Abstract
The coli surface antigen 26 (CS26) of enterotoxigenic Escherichia coli (ETEC) had been described as a putative adhesive pilus based on the partial sequence of the crsH gene, detected in isolates from children with diarrhea in Egypt. However, its production and activity as adherence determinant has not been experimentally addressed. The crsH was identified as a homolog of genes encoding structural subunits of ETEC colonization factors (CFs) CS12, CS18, and CS20. These CFs, along with the recently discovered CS30, belong to the γ2 family of pili assembled by the chaperone-usher pathway (CU pili). Further, the complete CS26 locus, crsHBCDEFG, was described in an O141 ETEC strain (ETEC 100664) obtained from a diarrhea case in The Gambia, during the Global Enterics Multicenter Study. Here, we report that CS26 is a pilus of ∼10 nm in diameter, with the capacity to increase the cell adherence of the non-pathogenic strain E. coli DH10B. As for other related pili, production of CS26 seems to be regulated by phase variation. Deletion of crsHBCDEFG in ETEC 100664 significantly decreased its adherence capacity, which was recovered by in trans complementation. Furthermore, CrsH was cross-recognized by polyclonal antibodies directed against the major structural subunit of CS20, CsnA, as determined by Western blotting and immunogold labeling. ETEC CS26+ strains were found to harbor the heat-labile enterotoxin only, within three different sequence types of phylogroups A and B1, the latter suggesting acquisition through independent events of horizontal transfer. Overall, our results demonstrate that CS26 is an adhesive pilus of human ETEC. In addition, cross-reactivity with anti-CsnA antibodies indicate presence of common epitopes in γ2-CFs.
Highlights
Enterotoxigenic Escherichia coli (ETEC) causes diarrhea in humans by secreting heat-labile toxin (LT) and/or heat-stable toxins (STh and STp) (Gomes et al, 2016)
The locus crsHBCDEFG [crs-short version,] was amplified from ETEC 100664, a strain obtained during the Global Enterics Multicenter Study (GEMS) (Kotloff et al, 2013; Del Canto et al, 2017), cloned in pEZ-BAC and introduced into E. coli DH10B, in order to determine if it confers adherence capacity
E. coli DH10B harboring either of the two versions displayed a significantly higher adherence capacity (p < 0.05) to intestinal Caco-2 cells compared to the control without vectors or harboring the empty bacmid, and this was observed in the case of ETEC 100664 (Figures 1B,C)
Summary
Enterotoxigenic Escherichia coli (ETEC) causes diarrhea in humans by secreting heat-labile toxin (LT) and/or heat-stable toxins (STh and STp) (Gomes et al, 2016). ETEC infections are responsible for millions of diarrhea cases worldwide and about 60,000 deaths every year, mainly in children under 5 years in developing countries (Khalil, 2017). Given that ETEC must adhere to epithelial cells to optimally induce a toxigenic effect, structures determining attachment are eligible targets for vaccine development (Dorsey et al, 2006; O’Ryan et al, 2015). The colonization factors (CFs), 23 functionally characterized proteinaceous surface pili, are the classical ETEC adherence determinants of which 18 are assembled by the chaperone-usher pathway (CU pili), a common mechanism to construct pili at surface of Gram-negative bacteria (Madhavan and Sakellaris, 2015; Del Canto et al, 2017). Chaperones are periplasmic proteins that bind and fold pilus structural subunits for assembly, which occurs at the usher, an outer membrane pore-forming platform. The most abundant and repetitive subunit in the pilus is known as the major structural subunit while others are considered as minor structural subunits (Busch and Waksman, 2012)
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