Type III Secretion Research Articles

Optimization of type 3 protein secretion in enteropathogenic Escherichia coli.

The type 3 secretion system (T3SS) is a protein export pathway common to Gram-negative pathogens. It comprises a trans-envelope syringe, the injectisome, with a cytoplasm-facing translocase channel. In enteropathogenic Escherichia coli, exported substrates are chaperone-delivered to the major translocase component, EscV, and cross the membrane in strict hierarchical manner, e.g. first 'translocators', then 'effectors'. The in vitro dissection of the T3SS and the determination of its structure are hampered by the low numbers of the injectisomes per cell. We have now defined an optimal M9 minimal medium and established that the per transcriptional regulator enhances the number of filamented cells, the number of injectisomes per cell and the secretion of T3S substrates. Our findings provide a valuable tool for further biochemical and biophysical analysis of the T3SS and suggest that additional improvement to maximize injectisome production is possible in future efforts.

Low-iron conditions induces the hypersensitive reaction and pathogenicity hrp genes expression in Xanthomonas and is involved in modulation of hypersensitive response and virulence.

Expression of hrp (hypersensitive reaction and pathogenicity) genes inside the host is crucial for virulence of phytopathogenic bacteria. The hrp genes encode components of type3 secretion system (T3SS), HR elicitors and several regulators, which are involved in the co-ordinated expression of hrp genes in the host environment and in hrp inducing chemically defined medium. However, little is known about specific host or environmental factors which may play a role in the induction of hrp gene expression. In this study, we show that iron-limiting condition elicits induced expression of hrp genes, including type3 secretion system (T3SS) and effectors (T3E). Expression analysis using qRT-PCR and promoter probe strains suggest significant induction in the expression of Hrp and T3S-associated genes of Xanthomonas campestris pv. campestris (Xcc) under low-iron condition, and is suppressed by exogenous supplementation of iron. Furthermore, we show that with exogenous iron supplementation, wild type Xcc exhibited reduced disease symptoms in host-plant, and exhibited significant reduction in HR and callose deposition in the non-host plants. Xanthomonas oryzae and oryzicola pathovars also exhibited the iron affect, albeit to a lesser extend compared with the Xcc. Overall, our results suggest that low-iron condition inside the host may play a crucial role in pathogenicity.

Chitin-induced T6SS in Vibrio cholerae is dependent on ChiS activation.

Vibrio cholerae regularly colonizes the chitinous exoskeleton of crustacean shells in the aquatic region. The type 6 secretion system (T6SS) in V. cholerae is an interbacterial killing device. This system is thought to provide a competitive advantage to V. cholerae in a polymicrobial community of the aquatic region under nutrient-poor conditions. V. cholerae chitin sensing is known to be initiated by the activation of a two-component sensor histidine kinase ChiS in the presence of GlcNAc2 (N,N'-diacetylchitobiose) residues generated by the action of chitinases on chitin. It is known that T6SS in V. cholerae is generally induced by chitin. However, the effect of ChiS activation on T6SS is unknown. Here, we found that ChiS inactivation resulted in impaired bacterial killing and reduced expression of T6SS genes. Active ChiS positively affected T6SS-mediated natural transformation in V. cholerae. ChiS depletion or inactivation also resulted in reduced colonization on insoluble chitin surfaces. Therefore, we have shown that V. cholerae colonization on chitinous surfaces activates ChiS, which promotes T6SS-dependent bacterial killing and horizontal gene transfer. We also highlight the importance of chitinases in T6SS upregulation.

