Secretion Of Cholera Toxin Research Articles

The Type II Secretory System Mediates Phage Infection in Vibrio cholerae.

Attachment and specific binding to the receptor on the host cell surface is the first step in the process of bacteriophage infection. The lytic phage VP2 is used in phage subtyping of the Vibrio cholerae biotype El Tor of the O1 serogroup; however, its infection mechanism is poorly understood. In this study, we aimed to identify its receptor on V. cholerae. The outer membrane protein EpsD in the type II secretory system (T2SS) was found to be related to VP2-specific adsorption to V. cholerae, and the T2SS inner membrane protein EpsM had a role in successful VP2 infection, although it was not related to adsorption of VP2. The tail fiber protein gp20 of VP2 directly interacts with EpsD. Therefore, we found that in V. cholerae, in addition to the roles of the T2SS as the transport apparatus of cholera toxin secretion and filamentous phage release, the T2SS is also used as the receptor for phage infection and probably as the channel for phage DNA injection. Our study expands the understanding of the roles of the T2SS in bacteria.

Cholera Toxin Encapsulated within Several Vibrio cholerae O1 Serotype Inaba Outer Membrane Vesicles Lacks a Functional B-Subunit.

Cholera toxin (CT), the major virulence factor of Vibrio cholerae, is an AB5 toxin secreted through the type II secretion system (T2SS). Upon secretion, the toxin initiates endocytosis through the interaction of the B pentamer with the GM1 ganglioside receptor on small intestinal cells. In addition to the release of CT in the free form, the bacteria secrete CT in association with outer membrane vesicles (OMVs). Previously, we demonstrated that strain 569B releases OMVs that encapsulate CT and which interact with host cells in a GM1-independent mechanism. Here, we have demonstrated that OMV-encapsulated CT, while biologically active, does not exist in an AB5 form; rather, the OMVs encapsulate two enzymatic A-subunit (CTA) polypeptides. We further investigated the assembly and secretion of the periplasmic CT and found that a major fraction of periplasmic CTA does not participate in the CT assembly process and instead is continuously encapsulated within the OMVs. Additionally, we found that the encapsulation of CTA fragments in OMVs is conserved among several Inaba O1 strains. We further found that under conditions in which the amount of extracellularly secreted CT increases, the concentration of OMV-encapsulated likewise CTA increases. These results point to a secondary mechanism for the secretion of biologically active CT that does not depend on the CTB-GM1 interaction for endocytosis.

Association of Vibrio cholerae 569B outer membrane vesicles with host cells occurs in a GM1-independent manner.

The primary virulence factor of Vibrio cholerae, cholera toxin (CT), initiates a pathway in epithelial cells that leads to the severe diarrhoea characteristic of cholera. Secreted CT binds to GM1 on the surface of host cells to facilitate internalisation. Many bacterial toxins, including CT, have been shown to be additionally delivered via outer membrane vesicles (OMVs). A fraction of the closely related heat labile toxin produced by enterotoxigenic Escherichia coli has been demonstrated to reside on the surface of OMVs, where it binds GM1 to facilitate OMV internalisation by host cells. In this work, we investigated whether OMV-associated CT is likewise trafficked to host cells in a GM1-dependent mechanism. We demonstrated that a majority of CT is secreted in its OMV-associated form and is located exclusively inside the vesicle. Therefore, the toxin is unable to bind GM1 on the host cell surface, and the OMVs are trafficked to the host cells in a GM1-independent mechanism. These findings point to a secondary, noncompeting mechanism for secretion and delivery of CT, beyond its well-studied secretion via a Type II secretion system and underscore the importance of focusing future studies on understanding this GM1-independent delivery mechanism to fully understand Vibrio cholerae pathogenesis.

Structure and Membrane Topography of the Vibrio-Type Secretin Complex from the Type 2 Secretion System of Enteropathogenic Escherichia coli.

