T3S System Research Articles

The Type II Secretion System of Legionella pneumophila Dampens the MyD88 and Toll-Like Receptor 2 Signaling Pathway in Infected Human Macrophages.

Previously, we reported that mutants of Legionella pneumophila lacking a type II secretion (T2S) system elicit higher levels of cytokines (e.g., interleukin-6 [IL-6]) following infection of U937 cells, a human macrophage-like cell line. We now show that this effect of T2S is also manifest upon infection of human THP-1 macrophages and peripheral blood monocytes but does not occur during infection of murine macrophages. Supporting the hypothesis that T2S acts to dampen the triggering of an innate immune response, we observed that the mitogen-activated protein kinase (MAPK) and nuclear transcription factor kappa B (NF-κB) pathways are more highly stimulated upon infection with the T2S mutant than upon infection with the wild type. By using short hairpin RNA to deplete proteins involved in specific pathogen-associated molecular pattern (PAMP) recognition pathways, we determined that the dampening effect of the T2S system was not dependent on nucleotide binding oligomerization domain (NOD)-like receptors (NLRs), retinoic acid-inducible protein I (RIG-I)-like receptors (RLRs), double-stranded RNA (dsRNA)-dependent protein kinase receptor (PKR), or TIR domain-containing adaptor inducing interferon beta (TRIF) signaling or an apoptosis-associated speck-like protein containing a CARD (ASC)- or caspase-4-dependent inflammasome. However, the dampening effect of T2S on IL-6 production was significantly reduced upon gene knockdown of myeloid differentiation primary response 88 (MyD88), TANK binding kinase 1 (TBK1), or Toll-like receptor 2 (TLR2). These data indicate that the L. pneumophila T2S system dampens the signaling of the TLR2 pathway in infected human macrophages. We also document the importance of PKR, TRIF, and TBK1 in cytokine secretion during L. pneumophila infection of macrophages.

Crystal structure of the type IV secretion system component CagX from Helicobacter pylori.

Helicobacter pylori, a Gram-negative bacterial pathogen prevalent in the human population, is the causative agent of severe gastric diseases. An H. pylori type IV secretion (T4S) system encoded by the cytotoxin-associated gene pathogenicity island (cagPAI) is responsible for communication with host cells. As a component of the cagPAI T4S system core complex, CagX plays an important role in virulence-protein translocation into the host cells. In this work, the crystal structure of the C-terminal domain of CagX (CagXct), which is a homologue of the VirB9 protein from the VirB/D4 T4S system, is presented. CagXct is only the second three-dimensional structure to be elucidated of a VirB9-like protein. Another homologue, TraO, which is encoded on the Escherichia coli conjugative plasmid pKM101, shares only 19% sequence identity with CagXct; however, there is a remarkable similarity in tertiary structure between these two β-sandwich protein domains. Most of the residues that are conserved between CagXct and TraO are located within the protein core and appear to be responsible for the preservation of this domain fold. The studies presented here will contribute to our understanding of different bacterial T4S systems.

The Predicted Lytic Transglycosylase HpaH from Xanthomonas campestris pv. vesicatoria Associates with the Type III Secretion System and Promotes Effector Protein Translocation.

The pathogenicity of the Gram-negative plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria depends on a type III secretion (T3S) system, which spans both bacterial membranes and translocates effector proteins into plant cells. The assembly of the T3S system presumably involves the predicted lytic transglycosylase (LT) HpaH, which is encoded adjacent to the T3S gene cluster. Bacterial LTs degrade peptidoglycan and often promote the formation of membrane-spanning macromolecular protein complexes. In the present study, we show that HpaH localizes to the bacterial periplasm and binds to peptidoglycan as well as to components of the T3S system, including the predicted periplasmic inner rod proteins HrpB1 and HrpB2 as well as the pilus protein HrpE. In vivo translocation assays revealed that HpaH promotes the translocation of various effector proteins and of early substrates of the T3S system, suggesting a general contribution of HpaH to type III-dependent protein export. Mutant studies and the analysis of reporter fusions showed that the N-terminal region of HpaH contributes to protein function and is proteolytically cleaved. The N-terminally truncated HpaH cleavage product is secreted into the extracellular milieu by a yet-unknown transport pathway, which is independent of the T3S system.