Characterization of s1289 in Biofilm Formation and Infection in S. flexneri

Shigella flexneri is an intracellular pathogen that invades colonic epithelial cells and causes bacterial dysentery, which accounts for approximately 165 million cases worldwide and 1 million associated deaths, annually. Shigella infections are predominant in third world countries with poor access to hygiene resources. It can be spread via person to person contact or by contaminated food or water. What is known about Shigella pathogenesis is that it can withstand the acidic environments of the digestive tract, evade immune responses, invade intestinal epithelium using a Type 3 Secretion System (T3SS) and acquire actin‐based motility by manipulating the host cell actin cytoskeleton. Another characteristic of the pathogen is its ability to disseminate between epithelial cells via the formation of plasma membrane protrusions that resolve into vacuole like protrusions (VLP's) and finally collapse and form a vacuole in the adjacent intestinal cell. Although much is known about how the pathogen invades and spreads into neighboring intestinal cells using the T3SS, there is not enough information on how Shigella transit through the intestinal tract and what events occur prior to infection of colonic cells. We decided to focus on the possibility of other extracellular components in the bacterium that could contribute to virulence. The gene s1289 in Shigella chromosomal DNA encodes for an outer membrane autotransporter protein that can possibly be linked with adhesion to host cells and may contribute to invasion. Recent research has shown that the presence of bile salts induces biofilm formation of S. flexneri. Formation of biofilm is an important determinant for increased adherence and virulence in E. coli strains, but has not been thoroughly investigated in S. flexneri. We decided to test the role of gene s1289 in the formation of S. flexneri biofilms and in adherence to intestinal cells. Our hypothesis was that if we were able to delete the gene s1289 from the bacterial genome, the new mutant strain would lose its capacity to form biofilms and to adhere to colonic epithelial cells. We created a construct displaying a disrupted version of s1289 in E. coli and introduced it in S. flexneri by conjugation. After allelic exchange by homologous recombination, we identified mutant strains displaying the disrupted version of s1289 in their genome. We determined biofilm thickness using confocal microscopy, tested for susceptibility of biofilm using antimicrobial agents, and investigated the ability of the mutant to infect HT‐29 intestinal cells. Although the Δs1289 mutant did form biofilm in presence of 0.4% bile salts, results showed that the structure of the biofilm exhibited reduced thickness compared to wild type. Preliminary infection results indicate a possible adhesion defect in mutant bacteria, therefore reducing invasion in colon cells. However, wild type and mutant biofilms demonstrated no difference in susceptibility to antimicrobials. We conclude that the S1289 protein potentially contributes to biofilm formation and adherence of S. flexneri to HT‐29 cells.Support or Funding InformationUPR‐PRISE Program. Funded by NIH‐NIGMS #2R25GM096955This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Tandem tyrosine phosphosites in the Enteropathogenic Escherichia coli chaperone CesT are required for differential type III effector translocation and virulence.

Enteropathogenic Escherichia coli (EPEC) use a type 3 secretion system (T3SS) for injection of effectors into host cells and intestinal colonization. Here, we demonstrate that the multicargo chaperone CesT has two strictly conserved tyrosine phosphosites, Y152 and Y153 that regulate differential effector secretion in EPEC. Conservative substitution of both tyrosine residues to phenylalanine strongly attenuated EPEC type 3 effector injection into host cells, and limited Tir effector mediated intimate adherence during infection. EPEC expressing a CesT Y152F variant were deficient for NleA effector expression and exhibited significantly reduced translocation of NleAinto host cells during infection. Other effectors were observed to be dependent on CesT Y152 for maximal translocation efficiency. Unexpectedly, EPEC expressing a CesT Y153F variant exhibited significantly enhanced effector translocation of many CesT-interacting effectors, further implicating phosphosites Y152 and Y153 in CesT functionality. A mouse infection model of intestinal disease using Citrobacter rodentium revealed that CesT tyrosine substitution variants displayed delayed colonization and were more rapidly cleared from the intestine. These data demonstrate genetically separable functions for tandem tyrosine phosphosites within CesT. Therefore, CesT via its C-terminal tyrosine phosphosites, has relevant roles beyond typical type III secretion chaperones that interact and stabilize effector proteins.

Xanthomonas citri T6SS mediates resistance to Dictyostelium predation and is regulated by an ECF σ factor and cognate Ser/Thr kinase.

