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Subtractive hybridization and identification of putative adhesins in a Shiga toxin-producing eae-negative Escherichia coli

¹ Programa de Microbiología y Micologia, ICBM, Facultad de Medicina, Universidad de Chile, Santiago, Chile
² Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555-1070, USA
³ Department of Clinical Laboratory Sciences, University of Texas Medical Branch, Galveston, TX 77555-1070, USA
⁴ Department of Pathology and Sealy Center for Vaccine Development, University of Texas Medical Branch, Galveston, TX 77555-1070, USA
⁵ Programa de Biologia Celular y Molecular, ICBM, Facultad de Medicina, Universidad de Chile, Santiago, Chile

Correspondence
Roberto M. VidalE-mail:
rvidal{at}med.uchile.cl
or
Alfredo G. Torres
altorres{at}utmb.edu

Adherence to epithelial cells by specific adhesins is a characteristic of Shiga toxin-producing Escherichia coli (STEC) strains. The eae-encoded protein intimin is the main adhesin implicated in intestinal colonization in vivo. We recently showed that STEC strains isolated in Chile displayed a wide variety of adhesins; here we demonstrate that some of these STEC strains are eae-negative and still adhere to epithelial cells at a level 100-fold higher than enterohaemorrhagic E. coli (EHEC) O157 : H7 prototype strain EDL933. This phenotype is associated with the presence of adherence factors different from the intimin protein. Subtractive hybridization between EHEC EDL933 and STEC eae-negative strain 472-1 was used to identify regions implicated in adhesion. In addition to the saa gene, we identified 18 specific genes in STEC 472-1, 16 of which had nucleotide identity to Salmonella ST46 phage genes; the two remaining ones shared identity to a gene encoding a hypothetical protein of uropathogenic E. coli. The DNA sequence of the STEC 472-1 psu-int region identified five open reading frames with homology to phage genes. We constructed mutant strains in the saa gene and the psu-int region to study the participation of these genes in the adherence to epithelial cells and our results demonstrated that STECsaa and STECpsu-int mutants displayed a 10-fold decrease in adherence as compared to the STEC 472-1 wild-type strain. Overall, our results suggest that STEC strain 472-1 adheres to epithelial cells in an eae-independent matter and that saa and psu-int participate in this adhesion process.

Supplementary material is available with the online version of this paper.