Introduction to Type III Secretion Systems.

Type III secretion (T3S) systems are found in a large number of gram-negative bacteria where they function to manipulate the biology of infected hosts. Hosts targeted by T3S systems are widely distributed in nature and are represented by animals and plants. T3S systems are found in diverse genera of bacteria and they share a common core structure and function. Effector proteins are delivered by T3S systems into targeted host cells without prior secretion of the effectors into the environment. Instead, an assembled translocon structure functions to translocate effectors across eukaryotic cell membranes. In many cases, T3S systems are essential virulence factors and in some instances they promote symbiotic interactions.

In Vivo Photo-Cross-Linking to Study T3S Interactions Demonstrated Using the Yersinia pestis T3S System.

Cross-linking of proteins is effective in determining protein-protein interactions. The use of photo-cross-linkers was developed to study protein interactions in several manners. One method involved the incorporation of photo-activatable cross-linking groups into chemically synthesized peptides. A second approach relies on incorporation of photo-activatable cross-linking groups into proteins using tRNAs with chemically bound photo-activatable amino acids with suppressor tRNAs translational systems to incorporate the tags into specific sites. A third system was made possible by the development of photoreactive amino acids that use the normal cellular tRNAs and aminoacyl tRNA synthetases. In this method, the third system is used to demonstrate its utility for the study of T3S system interactions. This method describes how two photo-activatable amino acids, photo-methionine and photo-leucine, that use the normal cellular machinery are incorporated into Yersinia pestis and used to study interactions in the T3S system. To demonstrate the system, the method was used to cross-link the T3S regulatory proteins LcrG and LcrV.

Isolation of Type III Secretion System Needle Complexes by Shearing.

Type III secretion (T3S) needle proteins are essential for the pathogenesis of many gram-negative bacteria. The needle component of the T3S system serves as the conduit for the translocation of effector proteins from the cytoplasm of many gram-negative bacteria into their target eukaryotic cells. Despite substantial advances that have been made in their characterization, a lot is still unknown about their interactions with other T3S system proteins and their roles in modulating host immune responses during infections. Critical to achieving this knowledge is the ability to isolate these needle proteins in their stable, native form. In this chapter, we describe a modified, streamlined isolation strategy for native forms of these T3S system needle proteins. We also present assays to detect the presence and quantification of these needle proteins.

Detection of Protein Interactions in T3S Systems Using Yeast Two-Hybrid Analysis.

Two-hybrid systems, sometimes termed interaction traps, are genetic systems designed to find and analyze interactions between proteins. The most common systems are yeast based (commonly Saccharomyces cerevisae) and rely on the functional reconstitution of the GAL4 transcriptional activator. Reporter genes, such as the lacZ gene of Escherichia coli (encodes β-galactosidase), are placed under GAL4-dependent transcriptional control to provide quick and reliable detection of protein interactions. In this method the use of a yeast-based two-hybrid system is described to study protein interactions between components of type III secretion systems.

Use of Transcriptional Control to Increase Secretion of Heterologous Proteins in T3S Systems.

Heterologous proteins can be produced in a bacterial host and purified from the cellular constituents. Secretion of the protein of interest to the extracellular space simplifies the purification process and is thought to alleviate toxicity problems associated with intracellular accumulation of the protein of interest. In this protocol, we describe a strategy to engineer protein secretion in a bacterial culture using transcriptional control. The transcription factor HilA is inducibly produced to control production of the secretion machine, and in turn signals the production and secretion of a protein of interest. This allows for high titer of secreted protein in optimized culturing conditions and the effect is observed with all proteins tested.

A Method for Characterizing the Type III Secretion System's Contribution to Pathogenesis: Homologous Recombination to Generate Yersinia pestis Type III Secretion System Mutants.

The type III (T3S) secretion system of many gram-negative bacteria is a surface-exposed protein secretion apparatus used to directly inject bacterial effector molecules into eukaryotic cells. These effector molecules contribute to bacterial pathogenesis in many ways, and have been shown to be crucial for infectivity. Here, we describe a protocol for using homologous recombination to generate T3S system mutants to assess the role of different T3S system proteins in bacterial pathogenesis.

