Type II secretory pathway for surface secretion of DraD invasin from the uropathogenic Escherichia coli Dr+ strain.
ABSTRACT: The virulence of the uropathogenic Escherichia coli Dr(+) IH11128 strain is associated with the presence of Dr fimbrial structures and a DraD invasin which can act as a fimbrial capping domain at the bacterial cell surface. However, a recent study suggests that the DraD protein is surface exposed in two forms: fimbria associated and fimbria nonassociated (prone to interaction with the N-terminal extension of the DraE protein located on the fimbrial tip). The actual mechanism of DraD surface secretion is presently unknown. We identified a previously unrecognized type II secretory pathway (secreton) in the uropathogenic E. coli Dr(+) strain which is well conserved among gram-negative bacteria and used mainly for secretion of virulence determinants. An active secreton is composed of 12 to 15 different proteins, among which GspD functions as an outer-membrane channel to permit extrusion of proteins in a folded state. Therefore, we inactivated the pathway by inserting the group II intron into a gspD gene of the type II secretion machinery by site-specific recombination. DraD secretion by the E. coli Dr(+) and gspD mutant strains was determined by immunofluorescence microscopy (with antibodies raised against DraD) and an assay of cell binding between bacteria and HeLa cells. The specificity of DraD-mediated bacterial binding for the integrin receptor was confirmed by examination of the adhesion of DraD-coated beads to HeLa cells in the presence and absence of alpha(5)beta(1) monoclonal antibodies. The investigations that we performed showed that type II secretion in E. coli Dr(+) strains leads to DraD translocation at the bacterial cell surfaces.
Project description:Several virulence-related genes have been described for prototype enteroaggregative Escherichia coli (EAEC) strain 042, which has been shown to cause diarrhea in human volunteers. Among these factors are the enterotoxins Pet and EAST and the fimbrial antigen aggregative adherence fimbria II (AAF/II), all of which are encoded on the 65-MDa virulence plasmid pAA2. Using nucleotide sequence analysis and insertional mutagenesis, we have found that the genes required for the expression of each of these factors, as well as the transcriptional activator of fimbrial expression AggR, map to a distinct cluster on the pAA2 plasmid map. The cluster is 23 kb in length and includes two regions required for expression of the AAF/II fimbria. These fimbrial biogenesis genes feature a unique organization in which the chaperone, subunit, and transcriptional activator lie in one cluster, whereas the second, unlinked cluster comprises a silent chaperone gene, usher, and invasin reminiscent of Dr family fimbrial clusters. This plasmid-borne virulence locus may represent an important set of virulence determinants in EAEC strains.
Project description:The Dr hemagglutinin of uropathogenic Escherichia coli mediates adherence to the upper urinary tract. E. coli strains which express this adhesin bind to the Dr blood group antigen and mediate mannose-resistant hemagglutination (MRHA). Chloramphenicol inhibits MRHA produced by the Dr hemagglutinin and may act as an analog for the tissue receptor at the adhesin-binding site. The nucleotide sequence of the Dr hemagglutinin fimbrial subunit was determined and found to have significant homology with that of F1845, a fimbrial adhesin associated with diarrhea, and with the afimbrial adhesin AFA-I of uropathogenic E. coli. Chimeric adhesin determinants consisting of the Dr structural subunit and F1845 accessory genes or of the F1845 structural subunit and Dr accessory genes were constructed. The Dr and F1845 determinants were shown to have a close structural relationship, with functional differences concentrated in the fimbrial subunit. Oligonucleotide-directed site-specific mutagenesis was used to facilitate construction of a hybrid adhesin subunit gene containing the amino terminus of F1845 fused to the carboxy terminus of the Dr structural gene. The resulting construct confers chloramphenicol-resistant hemagglutination when introduced into an E. coli strain expressing the cloned Dr hemagglutinin. The chloramphenicol sensitivity or resistant phenotype of MRHA produced by this family of adhesins is determined solely by the fimbrial subunit gene. Domains responsible for the chloramphenicol sensitivity of Dr-mediated MRHA reside within the amino-terminal portion of the fimbrial subunit.