The β-barrel assembly machinery (BAM) complex is the core machinery for the assembly of β-barrel membrane proteins, and inhibition of BAM complex activity is lethal to bacteria. Discovery of integral membrane proteins that are key to pathogenesis and yet do not require assistance from the BAM complex raises the question of how these proteins assemble into bacterial outer membranes. Here, we address this question through a structural analysis of the type 2 secretion system (T2SS) secretin from enteropathogenic Escherichia coli O127:H6 strain E2348/69. Long β-strands assemble into a barrel extending 17 Å through and beyond the outer membrane, adding insight to how these extensive β-strands are assembled into the E. coli outer membrane. The substrate docking chamber of this secretin is shown to be sufficient to accommodate the substrate mucinase SteC.IMPORTANCE In order to cause disease, bacterial pathogens inhibit immune responses and induce pathology that will favor their replication and dissemination. In Gram-negative bacteria, these key attributes of pathogenesis depend on structures assembled into or onto the outer membrane. One of these is the T2SS. The Vibrio-type T2SS mediates cholera toxin secretion in Vibrio cholerae, and in Escherichia coli O127:H6 strain E2348/69, the same machinery mediates secretion of the mucinases that enable the pathogen to penetrate intestinal mucus and thereby establish deadly infections.

Characterization of the gluconate utilization system ofVibrio choleraewith special reference to virulence modulation

Orthologs search identified that the Vibrio cholerae gluconate (Gnt) utilization system minimally consisted of the Entner-Doudoroff (ED) pathway (edd and eda) and three other genes, namely gntU, gntK and gntR This system appeared unique by genomic organization of component genes into two operons transcribed in opposite directions. In silico analysis indicated GntU as an inner-membrane protein functioning for transport and GntK as a kinase with cytosolic localization that generates Gnt6P, which is then metabolized through the ED pathway. Enzyme 6-phosphogluconate dehydratase encoded by edd converts Gnt6P to 2-keto-3-deoxy-6-phosphogluconate (KDPG), which is metabolized by the action of KDPG-aldolase encoded by eda Transcriptional upregulation of the Gnt utilization genes in the gntR mutant matched well to a predicted repressor role of GntR. GntR displayed DNA binding to a region in the promoters of two bi-directionally transcribed operons. Growth defect of mutants in Gnt-supplemented media confirmed obligate involvement of these genes in Gnt utilization and such defect was restored upon complementation. Defective Gnt utilization resulted in attenuation of colonization potential and reduction of cholera toxin secretion in V. cholerae The ED pathway mutants showed the highest level of virulence attenuation. Overall, this study established a minimal requirement of the V. cholerae Gnt utilization system, which played a critical role in pathogenesis.

Targeting aphA : a new high-throughput screening assay identifies compounds that reduce prime virulence factors of Vibrio cholerae.

A high-throughput screening (HTS) assay was developed for identifying compounds with inhibitory effect on aphA, one of the key regulators positively controlling Vibrio cholerae pathogenesis. An inhibitory effect on aphA was expected to lead to attenuation in the secretion of the major pathogenicity factors of V. cholerae, cholera toxin and toxin co-regulated pilus. The plasmid construct pAKSB was developed with a kanamycin resistance (KmR) gene under the control of the aphA -like promoter for conferring a KmR phenotype under aphA -expressing conditions. The HTS assay was performed to identify compounds with inhibitory effect on the growth of O139 V. cholerae MO10 carrying the construct pAKSB in growth medium containing Km (30 g ml-1), but not in its absence. Of 20 338 compounds screened, six compounds were identified to inhibit the pAKSB-induced KmR phenotype and these compounds caused transcriptional inhibition of aphA in V. cholerae O139 strain MO10 as well as variant V. cholerae O1 El Tor strain NM06-058. Of the three most active substances, compound 53760866 showed lowest half-maximal cytotoxicity in a eukaryotic cell viability assay and was characterized further. Compound 53760866 caused reduction in cholera toxin secretion and expression of TcpA in vitro. The in vitro virulence attenuation corroborated well in a suckling mouse model in vivo, which showed reduction of colonization by V. cholerae NM06-058 when co-administered with 53760866. The screening method and the compounds may lead to new preventive strategies for cholera by reducing the pathogenicity of V. cholerae .

ATP-association to intrabacterial nanotransportation system in Vibrio cholerae.

Vibrio cholerae colonizes the lumen of the proximal small intestine, which has an alkaline environment, and secretes cholera toxin (CT) through a type II secretion machinery. V. cholerae possesses the intrabacterial nanotransportation system (ibNoTS) for transporting CT from the inner portion toward the peripheral portion of the cytoplasm, and this system is controlled by extrabacterial pH. Association of ATP with ibNoTS has not yet been examined in detail. In this study, we demonstrated by immunoelectron microscopy that ibNoTS of V. cholerae under the extrabacterial alkaline condition was inhibited by ATP inhibitors, 2,4-dinitrophenol (DNP), a protonophore, or 8-amino-adenosine which produces inactive form of ATP. The inhibition of CT transport can be reversed by neutralization of DNP. Those inhibitions were associated with decrease of CT secretion by which ibNoTS followed. We propose that ATP closely associates with V. cholerae ibNoTS for transporting CT.