HrcU and HrpP are pathogenicity factors in the fire blight pathogen Erwinia amylovora required for the type III secretion of DspA/E.

BackgroundMany Gram-negative bacterial pathogens mediate host-microbe interactions via utilization of the type III secretion (T3S) system. The T3S system is a complex molecular machine consisting of more than 20 proteins. Collectively, these proteins translocate effectors across extracellular space and into the host cytoplasm. Successful translocation requires timely synthesis and allocation of both structural and secreted T3S proteins. Based on amino acid conservation in animal pathogenic bacteria, HrcU and HrpP were examined for their roles in regulation of T3S hierarchy.ResultsBoth HrcU and HrpP were shown to be required for disease development in an immature pear infection model and respective mutants were unable to induce a hypersensitive response in tobacco. Using in vitro western blot analyses, both proteins were also shown to be required for the secretion of DspA/E, a type 3 effector and an important pathogenicity factor. Via yeast-two hybridization (Y2H), HrpP and HrcU were revealed to exhibit protein-protein binding. Finally, all HrcU and HrpP phenotypes identified were shown to be dependent on a conserved amino acid motif in the cytoplasmic tail of HrcU.ConclusionsCollectively, these data demonstrate roles for HrcU and HrpP in regulating T3S and represent the first attempt in understanding T3S heirarchy in E. amylovora.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0702-y) contains supplementary material, which is available to authorized users.

The N terminus of Yersinia pestis’ YscF masks YscF’s naturally occurring PAMP, inhibiting inflammation and allowing for colonization in vivo

Abstract Type III secretion (T3S) systems, common to many Gram- bacteria, act as a needle to deliver virulence factors directly into eukaryotic cells, often modulating host immunity. Multimers of YscF polymerize to form the surface-located T3S needle-like structure. We have previously shown that YscF has PAMP properties, acting as both a TLR2 and TLR4 agonist. We have also shown that modulation of this PAMP activity in vitro is located in the N terminus of YscF. We hypothesized that the N terminus of YscF contributes to Y. pestis survival within the eukaryotic host. We generated a 15 amino acid N terminal truncation of YscF (delta15YscF) expressed in Y. pestis. The truncation did not affect T3S system translocation. The delta15YscF Y. pestis strain was able to secrete Yops, albeit at a level lower than wild type (wt) Y. pestis. As expected, wt Y. pestis colonized the spleen and liver, whereas delta15YscF Y. pestis exhibited significantly impaired colonization in both organs. Mice infected with delta15YscF Y. pestis developed significantly more inflammation compared to wt Y. pestis. At 3 days p.i., delta15YscF spleen weights were significantly heavier and spleen inflammation was accompanied by a significant enhancement of macrophage and neutrophil migration compared to wt mice. These data support a role for the N terminus of YscF contributing to host bacterial clearance. Truncating the N terminus may result in a PAMP which is thereby unmasked and able to induce an inflammatory response.

Type III-Dependent Translocation of HrpB2 by a Nonpathogenic hpaABC Mutant of the Plant-Pathogenic Bacterium Xanthomonas campestris pv. vesicatoria.

The plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria employs a type III secretion (T3S) system to translocate effector proteins into plant cells. The T3S apparatus spans both bacterial membranes and is associated with an extracellular pilus and a channel-like translocon in the host plasma membrane. T3S is controlled by the switch protein HpaC, which suppresses secretion and translocation of the predicted inner rod protein HrpB2 and promotes secretion of translocon and effector proteins. We previously reported that HrpB2 interacts with HpaC and the cytoplasmic domain of the inner membrane protein HrcU (C. Lorenz, S. Schulz, T. Wolsch, O. Rossier, U. Bonas, and D. Büttner, PLoS Pathog 4:e1000094, 2008, http://dx.doi.org/10.1371/journal.ppat.1000094). However, the molecular mechanisms underlying the control of HrpB2 secretion are not yet understood. Here, we located a T3S and translocation signal in the N-terminal 40 amino acids of HrpB2. The results of complementation experiments with HrpB2 deletion derivatives revealed that the T3S signal of HrpB2 is essential for protein function. Furthermore, interaction studies showed that the N-terminal region of HrpB2 interacts with the cytoplasmic domain of HrcU, suggesting that the T3S signal of HrpB2 contributes to substrate docking. Translocation of HrpB2 is suppressed not only by HpaC but also by the T3S chaperone HpaB and its secreted regulator, HpaA. Deletion of hpaA, hpaB, and hpaC leads to a loss of pathogenicity but allows the translocation of fusion proteins between the HrpB2 T3S signal and effector proteins into leaves of host and non-host plants. The T3S system of the plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria is essential for pathogenicity and delivers effector proteins into plant cells. T3S depends on HrpB2, which is a component of the predicted periplasmic inner rod structure of the secretion apparatus. HrpB2 is secreted during the early stages of the secretion process and interacts with the cytoplasmic domain of the inner membrane protein HrcU. Here, we localized the secretion and translocation signal of HrpB2 in the N-terminal 40 amino acids and show that this region is sufficient for the interaction with the cytoplasmic domain of HrcU. Our results suggest that the T3S signal of HrpB2 is required for the docking of HrpB2 to the secretion apparatus. Furthermore, we provide experimental evidence that the N-terminal region of HrpB2 is sufficient to target effector proteins for translocation in a nonpathogenic X. campestris pv. vesicatoria strain.

Editorial: Genomics and Effectomics of the Crop Killer Xanthomonas.

Phytopathogenic bacteria of the Xanthomonas genus cause severe diseases on hundreds of host plants, including economically important crops, such as bean, cabbage, cassava, citrus, hemp, pepper, rice, sugarcane, tomato, or wheat. Diseases occurring in nature comprise bacterial blight, canker, necrosis, rot, scald, spot, streak, or wilt. Xanthomonas spp., are distributed worldwide, and pathogenic and non-pathogenic strains are essentially found in association to plants. Some phytopathogenic strains are emergent or re-emergent and, consequently, dramatically impact agriculture, economy, and food safety. During the last decades, massive efforts were undertaken to decipher Xanthomonas biology. So far, more than 100 complete or draft genomes from diverse Xanthomonas species have been sequenced (http://www.xanthomonas.org), thus providing powerful tools to study genetic determinants triggering pathogenicity and adaptation to plant habitats. Xanthomonas spp., employ an arsenal of virulence factors to invade its host, including extracellular polysaccharides, plant cell wall-degrading enzymes, adhesins, and secreted effectors. In most xanthomonads, type III secretion (T3S) system and secreted effectors (T3Es) are essential to bacterial pathogenicity through the inhibition of plant immunity or the induction of plant susceptibility (S) genes, as reported for Transcription Activation-Like (TAL) effectors. Yet, toxins can also be major virulence determinants in some xanthomonads while non-pathogenic Xanthomonas species do live in sympatry with plant without any T3S systems nor T3Es. In a context of ever increasing international commercial exchanges and modifications of the climate, monitoring and regulating pathogens spread is of crucial importance for food security. A deep knowledge of the genomic diversity of Xanthomonas spp., is required for scientists to properly identify strains, to help preventing future disease outbreaks and to achieve knowledge-informed sustainable disease resistance in crops. This research topic published in the “Plant Biotic Interactions” section of Frontiers in Plant Science and Frontiers in Microbiology aims at illustrating several of the recent achievements of the Xanthomonas community. We collected twelve manuscripts dealing with comparative genomics or T3E repertoires, including five focusing on TAL effectors which we hope will contribute to advance research on plant pathogenic bacteria.

Acinetobacter baumannii Is Dependent on the Type II Secretion System and Its Substrate LipA for Lipid Utilization and In Vivo Fitness.

