Enterotoxigenic Escherichia coli CS21 pilus contributes to adhesion to intestinal cells and to pathogenesis under in vivo conditions.

Colonization surface antigens (CSs) represent key virulence-associated factors of enterotoxigenic Escherichia coli (ETEC) strains. They are required for gut colonization, the first step of the diarrhoeal disease process induced by these bacteria. One of the most prevalent CSs is CS21, or longus, a type IV pili associated with bacterial self-aggregation, protection against environmental stresses, biofilm formation and adherence to epithelial cell lines. The objectives of this study were to assess the role of CS21 in adherence to primary intestinal epithelial cells and to determine if CS21 contributes to the pathogenesis of ETEC infection in vivo. We evaluated adherence of a CS21-expressing wild-type ETEC strain and an isogenic CS21-mutant strain to pig-derived intestinal cell lines. To determine the role of CS21 in pathogenesis we used the above ETEC strains in a neonatal mice challenge infection model to assess mortality. Quantitative adherence assays confirmed that ETEC adheres to primary intestinal epithelial cells lines in a CS21-dependent manner. In addition, the CS21-mediated ETEC adherence to cells was specific as purified LngA protein, the CS21 major subunit, competed for binding with the CS21-expressing ETEC while specific anti-LngA antibodies blocked adhesion to intestinal cells. Neonatal DBA/2 mice died after intra-stomach administration of CS21-expressing strains while lack of CS21 expression drastically reduced the virulence of the wild-type ETEC strain in this animal model. Collectively these results further support the role of CS21 during ETEC infection and add new evidence on its in vivo relevance in pathogenesis.

Prime-boost vaccine regimen confers protective immunity to human-derived enterotoxigenic Escherichia coli.

Development of effective vaccines against diarrhea caused by enterotoxigenic Escherichia coli (ETEC) strains is still a priority for those living at or traveling to endemic regions. In this work, we evaluated the protective role of an anti-ETEC vaccine regimen based on parenteral priming with a DNA vaccine, pRECFA, followed by oral boosting with a recombinant attenuated Salmonella Typhimurium vaccine strain, HG3, both encoding the same antigen, the structural subunit (CfaB) of the ETEC CFA/I fimbriae. The DNA-priming Salmonella-boosting protocol enhanced both murine anti-CfaB serum IgG and fecal IgA antibody responses and increased the ability of serum antibodies to inhibit the adhesive properties of the CFA/I fimbriae expressed by live bacteria, as compared to mice immunized with only one vaccine type. Addition of a mucosal adjuvant (LTR192G) to the Salmonella vaccine strain further enhanced the synergic effects of the vaccine regimen on the induced CfaB-specific antibody responses. DBA/2 dams submitted to the prime-boost regimen transferred complete passive protection to suckling neonates challenged with a virulent ETEC strain. Detection of milk anti-CfaB IgA antibodies and protection conferred by vaccinated dams to neonates born from non-vaccinated dams indicated that secretion of antigen-specific IgA is the immune response induced by the protective vaccine regimen. These results demonstrate that priming with a DNA vaccine and boosting with a Salmonella strain enhances both quantitatively and qualitatively the antibody responses to the CfaB antigen and represents an alternative for either active or passive immunization approach to ETEC-associated diarrhea.