Inhibition of cAMP-Activated Intestinal Chloride Secretion by Diclofenac: Cellular Mechanism and Potential Application in Cholera

Cyclic AMP-activated intestinal Cl− secretion plays an important role in pathogenesis of cholera. This study aimed to investigate the effect of diclofenac on cAMP-activated Cl− secretion, its underlying mechanisms, and possible application in the treatment of cholera. Diclofenac inhibited cAMP-activated Cl− secretion in human intestinal epithelial (T84) cells with IC50 of ∼20 µM. The effect required no cytochrome P450 enzyme-mediated metabolic activation. Interestingly, exposures of T84 cell monolayers to diclofenac, either in apical or basolateral solutions, produced similar degree of inhibitions. Analyses of the apical Cl− current showed that diclofenac reversibly inhibited CFTR Cl− channel activity (IC50∼10 µM) via mechanisms not involving either changes in intracellular cAMP levels or CFTR channel inactivation by AMP-activated protein kinase and protein phosphatase. Of interest, diclofenac had no effect on Na+-K+ ATPases and Na+-K+-Cl− cotransporters, but inhibited cAMP-activated basolateral K+ channels with IC50 of ∼3 µM. In addition, diclofenac suppressed Ca2+-activated Cl− channels, inwardly rectifying Cl− channels, and Ca2+-activated basolateral K+ channels. Furthermore, diclofenac (up to 200 µM; 24 h of treatment) had no effect on cell viability and barrier function in T84 cells. Importantly, cholera toxin (CT)-induced Cl− secretion across T84 cell monolayers was effectively suppressed by diclofenac. Intraperitoneal administration of diclofenac (30 mg/kg) reduced both CT and Vibrio cholerae-induced intestinal fluid secretion by ∼70% without affecting intestinal fluid absorption in mice. Collectively, our results indicate that diclofenac inhibits both cAMP-activated and Ca2+-activated Cl− secretion by inhibiting both apical Cl− channels and basolateral K+ channels in intestinal epithelial cells. Diclofenac may be useful in the treatment of cholera and other types of secretory diarrheas resulting from intestinal hypersecretion of Cl−.

Multiple approaches towards understanding the type II secretion system

Transport of folded proteins across membranes is a feat accomplished by few biomacromolecular machines. One of the machineries able to do so is the sophisticated type II secretion system (T2SS). It can translocate key virulence factors from the bacterial periplasm into the lumen of the gut of the human host. A prime example is the secretion of cholera toxin by Vibrio cholerae. The T2SS consists of ~12 different proteins, most of these present in multiple copies, organized into three subassemblies: (i) the Inner Membrane Platform; (ii) the Pseudopilus in the periplasm, which acts most likely as a piston pushing exoproteins through the outer membrane pore; (iii) the Outer Membrane Complex, allowing passage of ~100 kDa folded proteins. We have determined crystal structures from more than a dozen T2SS domains, yet, a full understanding of the architecture and mechanism of action of the T2SS remains a formidable challenge. Our approaches include the use of "assistant-multimers" to promote recalcitrant multimer formation and of nanobodies to overcome reluctant crystal formation. The Inner Membrane Platform is interacting with the secretion ATPase GspE which most likely needs to be hexameric for full activity. Full-length GspE co-crystallized with its major partner, the cytoplasmic domain of GspL, revealed a tremendous flexibility of this ATPase, and, most unexpectedly, also the organization of the same linear arrangement of cyto-GspL domains throughout three entirely different crystal forms. Two very different hexamers of GspE were elucidated by linking the GspE subunit to the subunit of Hcp1, which successfully acted as an "assistant hexamer", inducing hexamer formation by GspE. The dodecameric nature of the ~ 850 kDa GspD, the major component of the Outer Membrane Complex, evident in earlier electron microscopy studies, was observed in the dodecameric ring-like helix in crystals of its N-terminal domain. The contacts between GspD and the inner-membrane protein GspC will be discussed as well as the remarkably frequent occurrence of dimers of Inner Membrane Platform domains. How dimers are co-assembled with an ATPase hexamer with C6 symmetry and the Outer Membrane Complex dodecamer with C12 symmetry remains one of the many fascinating outstanding questions of the T2SS.