Plant-associated bacteria of the genus Xanthomonas cause disease in a wide range of economically important crops. However, their ability to persist in the environment is still poorly understood. Predation by amoebas represents a major selective pressure to bacterial populations in the environment. In this study, we show that the X. citri type 6 secretion system (T6SS) promotes resistance to predation by the soil amoeba Dictyostelium discoideum. We found that an extracytoplasmic function (ECF) sigma factor (EcfK) is required for induction of T6SS genes during interaction with Dictyostelium. EcfK homologues are found in several environmental bacteria in association with a gene encoding a eukaryotic-like Ser/Thr kinase (pknS). Deletion of pknS causes sensitivity to amoeba predation and abolishes induction of T6SS genes. Phosphomimetic mutagenesis of EcfK identified a threonine residue (T51) that renders EcfK constitutively active in standard culture conditions. Moreover, susceptibility of ΔpknS to Dictyostelium predation can be overcome by expression of the constitutively active version EcfKT51E from a multicopy plasmid. Together, these results describe a new regulatory cascade in which PknS functions through activation of EcfK to promote T6SS expression. Our work reveals an important aspect of Xanthomonas physiology that affects its ability to persist in the environment.

Multitalented EspB of enteropathogenic Escherichia coli (EPEC) enters cells autonomously and induces programmed cell death in human monocytic THP-1 cells

Enteropathogenic Escherichia coli (EPEC) subvert host cell signaling pathways by injecting effector proteins via a Type 3 Secretion System (T3SS). The T3SS-dependent EspB protein is a multi-functional effector protein, which contributes to adherence and translocator pore formation and after injection exhibits several intracellular activities. In addition, EspB is also secreted into the environment. Effects of secreted EspB have not been reported thus far. As a surrogate for secreted EspB we employed recombinant EspB (rEspB) derived from the prototype EPEC strain E2348/69 and investigated the interactions of the purified protein with different human epithelial and immune cells including monocytic THP-1 cells, macrophages, dendritic cells, U-937, epithelial T84, Caco-2, and HeLa cells. To assess whether these proteins might exert a cytotoxic effect we monitored the release of lactate dehydrogenase (LDH) as well as propidium iodide (PI) uptake. For comparison, we also investigated several homologs of EspB such as IpaD of Shigella, and SipC, SipD, SseB, and SseD of Salmonella as purified recombinant proteins. Interestingly, cytotoxicity was only observed in THP-1 cells and macrophages, whereas epithelial cells remained unaffected. Cell fractionation and immune fluorescence experiments showed that rEspB enters cells autonomously, which suggests that EspB might qualify as a novel cell-penetrating effector protein (CPE). Using specific organelle tracers and inhibitors of signaling pathways we found that rEspB destroys the mitochondrial membrane potential – an indication of programmed cell death induction in THP-1 cells. Here we show that EspB not only constitutes an essential part of the T3SS-nanomachine and contributes to the arsenal of injected effector proteins but, furthermore, that secreted (recombinant) EspB autonomously enters host cells and selectively induces cell death in immune cells.

Minimal SPI1-T3SS effector requirement for Salmonella enterocyte invasion and intracellular proliferation in vivo.

Effector molecules translocated by the Salmonella pathogenicity island (SPI)1-encoded type 3 secretion system (T3SS) critically contribute to the pathogenesis of human Salmonella infection. They facilitate internalization by non-phagocytic enterocytes rendering the intestinal epithelium an entry site for infection. Their function in vivo has remained ill-defined due to the lack of a suitable animal model that allows visualization of intraepithelial Salmonella. Here, we took advantage of our novel neonatal mouse model and analyzed various bacterial mutants and reporter strains as well as gene deficient mice. Our results demonstrate the critical but redundant role of SopE2 and SipA for enterocyte invasion, prerequisite for transcriptional stimulation and mucosal translocation in vivo. In contrast, the generation of a replicative intraepithelial endosomal compartment required the cooperative action of SipA and SopE2 or SipA and SopB but was independent of SopA or host MyD88 signaling. Intraepithelial growth had no critical influence on systemic spread. Our results define the role of SPI1-T3SS effector molecules during enterocyte invasion and intraepithelial proliferation in vivo providing novel insight in the early course of Salmonella infection.

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.

Tracking Vibrio cholerae Cell-Cell Interactions during Infection Reveals Bacterial Population Dynamics within Intestinal Microenvironments

Vibrio cholerae is the causative agent of the diarrheal disease cholera. Although many V.cholerae virulence factors have been studied, the role of interbacterial interactions within the host gut and their influence on colonization are poorly understood. Here, we utilized the conjugative properties of a Vibrio-specific plasmid to serve as a quantifiable genetic marker for direct contact among V.cholerae cells in the infant rabbit model for cholera. In conjunction, we also quantified contact-dependent type 6 secretion system (T6SS)-mediated killing of co-infecting V.cholerae strains. Tracking these interbacterial interactions revealed that most contact-dependent cell-cell interactions among V.cholerae occur in specific intestinal microenvironments, notably the distal small intestine and cecum, and that the T6SS confers a competitive advantage within the middle small intestine. These results support amodel for V.cholerae gut colonization, which includes microenvironments where critical microbial-host and bacterial-bacterial interactions occur to facilitate colonization by this pathogen.

