analysis indicated that the chitinase of V. harveyi is homologous with human hexosamindase, indicating that the two enzymes, as well as other chitinases, are members of a phylogenically related group (56).V. cholerae is a human intestinal pathogen that resides in brackish and marine waters. In vitro experiments established that V. cholerae has the potential to use chitin as a sole source of carbon for growth (15). It is likely, therefore, that production of chitinase (29, 30, 42) by V. cholerae provides the bacterium with a readily available nutrient source in aquatic environments. Hydrolysis of chitin by V. cholerae is an extracellular process that requires expression of epsE, one of a cluster of genes in the eps locus (43, 46–48). Several proteins of V. cholerae are dependent on the eps system for extracellular transport, including cholera toxin (CT), an undefined protease, and a chitinase activity (43, 48). Although expression of chitinase activity has been reported for V. cholerae, the enzyme responsible for the activity has not been identified. To further characterize the extracellular chitinase of V. cholerae, we cloned a gene encoding chitinase activity. Here we report the nucleotide sequence of a cloned endochitinase gene and establish that the protein encoded by that gene is secreted to the extracellular environment by an eps-dependent mechanism.     

Multidrug Efflux Pump AcrAB of Salmonella typhimurium Excretes Only Those β-Lactam Antibiotics Containing Lipophilic Side Chains

We found that the previously reported SS-B drug-supersusceptible mutant of Salmonella typhimurium (S. Sukupolvi, M. Vaara, I. M. Helander, P. Viljanen, and P. H. Mäkelä, J. Bacteriol. 159:704–712, 1984) had a mutation in the acrAB operon. Comparison of this mutant with its parent strain and with an AcrAB-overproducing strain showed that the activity of the AcrAB efflux pump often produced significant resistance to β-lactam antibiotics in the complete absence of β-lactamase. The effect of AcrAB activity on resistance was more pronounced with agents containing more lipophilic side chains, suggesting that such compounds were better substrates for this pump. This correlation is consistent with the hypothesis that only those molecules that become at least partially partitioned into the lipid bilayer of the cytoplasmic membrane are captured by the AcrAB pump. According to this mechanism, the pump successfully excretes even those β-lactams that fail to traverse the cytoplasmic membrane, because these compounds are likely to become partitioned into the outer leaflet of the bilayer. Even the compounds with lipophilic side chains were shown to penetrate across the outer membrane relatively rapidly, if the pump was inactivated genetically or physiologically. The exclusion of such compounds, exemplified by nafcillin, from cells of the wild-type S. typhimurium was previously interpreted as the result of poor diffusion across the outer membrane (H. Nikaido, Biochim. Biophys. Acta 433:118–132, 1976), but it is now recognized as the consequence of efficient pumping out of entering antibiotics by the active efflux process.