Impact of solar irradiation on cholera toxin secretion by different strains of Vibrio cholerae

Cholera toxin is the aetiological agent of cholera – a deadly waterborne disease acquired through the consumption of untreated water contaminated with CTXФ bacteriophage harbouring strains of V. cholerae. Solar disinfection is a re-emerging technique that relies on the ultraviolet component of sunlight to inactivate the growth of Vibrio cholerae in water, rendering the water microbiologically safe for consumption. However, studies have shown that DNA damaging agents, such as ultraviolet light, induce the replication of the CTXФ bacteriophage with subsequent expression of the cholera toxin. In this study we investigated the impact of solar irradiation on the secretion of cholera toxin by toxigenic strains of V. cholerae in water. The cholera toxin ELISA assay, qualitative and quantitative real-time PCR as well as growth on solid media were used to determine cholera toxin secretion, DNA integrity and growth of the bacteria after 7 h and 31 h of solar irradiation. Solar irradiation in water reduced the integrity of DNA, inactivated the growth of V. cholerae and, most importantly, prevented the secretion of detectable levels of cholera toxin. This finding is encouraging for resource-poor communities that may rely on solar disinfection to alleviate the burden of cholera-related fatalities.

ToxR Recognizes a Direct Repeat Element in the toxT , ompU , ompT , and ctxA Promoters of Vibrio cholerae To Regulate Transcription

ToxR facilitates TcpP-mediated activation of the toxT promoter in Vibrio cholerae, initiating a regulatory cascade that culminates in cholera toxin secretion and toxin coregulated pilus expression. ToxR binds a region from -104 to -68 of the toxT promoter, from which ToxR recruits TcpP to the TcpP-binding site from -53 to -38. To precisely define the ToxR-binding site within the toxT promoter, promoter derivatives with single-base-pair transversions spanning the ToxR-footprinted region were tested for transcription activation and DNA binding. Nine transversions between -96 to -83 reduced toxT promoter activity 3-fold or greater, and all nine reduced the relative affinity of the toxT promoter for ToxR at least 2-fold, indicating that activation defects were due largely to reduced binding of ToxR to the toxT promoter. Nucleotides important for ToxR-dependent toxT activation revealed a consensus sequence of TNAAA-N(5)-TNAAA extending from -96 to -83, also present in other ToxR-regulated promoters. When these consensus nucleotides were mutated in the ompU, ompT, or ctxA promoters, ToxR-mediated regulation was disrupted. Thus, we have defined the core ToxR-binding site present in numerous ToxR-dependent promoters and we have precisely mapped the binding site for ToxR to a position three helical turns upstream of TcpP in the toxT promoter.

Expression and Secretion of Cholera Toxin B Subunit in Lactobacilli

Lactic acid bacteria (LAB) are used in various fields, including in food and medical supplies. There has been a great deal of research into vaccine development using LAB as carriers due to their "generally recognized as safe" status. Cholera is an infectious disease that causes diarrhea due to cholera toxin (CT) produced by Vibrio cholerae. The pentameric cholera toxin B (CTB) subunit has no toxicity, and is used as an antigen in cholera vaccines and as a delivery molecule in vaccines to various diseases. In this study, we generated recombinant LAB expressing and secreting CTB. Here, we first report that CTB expressed and secreted from LAB bound to GM1 ganglioside. The secreted CTB was purified, and its immunogenicity was determined by intranasal administration into mice. The results of the present study suggested that it may be useful as the basis of a new oral cholera vaccine combining LAB and CTB.

Nanotransportation system for cholera toxin in Vibrio cholerae 01

Vibrio cholerae (V. cholerae) cholera toxin (CT), which causes a severe watery diarrheal illness, is secreted via the type II secretion machinery; it remains unclear, however, how this toxin is transported toward the machinery. In this study, we determined that the pH-dependent intrabacterial transport system correlates with the priming of CT secretion by V. cholerae. The secretion and production of V. cholerae treated at different pHs were examined by enzyme immunoassay. The localization of the CT was analyzed by immunoelectron microscopy. The CT secretion level rapidly increases in the alkaline-pH-treated V. cholerae but does so more slowly in neutral- and acidic-pH-treated V. cholerae. The CT was found to be densely localized near the membrane in the alkaline-pH-treated bacterial cytoplasm, suggesting that the CT shifts from the center to the peripheral portion of the cytoplasm following an extracellular rise in pH. The shift was observed in V. cholerae treated at alkaline pH for more than 10 min. The pH treatment did not enhance CT production at the same stage at which secretion and intrabacterial transport of the CT were enhanced. We propose that V. cholerae possesses a pH-dependent intrabacterial nanotransportation system that probably accelerates priming for CT secretion.

