Identification and Characterisation of Novel Autotransporters of Enterohaemorrhagic Escherichia coli O157:H7

Enterohemorrhagic Escherichia coli (EHEC) are a subgroup of Shiga toxin producing E. coli that cause gastrointestinal disease with the potential for life-threatening sequelae. Cattle serve as the natural reservoir for EHEC and outbreaks occur sporadically as a result of contaminated beef products and other farming products. E. coli O157:H7 was the first EHEC strain described and has been responsible for hundreds of outbreaks in Canada, Europe, Japan and the U.S.A. since 1982. While certain EHEC virulence mechanisms have been extensively studied, the factors that mediate host colonisation are poorly defined. Autotransporter (AT) proteins have been identified in many Gram-negative pathogens and are unique in that their primary sequence is sufficient to direct their transport across the bacterial membrane system. Many characterised members are associated with virulence. Using conserved AT motifs as a search tool five putative AT proteins were identified in the EHEC O157:H7 EDL933 genome. The genes encoding these proteins (z0402/ehaA, z0469/ehaB, z3487/ehaC, z3948/ehaD and z5029/ehaG) were PCR amplified, cloned and expressed in an E. coli K-12 MG1655flu background. Characterisation revealed that ehaA, ehaB, ehaD and ehaG encode proteins associated with an increase in biofilm formation. EhaA, EhaB and EhaG were found to mediate biofilm formation under continuous flow conditions when expressed in E. coli K-12. Over-expression of either EhaA or EhaG in E. coli K-12 resulted in the formation of large cell aggregates. Three of the AT proteins were found to mediate adhesion when over-expressed in E. coli K-12. The EhaA AT protein mediated binding to primary epithelial cells of the bovine terminal rectum. EhaB promoted adhesion to the extracellular matrix (ECM) proteins laminin and collagen I and was recognised by IgA serum taken from calves challenged with E. coli O157:H7. EhaG is a member of the trimeric autotransporter adhesin (TAA) sub-group of AT proteins and mediated adhesion to colorectal adenocacinoma (Caco-2) epithelial cells. Our results suggest that EhaA, EhaB, EhaD and EhaG may contribute to adhesion, colonisation and biofilm formation by E. coli O157:H7. This study also used a bioinformatic approach to identify AT encoding genes in available E. coli genomes. We identified 156 AT encoding genes in 18 E. coli genomes queried. Alignment and analysis of these proteins identified three broad groups, the serine protease autotransporters of enterobacteriacae (SPATEs), the trimeric autotransporter adhesins (TAAs) and AIDA-I type AT vii proteins. The latter group consisted of a further ten sub-groups. The results demonstrated that E. coli strains encode multiple AT proteins, many of which may have some degree of functional redundancy