Characterization of oligomeric assembly of colonization factor CS6 from enterotoxigenic Escherichia coli

Sabui, Subrata; Debnath, A. K.; Ghosal, Abhisek; Wajima, Takeaki; Hamabata, Takashi; Ramamurthy, T.; Ghosh, Avishek; Basak, Soumen; Chatterjee, Nando K.

doi:10.1099/mic.0.000180

Cited by 5 publications

(6 citation statements)

References 31 publications

(37 reference statements)

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“…Expression, purification, and characterization of "homologous donor strandcomplemented" CS6-derived recombinant proteins. It has been observed that purified CS6 (21) as well as its CssA and CssB subunits (S. J. Savarino, unpublished data) form oligomeric complexes in solution. This has also been described for the class 5 major fimbrial subunit CfaB, which multimerizes via donor strand interactions between two subunits (27).…”

Section: Resultsmentioning

confidence: 98%

“…Instead, CS6 is afimbrial in structure, associating closely with the bacterial cell surface instead of extending from the surface as is typical of the fimbrial CFs (11). Furthermore, it is made up of two structural subunits, CssA and CssB, in a 1:1 ratio (21). The bioassembly of CS6 is encoded by a plasmid-associated operon consisting of four genes (cssABCD) that is transcribed as a single transcript.…”

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confidence: 99%

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Biochemical and Immunological Evaluation of Recombinant CS6-Derived Subunit Enterotoxigenic Escherichia coli Vaccine Candidates

et al. 2019

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CS6, a prevalent surface antigen expressed in nearly 20% of clinical enterotoxigenic Escherichia coli (ETEC) isolates, is comprised of two major subunit proteins, CssA and CssB. Using donor strand complementation, we constructed a panel of recombinant proteins of 1 to 3 subunits that contained combinations of CssA and/or CssB subunits and a donor strand, a C-terminal extension of 16 amino acids that was derived from the N terminus of either CssA or CssB. While the entire panel of recombinant proteins could be obtained as soluble, folded proteins, it was observed that the proteins possessing a heterologous donor strand, derived from the CS6 subunit different from the C-terminal subunit, had the highest degree of physical and thermal stability. Immunological characterization of the proteins, using a murine model, demonstrated that robust anti-CS6 immune responses were generated from fusions containing both CssA and CssB. Proteins containing only CssA were weakly immunogenic. Heterodimers, i.e., CssBA and CssAB, were sufficient to recapitulate the anti-CS6 immune response elicited by immunization with CS6, including the generation of functional neutralizing antibodies, as no further enhancement of the response was obtained with the addition of a third CS6 subunit. Our findings here demonstrate the feasibility of including a recombinant CS6 subunit protein in a subunit vaccine strategy against ETEC.

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Section: Resultsmentioning

confidence: 98%

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confidence: 99%

Biochemical and Immunological Evaluation of Recombinant CS6-Derived Subunit Enterotoxigenic Escherichia coli Vaccine Candidates

et al. 2019

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“…Thereafter, the CssA C -CssB N interaction is then driven in the presence of CssD through DSE. The final outcome is a spherical, hetero-oligomeric complex of (CssA-CssB) n on the bacterial outer surface (19).…”

Section: Discussionmentioning

confidence: 99%

“…Since CS6 forms concentration-dependent oligomers in solution (10,19), we analyzed the oligomer formation of those mutant CS6 proteins that were expressed on the bacterial cell surface at reduced levels. Three CssA mutC proteins, i.e., the G141A, V149A, and Y151A mutants, showed disruption in the oligomeric pattern, whereas the other two mutant proteins, i.e., the I142A and Y152A mutants, did not show any oligomeric pattern.…”

Section: Effects Of Alanine Substitutions On Cs6 Oligomerizationmentioning

confidence: 99%

Functional Role of N- and C-Terminal Amino Acids in the Structural Subunits of Colonization Factor CS6 Expressed by Enterotoxigenic Escherichia coli

et al. 2016

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CS6 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. Enterotoxigenic Escherichia coli (ETEC) infection is the most common cause of infantile diarrhea, which accounts for approximately 210 million episodes and 380,000 deaths annually (1). Plasmid-encoded colonization factors (CFs) and heat-labile and heat-stable (LT and ST, respectively) enterotoxins are the major determinants of ETEC virulence. Colonization is the prerequisite step in initiating the virulence mediated by the interaction of CFs with their cognate receptors on the intestinal epithelium. So far, 25 different CFs have been identified and subdivided on the basis of antigenicity, molecular weight, and structural morpholo...

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“…In contrast to the other ETEC CFs that are fimbrial, CS6 is afimbrial. CS6 consists of two tightly-associated subunits CssA and CssB, organised in an equal (1:1) ratio, to form an external array of higher-order oligomers [9].…”

Section: Introductionmentioning

confidence: 99%

Iron influences the expression of colonization factor CS6 of enterotoxigenic Escherichia coli

et al. 2021

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Enterotoxigenic Escherichia coli (ETEC) is a major pathogen of acute watery diarrhoea. The pathogenicity of ETEC is linked to adherence to the small intestine by colonization factors (CFs) and secretion of heat-labile enterotoxin (LT) and/or heat-stable enterotoxin (ST). CS6 is one of the most common CFs in our region and worldwide. Iron availability functions as an environmental cue for enteropathogenic bacteria, signalling arrival within the human host. Therefore, iron could modify the expression of CS6 in the intestine. The objective of this study was to determine the effect of iron availability on CS6 expression in ETEC. This would help in understanding the importance of iron during ETEC pathogenesis. ETEC strain harbouring CS6 was cultured under increasing concentrations of iron salt to assess the effect on CS6 RNA expression by quantitative RT-PCR, protein expression by ELISA, promoter activity by β-galactosidase activity, and epithelial adhesion on HT-29 cells. RNA expression of CS6 was maximum in presence of 0.2 mM iron (II) salt. The expression increased by 50-fold, which also reduced under iron-chelation conditions and an increased iron concentration of 0.4 mM or more. The surface expression of CS6 also increased by 60-fold in presence of 0.2 mM iron. The upregulation of CS6 promoter activity by 25-fold under this experimental condition was in accordance with the induction of CS6 RNA and protein. This increased CS6 expression was independent of ETEC strains. Bacterial adhesion to HT-29 epithelial cells was also enhanced by five-fold in the presence of 0.2 mM iron salt. These findings suggest that CS6 expression is dependent on iron concentration. However, with further increases in iron concentration beyond 0.2 mM CS6 expression is decreased, suggesting that there might be a strong regulatory mechanism for CS6 expression under different iron concentrations.

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Characterization of oligomeric assembly of colonization factor CS6 from enterotoxigenic Escherichia coli

Cited by 5 publications

References 31 publications

Biochemical and Immunological Evaluation of Recombinant CS6-Derived Subunit Enterotoxigenic Escherichia coli Vaccine Candidates

Biochemical and Immunological Evaluation of Recombinant CS6-Derived Subunit Enterotoxigenic Escherichia coli Vaccine Candidates

Functional Role of N- and C-Terminal Amino Acids in the Structural Subunits of Colonization Factor CS6 Expressed by Enterotoxigenic Escherichia coli

Iron influences the expression of colonization factor CS6 of enterotoxigenic Escherichia coli

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