Specificity in Transmembrane Helix-Helix Interactions Mediated by Aromatic Residues

Aromatic residues have been previously shown to mediate the self-assembly of different soluble proteins through pi-pi interactions (McGaughey, G. B., Gagne, M., and Rappe, A. K. (1998) J. Biol. Chem. 273, 15458-15463). However, their role in transmembrane (TM) assembly is not yet clear. In this study, we performed statistical analysis of the frequency of occurrence of aromatic pairs in a bacterial TM data base that provided an initial indication that the appearance of a specific aromatic pattern, Aromatic-XX-Aromatic, is not coincidental, similar to the well characterized QXXS motif. The QXXS motif was previously shown to be both critical and sufficient for stabilizing TM self-assembly. Using the ToxR system, we monitored the dimerization propensities of TM domains that contain mutations of interacting residues to aromatic amino acids and demonstrated that aromatic residues can adequately stabilize self-association. Importantly, we have provided an example of a natural TM domain, the cholera toxin secretion protein EpsM, whose TM self-assembly is mediated by an aromatic motif (WXXW). This is, in fact, the first evidence that aromatic residues are involved in the dimerization of a wild type TM domain. The association mediated by aromatic residues was found to be sensitive to the TM sequence, suggesting that aromatic residue motifs can provide a general means for specificity in TM assembly. Molecular dynamics provided a structural explanation for this backbone sequence sensitivity.

Differential expression of intestinal membrane transporters in cholera patients

Vibrio cholerae causes the cholera disease through secretion of cholera toxin (CT), resulting in severe diarrhoea by modulation of membrane transporters in the intestinal epithelium. Genes encoding membrane-spanning transporters identified as being differentially expressed during cholera disease in a microarray screening were studied by real-time PCR, immunohistochemistry and in a CaCo-2 cell model. Two amino acid transporters, SLC7A11 and SLC6A14, were upregulated in acute cholera patients compared to convalescence. Five other transporters were downregulated; aquaporin 10, SLC6A4, TRPM6, SLC23A1 and SLC30A4, which have specificity for water, serotonin (5-HT), magnesium, vitamin C and zinc, respectively. The majority of these changes appear to be attempts of the host to counteract the secretory response. Our results also support the concept that epithelial cells are involved in 5-HT signalling during acute cholera.

Synergistic stimulation of EpsE ATP hydrolysis by EpsL and acidic phospholipids

EpsE is a cytoplasmic component of the type II secretion system in Vibrio cholerae. Through ATP hydrolysis and an interaction with the cytoplasmic membrane protein EpsL, EpsE supports secretion of cholera toxin across the outer membrane. In this study, we have determined the effect of the cytoplasmic domain of EpsL (cyto-EpsL) and purified phospholipids on the ATPase activity of EpsE. Acidic phospholipids, specifically cardiolipin, bound the copurified EpsE/cyto-EpsL complex and stimulated its ATPase activity 30-130-fold, whereas the activity of EpsE alone was unaffected. Removal of the last 11 residues (residues 243-253) from cyto-EpsL prevented cardiolipin binding as well as stimulation of the ATPase activity of EpsE. Further mutagenesis of the C-terminal region of the EpsL cytoplasmic domain adjacent to the predicted transmembrane helix suggested that this region participates in fine tuning the interaction of EpsE with the cytoplasmic membrane and influences the oligomerization state of EpsE thereby stimulating its ATPase activity and promoting extracellular secretion in V. cholerae.