Burkholderia cenocepacia Induces Macropinocytosis to Enter Macrophages.

Burkholderia cenocepacia is an opportunistic pathogen that infects individuals with cystic fibrosis, chronic granulomatous disease, and other immunocompromised states. B. cenocepacia survives in macrophages in membrane-bound vacuoles; however, the mechanism by which B. cenocepacia gains entry into macrophages remains unknown. After macrophage internalization, survival of B. cenocepacia within a bacteria-containing membrane vacuole (BcCV) is associated with its ability to arrest the maturation of the BcCV. In this study, we show that B. cenocepacia induces localized membrane ruffling, macropinocytosis, and macropinosomes-like compartments upon contact with the macrophage. The Type 3 Secretion System (T3SS) of B. cenocepacia contributes to macrophage entry and macropinosome-like compartment formation. These data demonstrate the ability of Burkholderia to enter macrophages through the induction of macropinocytosis.

Salmonella Translocated Effectors Recruit OSBP1 to the Phagosome to Promote Vacuolar Membrane Integrity

Intracellular Salmonella use a type III secretion system (TTSS) to translocate effector proteins across the phagosome membrane to promote vacuole membrane tubulation, resulting in intracellular survival. This work demonstrates that the effector SseJ binds the eukaryotic lipid transporter oxysterol binding protein 1 (OSBP1). SseJ directs OSBP1 to the endosomal compartment in a manner dependent on the TTSS located on Salmonella pathogenicity island 2 (SPI2) and is mediated by both SseJ and another OSBP1-binding SPI2 translocated effector, the deubiquitinase SseL. Only deletion of both SseJ and SseL reduced vacuolar integrity with increased bacteria released into the eukaryotic cytoplasm, indicating that their combined activities are necessary for vacuole membrane stability. Cells deleted for the OSBP1-binding proteins VAPA/B also demonstrate loss of vacuole integrity, consistent with the hypothesis that OSBP1 recruitment is required to provide membrane for a vacuole undergoing endosomal tubulation, cholesterol esterification, and other SPI2-mediated alterations that promote Salmonellae intracellular survival.

XopR TTSS-effector regulates in planta growth, virulence of Indian strain of Xanthomonas oryzae pv. oryzae via suppressing reactive oxygen species production and cell wall-associated rice immune responses during blight induction.

Xanthomonas oryzae pv. oryzae (Xoo) causing bacterial blight of rice is a global problem in rice production. Phytopathogenic Xanthomonads overpower PAMP-triggered immunity (PTI) through secreting effectors via type III secretion system (TTSS). We previously screened the TTSS effector repository of an Indian strain of Xoo (race 4), a predominant strain from north-west India that contains 21 Xop and 18 TALE effectors. Here, we demonstrate that Xoo race 4 employs XopR for in planta colonisation, virulence and for the suppression of cell wall-associated immune responses in its natural host. XopR null mutant (Xoo ΔxopR) produced 2.6-fold less-severe lesion as compared with Xoo wild type. Xoo ΔxopR showed 1.58-fold reduced colonisation compared with wild indicating that XopR is required for maximum colonisation in rice. Xoo ΔxopR produced 3.8-fold more callose deposits compared with wild. Xoo ΔxopR caused significantly higher production of ROS in rice. RT-qPCR expression analysis of immune responsive genes of rice indicated 10- to 43-fold upregulation upon challenged inoculation with Xoo ΔxopR over wild. Altogether, our study revealed that XopR of Indian Xoo strain supports its in planta growth and contributes immensely for successful blight development through suppressing defence related events like reactive oxygen species production, callose deposition and transcript abundance of immune responsive genes during rice::Xoo interaction.