Gram-negative bacteria express a number of sophisticated secretion systems to transport virulence factors across the cell envelope, including the type II secretion (T2S) system. Genes for the T2S components GspC through GspN and PilD are conserved among isolates of Acinetobacter baumannii, an increasingly common nosocomial pathogen that is developing multidrug resistance at an alarming rate. In contrast to most species, however, the T2S genes are dispersed throughout the genome rather than linked into one or two operons. Despite this unique genetic organization, we show here that the A. baumannii T2S system is functional. Deletion of gspD or gspE in A. baumannii ATCC 17978 results in loss of secretion of LipA, a lipase that breaks down long-chain fatty acids. Due to a lack of extracellular lipase, the gspD mutant, the gspE mutant, and a lipA deletion strain are incapable of growth on long-chain fatty acids as a sole source of carbon, while their growth characteristics are indistinguishable from those of the wild-type strain in nutrient-rich broth. Genetic inactivation of the T2S system and its substrate, LipA, also has a negative impact on in vivo fitness in a neutropenic murine model for bacteremia. Both the gspD and lipA mutants are outcompeted by the wild-type strain as judged by their reduced numbers in spleen and liver following intravenous coinoculation. Collectively, our findings suggest that the T2S system plays a hitherto-unrecognized role in in vivo survival of A. baumannii by transporting a lipase that may contribute to fatty acid metabolism. Infections by multidrug-resistant Acinetobacter baumannii are a growing health concern worldwide, underscoring the need for a better understanding of the molecular mechanisms by which this pathogen causes disease. In this study, we demonstrated that A. baumannii expresses a functional type II secretion (T2S) system that is responsible for secretion of LipA, an extracellular lipase required for utilization of exogenously added lipids. The T2S system and the secreted lipase support in vivo colonization and thus contribute to the pathogenic potential of A. baumannii.

Chlamydia trachomatis Type III Secretion Proteins Regulate Transcription.

The Scc4 protein (CT663) of the pathogenic bacterium Chlamydia has been described as a type III secretion (T3S) chaperone as well as an inhibitor of RNA polymerase. To examine if these roles are connected, we first investigated physical interactions between Chlamydia trachomatis Scc4 and the T3S chaperone Scc1 and a T3S substrate, CopN. In a yeast 3-hybrid assay, Scc4, Scc1, and CopN were all required to detect an interaction, which suggests that these proteins form a trimolecular complex. We also detected interactions between any two of these three T3S proteins in a pulldown assay using only recombinant proteins. We next determined whether these interactions affected the function of Scc4 as an inhibitor of RNA transcription. Using Escherichia coli as a heterologous in vivo system, we demonstrated that expression of C. trachomatis Scc4 led to a drastic decrease in transcript levels for multiple genes. However, coexpression of Scc4 with Scc1, CopN, or both alleviated Scc4-mediated inhibition of transcription. Scc4 expression also severely impaired E. coli growth, but this growth defect was reversed by coexpression of Scc4 with Scc1, CopN, or both, suggesting that the inhibitory effect of Scc4 on transcription and growth can be antagonized by interactions between Scc4, Scc1, and CopN. These findings suggest that the dual functions of Scc4 may serve as a bridge to link T3S and the regulation of gene expression in Chlamydia. This study investigates a novel mechanism for regulating gene expression in the pathogenic bacterium Chlamydia. The Chlamydia type III secretion (T3S) chaperone Scc4 has been shown to inhibit transcription by RNA polymerase. This study describes physical interactions between Scc4 and the T3S proteins Scc1 and CopN. Furthermore, Chlamydia Scc1 and CopN antagonized the inhibitory effects of Scc4 on transcription and growth in a heterologous Escherichia coli system. These results provide evidence that transcription in Chlamydia can be regulated by the T3S system through interactions between T3S proteins.

Xanthomonas campestris pv. vesicatoria Secretes Proteases and Xylanases via the Xps Type II Secretion System and Outer Membrane Vesicles.