Project description:Escherichia coli is the leading cause of urinary tract infections (UTIs), one of the most common infections in humans. P fimbria was arguably the first proposed virulence factor for uropathogenic E. coli, based on the capacity of E. coli isolated from UTIs to adhere to exfoliated epithelial cells in higher numbers than fecal strains of E. coli. Overwhelming epidemiologic evidence has been presented for involvement of P fimbriae in colonization. It has been difficult, however, to demonstrate this requirement for uropathogenic strains in animal models of infections or in humans. In this study, a signature-tagged mutagenesis screen identified a P-fimbrial gene (papC) and 18 other genes as being among those required for full fitness of cystitis isolate E. coli F11. A P-fimbrial mutant was outcompeted by the wild-type strain in cochallenge in the murine model of ascending UTI, and this colonization defect could be complemented with the cloned pap operon. To our knowledge, this study is the first to fulfill molecular Koch's postulates in which a pathogenic strain was attenuated by mutation of pap genes and then complemented to restore fitness, confirming P fimbria as a virulence factor in a pathogenic clinical isolate.
Project description:Uropathogenic Escherichia coli strain J96 carries multiple determinants for fimbrial adhesins. The regulatory protein PapB of P fimbriae has previously been implicated in potential coregulatory events. The focB gene of the F1C fimbria determinant is highly homologous to papB; the translated sequences share 81% identity. In this study we investigated the role of PapB and FocB in regulation of the F1C fimbriae. By using gel mobility shift assays, we showed that FocB binds to sequences in both the pap and foc operons in a somewhat different manner than PapB. The results of both in vitro cross-linking and in vivo oligomerization tests indicated that FocB could function in an oligomeric fashion. Furthermore, our results suggest that PapB and FocB can form heterodimers and that these complexes can repress expression of the foc operon. The effect of FocB on expression of type 1 fimbriae was also tested. Taken together, the results that we present expand our knowledge about a regulatory network for different adhesin gene systems in uropathogenic E. coli and suggest a hierarchy for expression of the fimbrial adhesins.
Project description:The enterotoxigenic Escherichia coli (ETEC) pathotype, characterized by the prototypical strain H10407, is a leading cause of morbidity and mortality in the developing world. A major virulence factor of ETEC is the type II secretion system (T2SS) responsible for secretion of the diarrheagenic heat-labile enterotoxin (LT). In this study, we have characterized the two type II secretion systems, designated alpha (T2SS(?)) and beta (T2SS(?)), encoded in the H10407 genome and describe the prevalence of both systems in other E. coli pathotypes. Under laboratory conditions, the T2SS(?) is assembled and functional in the secretion of LT into culture supernatant, whereas the T2SS(?) is not. Insertional inactivation of the three genes located upstream of gspC(?) (yghJ, pppA, and yghG) in the atypical T2SS(?) operon revealed that YghJ is not required for assembly of the GspD(?) secretin or secretion of LT, that PppA is likely the prepilin peptidase required for the function of T2SS(?), and that YghG is required for assembly of the GspD(?) secretin and thus function of the T2SS(?). Mutational and physiological analysis further demonstrated that YghG (redesignated GspS(?)) is a novel outer membrane pilotin protein that is integral for assembly of the T2SS(?) by localizing GspD(?) to the outer membrane, whereupon GspD(?) forms the macromolecular secretin multimer through which T2SS(?) substrates are translocated.
Project description:Type II secretion systems (T2SSs) are critical for secretion of many proteins from Gram-negative bacteria. In the T2SS, the outer membrane secretin GspD forms a multimeric pore for translocation of secreted proteins. GspD and the inner membrane protein GspC interact with each other via periplasmic domains. Three different crystal structures of the homology region domain of GspC (GspC(HR)) in complex with either two or three domains of the N-terminal region of GspD from enterotoxigenic Escherichia coli show that GspC(HR) adopts an all-? topology. N-terminal ?-strands of GspC and the N0 domain of GspD are major components of the interface between these inner and outer membrane proteins from the T2SS. The biological relevance of the observed GspC-GspD interface is shown by analysis of variant proteins in two-hybrid studies and by the effect of mutations in homologous genes on extracellular secretion and subcellular distribution of GspC in Vibrio cholerae. Substitutions of interface residues of GspD have a dramatic effect on the focal distribution of GspC in V. cholerae. These studies indicate that the GspC(HR)-GspD(N0) interactions observed in the crystal structure are essential for T2SS function. Possible implications of our structures for the stoichiometry of the T2SS and exoprotein secretion are discussed.