Molecular detection of Escherchia coli carrying uropathogenic-specific-protein and typeⅢ secretion system from patients with urinary tract infection

Molecular detection of Escherchia coli carrying uropathogenic-specific-protein and typeⅢ secretion system from patients with urinary tract infection

Integration of QsvR into the Quorum Sensing Circuit of <i>Vibrio parahaemolyticus</i>

Vibrio parahaemolyticus, the leading cause of seafood-associated gastroenteritis worldwide, requires the two type-III secretion systems (T3SS1 and T3SS2) and a thermostable direct hemolysin (encoded by tdh1 and tdh2) to infect the small intestine. The tdh genes and the T3SS2 gene cluster constitute an 80-kb pathogenicity island known as Vp-PAI located on the chromosome II. Expression of T3SS1 and Vp-PAI is regulated in a quorum sensing (QS)-dependent manner but lacks the detailed regulatory mechanisms. Herein, we show that three factors (QS regulators AphA and OpaR and novel regulator QsvR) form a complex regulatory network to regulate the T3SS1 and Vp-PAI genes. At a low cell density, the expression of the T3SS1 genes is induced by the direct binding of AphA to the exsBAD-vscBCD operon, while Vp-PAI expression is repressed. At a high cell density (HCD), the bacterium turns off T3SS1 expression by replacing AphA with OpaR, triggering the induction of Vp-PAI. Furthermore, QsvR binds to the regulatory regions of all the tested Vp-PAI genes to activate their transcription at HCD. Taken together, our data highlight how multiple QS regulators contribute to the pathogenicity of V. parahaemolyticus by precisely controlling the expression of major virulence determinants during different stages of infection. Funding Statement: This work was supported by the National Natural Science Foundation of China (grant numbers 31671290 and 81601809) Declaration of Interests: The authors declared that they have no conflicts of interest to this work. Ethics Approval Statement: All the animal experiments were approved by the Committee on Animal Research of the Academy of Military Medical Sciences and carried out per the approved guideline.

Pathotyping of diarrhoeagenic cattle Escherichia coli strains isolated in the Province of Tehran, Iran.

An oligonucleotide DNA microarray targeting 348 virulence factors and genetic markers was used in the pathotyping, serotyping and phylogrouping of 51 Escherichia coli strains isolated from faecal samples. The samples were collected from diarrhoeic 1 to 30 days old calves located at 14 farms in the Tehran province, Iran. Positive microarray signals for genes encoding the Locus of Enterocyte E acement (LEE), the Type III Secretion System (TTSS), and the absence of EPEC adherence factor (EAF) permitted the pathotyping of 25 strains as atypical Enteropathogenic (aEPEC) or Enterohaemorrhagic Escherichia coli (EHEC). The lack of LEE and TTSS-associated genes distinguished the remaining 26 strains, which were classi ed as Extraintestinal pathogenic E. coli (ExPEC). Atypical EPEC belonged to phylogroup B1 and possessed a LEE pro le tir-1, eae(beta), espA-1, espB-3. The EHEC strains primarily belonged to the B1 phylogroup type-O26 and possessed either a LEE pro le tir-1, eae(beta), espA-1, espB-3, or a B1 type-O111, LEE tir-3, eae(gamma), espA-1, espB-2. ExPEC-typed strains generally harboured genes localised to the constant region of Colicin V plasmid (pColV), including increased serum survival factor (iss), complement resistance protein (traT), aerobactin operon (iucD), and the siderophore receptor (iroN). The microarray platform used in this study is well suited to accurately and rapidly type attaching and e acing E. coli (AEEC-types), thus providing a database for the meta-analysis of ExPEC-typed strains.

ExoY, an actin-activated nucleotidyl cyclase toxin from P. aeruginosa: A minireview

ExoY is one of four well-characterized Pseudomonas aeruginosa type 3 secretion system (T3SS) effectors. It is a nucleotidyl cyclase toxin that is inactive inside the bacteria, but becomes potently activated once it is delivered into the eukaryotic target cells. Recently, filamentous actin was identified as the eukaryotic cofactor that stimulates specifically ExoY enzymatic activity by several orders of magnitude. In this review, we discuss recent advances in understanding the biochemistry of nucleotidyl cyclase activity of ExoY and its regulation by interaction with filamentous actin.

