Genetic diversity of heat labile toxin (LT) expressed by enterotoxigenic Escherichia coli (ETEC) strains isolated from humans

Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil, Department of Microbiology, Immunology and Parasitology, Federal University of São Paulo, São Paulo, Brazil, Division of Technological Development, Butantan Institute, São Paulo, Brazil

^* To whom correspondence should be addressed. Email: lcsf{at}usp.br.

	Abstract

The natural diversity of the elt operon, encoding the heat labile toxin (LT-I or LT), carried by enterotoxigenic Escherichia coli (ETEC) strains isolated from humans, was investigated. For many years, LT was supposed to be represented by a rather conserved toxin and one derivative, produced by the reference H10407 strain, was intensively studied either as virulent factor or as a vaccine adjuvant. Amplicons encompassing the two LT-encoding genes (eltA and eltB) of 51 human-derived ETEC strains, either producing LT⁺ (25) only or LT⁺/ST⁺ (26), isolated from asymptomatic (24) or diarrheic (27) subjects, were submitted to restriction fragment length polymorphism (RFLP) analysis and DNA sequencing. Seven polymorphic RFLP types were detected with six (BsaI, DdeI, HhaI, HincII, HphI, and MspI) restriction enzymes with regard to the H10407 strain. Additionally, the single nucleotide polymorphic analysis revealed 50 base changes in the elt operon, including 21 polymorphic sites at eltA and 9 at eltB. Based on the deduced amino acid sequences, 16 LT types were identified including LT1, expressed by the H10407 strain and 23 strains other belonging to seven different serotypes, and LT2, expressed by 11 strains of six different serotypes. In vitro experiments carried out with purified toxins indicated that no significant differences in GM1-binding affinity could be detected among LT1, LT2, and LT4. However, LT4, but not those belonging to other toxin types, showed reduced toxic activities measured either in vitro, with cultured cells (Y-1 cells), or in vivo in rabbit ligated ileal loops. Collectively, these results indicate that the natural diversity of LT produced by wild-type ETEC strains isolated from human hosts is considerably larger than previously assumed and may impact the pathogenesis of the strains and the epidemiology of the disease.