Project description:Enteroaggregative Escherichia coli (EAEC) has been implicated as an agent of pediatric diarrhea in the developing world. We have shown previously that EAEC adheres to HEp-2 cells by virtue of a plasmid-encoded fimbrial adhesin designated aggregative adherence fimbria I (AAF/I), the genes for which have been cloned and sequenced. However, not all EAEC strains express AAF/I. Using TnphoA mutagenesis, we have characterized a novel fimbria (designated AAF/II) which mediates HEp-2 adherence of the human-pathogenic strain 042. AAF/II is 5 nm in diameter and does not bind AAF/I antiserum, as determined by immunogold transmission electron microscopy. TnphoA identified a gene (designated aafA) which bears significant homology to aggA, the fimbrial subunit of AAF/I (25% identity and 47% similarity at the amino acid level). When hyperexpressed and purified by polyhistidine tagging, the AafA protein assembled into 5-nm-diameter filaments which bound anti-AAF/II antiserum. The cloned aafA gene complemented a mutation in the aggA gene to confer fimbrial expression from the AAF/I gene cluster, manifesting phenotypes characteristic of AAF/II but not AAF/I. The aafA mutant did not adhere to human intestinal tissue in culture, suggesting a role for AAF/II in intestinal colonization. By using DNA probes for AAF/I and AAF/II derived from fimbrial biosynthesis genes, we show that AAF/I and AAF/II are each found in only a minority of EAEC strains, suggesting that still more EAEC adhesins exist. Our data suggest that AAF adhesins represent a new family of fimbrial adhesins which mediate aggregative adherence in EAEC.
Project description: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.
Project description:Uropathogenic Escherichia coli strains utilize a variety of adherence factors that assist in colonization of the host urinary tract. TosA (type one secretion A) is a nonfimbrial adhesin that is predominately expressed during murine urinary tract infection (UTI), binds to kidney epithelial cells, and promotes survival during invasive infections. The tosRCBDAEF operon encodes the secretory machinery necessary for TosA localization to the E. coli cell surface, as well as the transcriptional regulator TosR. TosR binds upstream of the tos operon and in a concentration-dependent manner either induces or represses tosA expression. TosR is a member of the PapB family of fimbrial regulators that can participate in cross talk between fimbrial operons. TosR also binds upstream of the pap operon and suppresses PapA production. However, the scope of TosR-mediated cross talk is understudied and may be underestimated. To quantify the global effects of TosR-mediated regulation on the E. coli CFT073 genome, we induced expression of tosR, collected mRNA, and performed high-throughput RNA sequencing (RNA-Seq). These findings show that production of TosR affected the expression of genes involved with adhesins, including P, F1C, and Auf fimbriae, nitrate-nitrite transport, microcin secretion, and biofilm formation.IMPORTANCE Uropathogenic E. coli strains cause the majority of UTIs, which are the second most common bacterial infection in humans. During a UTI, bacteria adhere to cells within the urinary tract, using a number of different fimbrial and nonfimbrial adhesins. Biofilms can also develop on the surfaces of catheters, resulting in complications such as blockage. In this work, we further characterized the regulator TosR, which links both adhesin production and biofilm formation and likely plays a crucial function during UTI and disseminated infection.
Project description:Uropathogenic Proteus mirabilis produces at least four types of fimbriae. Amino acid sequences from two peptides, derived by tryptic digestion of the structural subunit of one type of these fimbriae, the ambient-temperature fimbriae, were determined: NVVPGQPSSTQ and LIEGENQLNYNA. PCR primers, based on these sequences and that of the N terminus, were used to amplify a 359-bp fragment. A cosmid clone, isolated from a P. mirabilis genomic library by hybridization with the 359-bp PCR product, was used to determine the nucleotide sequence of the atf gene cluster. A 3,903-bp region encodes three polypeptides: AtfA, the structural subunit; AtfB, the chaperone; and AtfC, the outer membrane molecular usher. No fimbria-related genes are evident either 5' or 3' to the three contiguous genes. AtfA demonstrates significant amino acid sequence identity with type 1 major fimbrial subunits of several enteric species. The 359-bp PCR product hybridized strongly with all Proteus isolates (n = 9) and 25% of 355 Escherichia coli isolates but failed to hybridize with any of 26 isolates among nine other uropathogenic species. Ambient-temperature fimbriae of P. mirabilis may represent a novel type of fimbriae of enteric species.