CS6 is the predominant colonization factor of enterotoxigenic Escherichia coli (ETEC). We report the existence of multiple CS6 subtypes caused by natural point mutations in cssA and cssB, the structural genes for CS6. The subtype AIBI was mostly associated with ETEC isolated from diarrhoeal cases, whereas AIIBII was mostly found in asymptomatic controls. Here we explore the rationale behind this association. ETEC isolates expressing AIIBII showed weaker adherence to intestinal epithelial cells compared with ETEC expressing AIBI. AIIBII expression on the ETEC cell surface was threefold less than AIBI. We found that alanine at position 37 in CssAII, in conjunction with asparagine at position 97 in CssBII, was responsible for the decreased levels of AIIBII on the bacterial surface. In addition, purified AIIBII showed fourfold less mucin binding compared with AIBI. The asparagine at position 97 in CssBII was also accountable for the decreased mucin binding by AIIBII. Reduced fluid accumulation and colonization occurred during infection with ETEC expressing AIIBII in animal models. Together these results indicate that the differential adherence between AIBI and AIIBII was a cumulative effect of decreased surface-level expression and mucin binding of AIIBII due to two specific amino acid variations. As a consequence, ETEC expressing these two subtypes displayed differential pathogenicity. We speculate that this might explain the subjective association of AIBI with ETEC from diarrhoeal cases and AIIBII with asymptomatic controls.
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