Role of mast cells and pro-inflammatory mediators on the intestinal secretion induced by cholera toxin

Recent data suggest that diarrhea caused by Vibrio cholerae involves a pro-inflammatory mediators release, such as cytokines, prostaglandin and nitric oxide. The aim of this study was to investigate the role of mast cells and their mediators in the intestinal secretion induced by cholera toxin. We examined the dose responses, time course and role of mast cells and pro-inflammatory mediators in cholera toxin intestinal secretory response, in vivo. Cholera toxin caused a dose-dependent secretion, in ligated small intestine loops, at 18 h. Rats treated with 48/80 compound or ketotifen had a significant decrease in the intestinal secretory response. Cholera toxin secretion was significantly reduced by an unspecific histamine/serotonin receptor antagonist, histamine receptor antagonist, phospholipase A 2 and cyclooxygenase inhibitors, platelet-activating factor (PAF) receptor antagonists and TNF-α synthesis blockers. On the other hand, pretreatment with a specific serotonin receptor antagonist and lipoxygenase inhibitors failed to block this effect. Analysis of the intestinal fluid from rats injected with cholera toxin, revealed that cholera toxin induces the release of IL-1β and TNF-α into fluid. The data suggest that, at least in part, mast cells are involved in cholera toxin-induced secretion, as well as point to the importance of histamine, prostaglandins, PAF, IL-1β and TNF-α in this process.

Identification of a protein secretory pathway for the secretion of heat-labile enterotoxin by an enterotoxigenic strain of Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) is an enteric pathogen that causes cholera-like diarrhea in humans and animals. ETEC secretes a heat-labile enterotoxin (LT), which resembles cholera toxin, but the actual mechanism of LT secretion is presently unknown. We have identified a previously unrecognized type II protein secretion pathway in the prototypic human ETEC strain, H10407 (serotype O78:H11). The genes for this pathway are absent from E. coli K-12, although examination of the K-12 genome suggests that it probably once possessed them. The secretory pathway bears significant homology at the amino acid level to the type II protein secretory pathway required by Vibrio cholerae for the secretion of cholera toxin. With this in mind, we determined whether the homologous pathway of E. coli H10407 played a role in the secretion of LT. To this end, we inactivated the pathway by inserting a kanamycin-resistance gene into one of the genes (gspD) of the type II secretion pathway by homologous recombination. LT secretion by E. coli H10407 and the gspD mutant was assayed by enzyme immunoassay, and its biological activity was assessed by using Y-1 adrenal cells. This investigation showed that the protein secretory pathway is functional and necessary for the secretion of LT by ETEC. Our findings have revealed the mechanism for the secretion of LT by ETEC, which previously was unknown, and provide further evidence of close biological similarities of the LT and cholera toxin.

Directed polar secretion of protease from single cells of Vibrio cholerae via the type II secretion pathway.

Bacteria have long been thought of as little more than sacks of homogeneously distributed enzymes. However, recent cytological studies indicate that bacteria are compartmentalized with proteins involved in processes such as cell division, motility, chemotaxis, and development located at distinct sites. We have used the green fluorescent protein as a reporter to determine the cellular distribution of the extracellular protein secretion (eps)-encoded type II secretion complex responsible for extracellular secretion of cholera toxin and hemagglutinin/protease in Vibrio cholerae. Real-time monitoring of green fluorescent protein fused to EpsM in living cells indicated that, like the single polar flagellum, the Eps complex is located at the old pole after cell division. Eps-dependent protease secretion was also visualized in single cells by fluorescence microscopy by using intramolecularly quenched casein. This analysis demonstrated that active protease secretion is focused at the poles and colocalizes with the site of the polar Eps apparatus. These results suggest that the type II secretion complex is responsible for directed delivery of virulence factors during cholera pathogenesis.

Characterization of the multimeric Eps complex required for cholera toxin secretion

Vibrio cholerae causes diarrheal disease through colonization of the small intestine. A critical aspect of V. cholerae pathogenesis is its ability to actively secrete cholera toxin to the extracellular environment. This occurs via the type II secretion pathway, where the toxin subunits are first transported to the periplasm through the Sec pathway. Following folding and assembly the toxin is then translocated across the outer membrane by a specialized Extracellular Protein Secretion (Eps) machinery encoded by at least 13 genes. Although the Eps proteins are believed to form a secretion apparatus that spans both membranes, cholera toxin is thought to engage this complex first in the periplasm. In order to determine the organization of the Eps apparatus and to understand the mechanism of secretion, the Eps apparatus has been dissected and three of the components, EpsE, EpsL and EpsM, have been purified and characterized. They were shown to form a stable, multiprotein complex spanning the cytoplasmic membrane.