Epidemiology of Campylobacter Infections among Children in Egypt.

Campylobacter is a frequently isolated bacterial pathogen among children with diarrhea. Data are lacking on the distribution and spectrum of disease associated with Campylobacter species and Campylobacter jejuni capsular polysaccharide (CPS) types. This information is essential because current vaccine research seeks to target specific CPS types. An effective CPS-conjugate vaccine will need to cover CPS types that are both common and associated with severe disease. The US Naval Medical Research Unit-3 conducted several prospective cohort studies researching diarrheal disease in Egypt from 1995 to 2003. In total, 1,057 children were enrolled and followed to a maximum age of 36 months. We analyzed Campylobacter-positive stool samples that were collected while subjects were symptomatic, along with corresponding clinical data. Of 441 Campylobacter isolates, 322 represented primary infections (189 C. jejuni, 127 Campylobacter coli, six unspeciated). There were 19 C. jejuni CPS types identified; eight accounted for 63.5% of primary C. jejuni infections. We also screened for the presence of the type-6 secretion system (T6SS), a putative virulence determinant. The T6SS was found in 18.0% of C. coli isolates and 57.6% of C. jejuni isolates (P < 0.001), and was not uniformly distributed among CPS types (P < 0.001). Strains with the T6SS were not associated with more severe disease. Clinical presentations across species and CPS types appeared similar. This study adds to the growing epidemiological data and also provides some analysis of the clinical spectrum associated with infection by specific Campylobacter species, C. jejuni capsule types, and possible virulence determinants.

The inner-rod component of Shigella flexneri type 3 secretion system, MxiI, is involved in the transmission of the secretion activation signal by its interaction with MxiC.

The virulence of Shigella mainly resides in the use of a Type 3 Secretion System (T3SS) to inject several proteins inside the host cell. Three categories of proteins are hierarchically secreted: (1) the needle components (MxiH and MxiI), (2) the translocator proteins which form a pore (translocon) inside the host cell membrane, and (3) the effectors interfering with the host cell signaling pathways. In the absence of host cell contact, the T3SS is maintained in an “off” state by the presence of a tip complex. We have previously identified a gatekeeper protein, MxiC, which sequesters effectors inside the bacteria probably by interacting with MxiI, the inner‐rod component. Upon cell contact and translocon insertion, a signal is most likely transmitted from the top of the needle to the base, passing through the needle and allowing effectors release. However, the molecular mechanism underlying the transmission of the activation signal through the needle is still poorly understood. In this work, we investigate the role of MxiI in the activation of the T3SS by performing a mutational study. Interestingly we have shown that mutations of a single residue in MxiI (T82) induce an mxiC‐like phenotype and prevent the interaction with MxiC. Moreover, we have shown that the L26A mutation significantly reduces T3 secretion. The L26A mutation impairs the interaction between MxiI and Spa40, a keystone component of the switch between needle assembly and translocators secretion. The L26A mutation also sequesters MxiC. All these results highlight the crucial role of MxiI in regulating the secretion and transmitting the activation signal of the T3SS.

Type-III secretion pore formed by flagellar protein FliP.

During assembly of the bacterial flagellum, protein subunits that form the exterior structures are exported through a specialized secretion apparatus energized by the proton gradient. This category of protein transport, together with the similar process that occurs in the injectisomes of gram-negative pathogens, is termed type-III secretion. The membrane-embedded part of the flagellar export apparatus contains five essential proteins: FlhA, FlhB, FliP, FliQ and FliR. Here, we have undertaken a variety of experiments that together support the proposal that the protein-conducting conduit is formed primarily, and possibly entirely, by FliP. Chemical modification experiments demonstrate that positions near the center of certain FliP trans-membrane (TM) segments are accessible to polar reagents. FliP expression sensitizes cells to a number of chemical agents, and mutations at predicted channel-facing positions modulate this effect. Multiple assays are used to show that FliP suffices to form a channel that can conduct a variety of medium-sized, polar molecules. Conductance properties are strongly modulated by mutations in a methionine-rich loop that is predicted to lie at the inner mouth of the channel, which might form a gasket around cargo molecules undergoing export. The results are discussed in the framework of an hypothesis for the architecture and action of the cargo-conducting part of the type-III secretion apparatus.