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Characterization and studies of the cellular interaction of native colonization factor CS6 purified from a clinical isolate of enterotoxigenic Escherichia coli.

ABSTRACT: CS6 is a widely expressed colonization factor of enterotoxigenic Escherichia coli (ETEC). To date, CS6 has not been well characterized in its native state. Here, we purified CS6 for the first time from an ETEC clinical isolate. Purified CS6 was composed of two structural subunits, CssA and CssB, which were present in equal amounts and tightly linked through noncovalent, detergent-stable association. The CssA subunit was poorly immunogenic, whereas CssB was highly immunogenic. Although the predicted molecular mass of CssA is 15 kDa, the purified CssA has an effective molecular mass of 18.5 kDa due to fatty acid modification. When purified CS6 was screened for its ability to bind with different extracellular matrix proteins, fibronectin (Fn) was found to interact with CS6 as well as CssA in a dose-dependent and saturable manner. This interaction was inhibited both by a synthetic peptide corresponding to the C-terminal hydrophilic, surface-exposed region of CssA (positions 112 to 126) and by the antibody derived against this region. Enzyme-linked immunosorbent assay results showed that CssA interacted with the 70-kDa N-terminal domain of Fn. The modifications on CssA probably do not play a role in Fn binding. Preincubation of INT 407 cells with CssA, but not CssB, inhibited ETEC binding to these cells. The results suggested that CS6-expressing ETEC binds to Fn of INT 407 cells through the C-terminal region of CssA. Purified CS6 was found to colocalize with Fn along the junctions of INT 407 cells. Based on the results obtained, we propose that CS6-expressing ETEC binds to the intestinal cells through Fn for colonization.

SUBMITTER: Ghosal A

PROVIDER: S-EPMC2681727 | biostudies-literature | 2009 May

REPOSITORIES: biostudies-literature

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Characterization and studies of the cellular interaction of native colonization factor CS6 purified from a clinical isolate of enterotoxigenic Escherichia coli.

Ghosal Abhisek A Bhowmick Rudra R Banerjee Rajat R Ganguly Sandipan S Yamasaki S S Ramamurthy T T Hamabata T T Chatterjee Nabendu Sekhar NS

Infection and immunity 20090223 5

CS6 is a widely expressed colonization factor of enterotoxigenic Escherichia coli (ETEC). To date, CS6 has not been well characterized in its native state. Here, we purified CS6 for the first time from an ETEC clinical isolate. Purified CS6 was composed of two structural subunits, CssA and CssB, which were present in equal amounts and tightly linked through noncovalent, detergent-stable association. The CssA subunit was poorly immunogenic, whereas CssB was highly immunogenic. Although the predic ...[more]

PMID: 19237522

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Project description:UnlabelledCS6 is a common colonization factor expressed by enterotoxigenic Escherichia coli It is a two-subunit protein consisting of CssA and CssB in an equal stoichiometry, assembled via the chaperone-usher pathway into an afimbrial, oligomeric assembly on the bacterial cell surface. A recent structural study has predicted the involvement of the N- and C-terminal regions of the CS6 subunits in its assembly. Here, we identified the functionally important residues in the N- and C-terminal regions of the CssA and CssB subunits during CS6 assembly by alanine scanning mutagenesis. Bacteria expressing mutant proteins were tested for binding with Caco-2 cells, and the results were analyzed with respect to the surface expression of mutant CS6. In this assay, many mutant proteins were not expressed on the surface while some showed reduced expression. It appeared that some, but not all, of the residues in both the N and C termini of CssA and CssB played an important role in the intermolecular interactions between these two structural subunits, as well as chaperone protein CssC. Our results demonstrated that T20, K25, F27, S36, Y143, and V147 were important for the stability of CssA, probably through interaction of CssC. We also found that I22, V29, and I33 of CssA and G154, Y156, L160, V162, F164, and Y165 of CssB were responsible for CssA-CssB intermolecular interactions. In addition, some of the hydrophobic residues in the C terminus of CssA and the N terminus of CssB were involved in the stabilization of higher-order complex formation. Overall, the results presented here might help in understanding the pathway used to assemble CS6 and predict its structure.ImportanceUnlike most other colonization factors, CS6 is nonfimbrial, and in a sense, its subunit composition and assembly are also unique. Here we report that both the N- and C-terminal amino acid residues of CssA and CssB play a critical role in the intermolecular interactions between them and assembly proteins. We found mainly that alternate hydrophobic residues present in these motifs are essential for the interaction between the structural subunits, as well as the chaperone and usher assembly proteins. Our results indicate the involvement of the side chains of identified amino acids in CS6 assembly. This study adds a step toward understanding the interactions between structural subunits of CS6 and assembly proteins during CS6 biogenesis.

