Allelic variation in colonization factor CS6 of enterotoxigenic Escherichia coli isolated from patients with acute diarrhoea and controls

Abstract

Colonization factor antigens (CFAs) are important virulence factors in enterotoxigenic Escherichia coli (ETEC). Using a multiplex PCR and RT-PCR, this study tested the presence of common colonization factor-encoding genes and their expression in 50 ETEC strains isolated from stool specimens. The samples were from patients (children) with acute diarrhoea (cases) admitted to the Infectious Disease Hospital (Kolkata, India) and from normal children (controls) under 5 years of age from the community. The results indicated that coli surface antigen 6 (CS6) was the most prevalent CFA (78 %) expressed by these ETEC strains. Sequence analysis of both of the CS6 structural genes, i.e. cssA and cssB, in different ETEC isolates revealed the presence of point mutations in a systematic fashion. Based on the analysis of these variations, it was found that CssA had three alleles and CssB had two. Based on the allelic variations, subtyping of CS6 into AIBI, AIIBII, AIIIBI, AIBII and AIIIBII is proposed. The point mutations in the different alleles were reflected in a partial alteration in the secondary structure of both subunits, as determined by computational analysis. The functional significance of these changes was confirmed with cellular binding studies in Caco-2 cells with representative ETEC isolates. CS6 with AI or AIII allelic subtypes showed a higher binding capacity than AII, whereas BI showed stronger binding than BII. The AII and BII alleles were mostly detected in controls rather than in cases. The antibody specificity of BI and BII also varied due to alteration of the amino acids. Thus, CS6 variants are formed as a result of different allelic combinations of CssA and CssB, and these changes at the functional level might be important in the development of an effective ETEC vaccine.

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

“…Our previous studies also demonstrated that CS6 is one of the most prevalent CFs among the clinical ETEC isolates obtained from hospitalized diarrhoeal patients (Ghosal et al, 2007;Sabui et al, 2010). Based on the high prevalence of CS6 positive ETEC, CS6 has been proposed as an important vaccine candidate (de Lorimier et al, 2003;Ghosal et al, 2007;Sabui et al, 2010).…”

Real-Time PCR-Based Mismatch Amplification Mutation Assay for Specific Detection of CS6-Expressing Allelic Variants of Enterotoxigenic Escherichia coli and Its Application in Assessing Diarrheal Cases and Asymptomatic Controls

“…The structural genes of CS6 are cssA and cssB, which exhibit variation in different CS6-expressing ETEC isolates (16,20). Recently, we documented that cssA has three alleles (AI, AII, and AIII) and cssB has two alleles (BI and BII).…”

“…Due to the increase in the incidence of ETEC-mediated diarrheal disease, considerable attention has been paid to the development of simple, sensitive, and less expensive molecular methods for rapid identification of the pathogen. Currently, CFAs are detected by conventional PCR assay, replacing the conventional immunological tests (7,12,13,16,17).The CS6 operon is composed of four genes, cssA, cssB, cssC, and cssD. The structural genes of CS6 are cssA and cssB, which exhibit variation in different CS6-expressing ETEC isolates (16,20).…”

“…Currently, CFAs are detected by conventional PCR assay, replacing the conventional immunological tests (7,12,13,16,17).…”

“…Due to the increase in the incidence of ETEC-mediated diarrheal disease, considerable attention has been paid to the development of simple, sensitive, and less expensive molecular methods for rapid identification of the pathogen. Currently, CFAs are detected by conventional PCR assay, replacing the conventional immunological tests (7,12,13,16,17).…”