Many plant-pathogenic bacteria utilize type II secretion (T2S) systems to secrete degradative enzymes into the extracellular milieu. T2S substrates presumably mediate the degradation of plant cell wall components during the host-pathogen interaction and thus promote bacterial virulence. Previously, the Xps-T2S system from Xanthomonas campestris pv. vesicatoria was shown to contribute to extracellular protease activity and the secretion of a virulence-associated xylanase. The identities and functions of additional T2S substrates from X. campestris pv. vesicatoria, however, are still unknown. In the present study, the analysis of 25 candidate proteins from X. campestris pv. vesicatoria led to the identification of two type II secreted predicted xylanases, a putative protease and a lipase which was previously identified as a virulence factor of X. campestris pv. vesicatoria. Studies with mutant strains revealed that the identified xylanases and the protease contribute to virulence and in planta growth of X. campestris pv. vesicatoria. When analyzed in the related pathogen X. campestris pv. campestris, several T2S substrates from X. campestris pv. vesicatoria were secreted independently of the T2S systems, presumably because of differences in the T2S substrate specificities of the two pathogens. Furthermore, in X. campestris pv. vesicatoria T2S mutants, secretion of T2S substrates was not completely absent, suggesting the contribution of additional transport systems to protein secretion. In line with this hypothesis, T2S substrates were detected in outer membrane vesicles, which were frequently observed for X. campestris pv. vesicatoria. We, therefore, propose that extracellular virulence-associated enzymes from X. campestris pv. vesicatoria are targeted to the Xps-T2S system and to outer membrane vesicles. The virulence of plant-pathogenic bacteria often depends on TS2 systems, which secrete degradative enzymes into the extracellular milieu. T2S substrates are being studied in several plant-pathogenic bacteria, including Xanthomonas campestris pv. vesicatoria, which causes bacterial spot disease in tomato and pepper. Here, we show that the T2S system from X. campestris pv. vesicatoria secretes virulence-associated xylanases, a predicted protease, and a lipase. Secretion assays with the related pathogen X. campestris pv. campestris revealed important differences in the T2S substrate specificities of the two pathogens. Furthermore, electron microscopy showed that T2S substrates from X. campestris pv. vesicatoria are targeted to outer membrane vesicles (OMVs). Our results, therefore, suggest that OMVs provide an alternative transport route for type II secreted extracellular enzymes.

Molecular ruler determines needle length for the Salmonella Spi-1 injectisome

The type-III secretion (T3S) systems of bacteria are part of self-assembling nanomachines: the bacterial flagellum that enables cells to propel themselves through liquid and across hydrated surfaces, and the injectisome that delivers pathogenic effector proteins into eukaryotic host cells. Although the flagellum and injectisome serve different purposes, they are evolutionarily related and share many structural similarities. Core features to these T3S systems are intrinsic length control mechanisms for external cellular projections: the hook of the flagellum and the injectisome needle. We present evidence that the Spi-1 injectisome, like the Salmonella flagellar hook, uses a secreted molecular ruler, InvJ, to determine needle length. This result supports a universal length control mechanism using molecular rulers for T3S systems.

PscI is a type III secretion needle anchoring protein with in vitro polymerization capacities.

The export of bacterial toxins across the bacterial envelope requires the assembly of complex, membrane-embedded protein architectures. Pseudomonas aeruginosa employs type III secretion (T3S) injectisome to translocate exotoxins directly into the cytoplasm of a target eukaryotic cell. This multi-protein channel crosses two bacterial membranes and extends further as a needle through which the proteins travel. We show in this work that PscI, proposed to form the T3S system (T3SS) inner rod, possesses intrinsic properties to polymerize into flexible and regularly twisted fibrils and activates IL-1β production in mouse bone marrow macrophages in vitro. We also found that point mutations within C-terminal amphipathic helix of PscI alter needle assembly in vitro and T3SS function in cell infection assays, suggesting that this region is essential for an efficient needle assembly. The overexpression of PscF partially compensates for the absence of the inner rod in PscI-deficient mutant by forming a secretion-proficient injectisome. All together, we propose that the polymerized PscI in P. aeruginosa optimizes the injectisome function by anchoring the needle within the envelope-embedded complex of the T3S secretome and - contrary to its counterpart in Salmonella - is not involved in substrate switching.

Structural studies of T4S systems by electron microscopy

Type IV secretion (T4S) systems are large dynamic nanomachines that transport DNA and/or proteins through the membranes of bacteria. Analysis of T4S system architecture is an extremely challenging task taking into account their multi protein organisation and lack of overall global symmetry. Nonetheless the last decade demonstrated an amazing progress achieved by X-ray crystallography and cryo-electron microscopy. In this review we present a structural analysis of this dynamic complex based on recent advances in biochemical, biophysical and structural studies.

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.