Project description:Enterotoxigenic Escherichia coli (ETEC) is a significant cause of childhood diarrhea and is a leading cause of traveler's diarrhea. ETEC strains encoding the heat-stable enterotoxin (ST) are more often associated with childhood diarrhea than ETEC strains that encode only the heat-labile enterotoxin (LT). Colonization factors (CFs) also have a demonstrated role in ETEC virulence, and two of the most prevalent CFs among ETEC that have caused diarrhea are colonization factor antigen I (CFA/I) and CS6. In the current report, we describe the genomes of 269 CS6- or CFA/I-encoding ST-only ETEC isolates that were associated with human diarrhea. While the CS6 and CFA/I ETEC were identified in at least 13 different ETEC genomic lineages, a majority (85%; 229/269) were identified in only six lineages. Complete genome sequencing of selected isolates demonstrated that a conserved plasmid contributed to the dissemination of CFA/I whereas at least five distinct plasmids were involved in the dissemination of ST and/or CS6. Additionally, there were differences in gene content between CFA/I and CS6 ETEC at the phylogroup and lineage levels and in association with their geographic location of isolation as well as lineage-related differences in ST production. Thus, we demonstrate that genomically diverse E. coli strains have acquired ST, as well as CFA/I or CS6, via one or more plasmids and that, in some cases, isolates of a particular lineage or geographic location have undergone additional modifications to their genome content. These findings will aid investigations of virulence and the development of improved diagnostics and vaccines against this important human diarrheal pathogen. IMPORTANCE Comparative genomics and functional characterization were used to analyze a global collection of CFA/I and CS6 ST-only ETEC isolates associated with human diarrhea, demonstrating differences in the genomic content of CFA/I and CS6 isolates related to CF type, lineage, and geographic location of isolation and also lineage-related differences in ST production. Complete genome sequencing of selected CFA/I and CS6 isolates enabled descriptions of a highly conserved ST-positive (ST+) CFA/I plasmid and of at least five diverse ST and/or CS6 plasmids among the CS6 ETEC isolates. There is currently no approved vaccine for ST-only ETEC, or for any ETEC for that matter, and as such, the current report provides functional verification of ST and CF production and antimicrobial susceptibility testing and an in-depth genomic characterization of a collection of isolates that could serve as representatives of CFA/I- or CS6-encoding ST-only ETEC strains for future studies of ETEC pathogenesis, vaccine studies, and/or clinical trials.

Project description:IntroductionEnterotoxigenic Escherichia coli (ETEC) is a common cause of infectious diarrhoea and a leading cause of morbidity and mortality in children living in resource-limited settings. It is also the leading cause of travellers' diarrhoea among civilian and military travellers. Its dual importance in global public health and travel medicine highlights the need for an effective vaccine. ETEC express colonization factors (CFs) that mediate adherence to the small intestine. An epidemiologically prevalent CF is coli surface antigen 6 (CS6). We assessed the safety and immunogenicity of a CS6-targeted candidate vaccine, CssBA, co-administered intramuscularly with the double-mutant heat-labile enterotoxin, dmLT [LT(R192G/L211A)].MethodsThis was an open-label trial. Fifty subjects received three intramuscular injections (Days 1, 22 and 43) of CssBA alone (5 µg), dmLT alone (0.1 µg) or CssBA (5, 15, 45 µg) + dmLT (0.1 and 0.5 µg). Subjects were actively monitored for adverse events for 28 days following the third vaccination. Antibody responses (IgG and IgA) were characterized in the serum and from lymphocyte supernatants (ALS) to CS6 and the native ETEC heat labile enterotoxin, LT.ResultsAcross all dose cohorts, the vaccine was safe and well-tolerated with no vaccine-related severe or serious adverse events. Among vaccine-related adverse events, a majority (98%) were mild with 79% being short-lived vaccine site reactions. Robust antibody responses were induced in a dose-dependent manner with a clear dmLT adjuvant effect. Response rates in subjects receiving 45 µg CssBA and 0.5 µg dmLT ranged from 50 to 100% across assays.ConclusionThis is the first study to demonstrate the safety and immunogenicity of CssBA and/or dmLT administered intramuscularly. Co-administration of the two components induced robust immune responses to CS6 and LT, paving the way for future studies to evaluate the efficacy of this vaccine target and development of a multivalent, subunit ETEC vaccine.

Dataset Information

Characterization and studies of the cellular interaction of native colonization factor CS6 purified from a clinical isolate of enterotoxigenic Escherichia coli.

Publications

Characterization and studies of the cellular interaction of native colonization factor CS6 purified from a clinical isolate of enterotoxigenic Escherichia coli.

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