Novel type of pilus associated with a Shiga-toxigenic E. coli hybrid pathovar

A large outbreak 2011 in Germany was caused by a locus of enterocyte effacement (LEE)-negative enterohemorrhagic E. coli (EHEC) of the serotype O104:H4. This strain harbors markers characteristic for both EHEC and for enteroaggregative E. coli (EAEC), including aggregative adhesion fimbriae (AAF) genes. Such seldom described EHEC/EAEC hybrids show a high pathogenicity due to the combination of genes promoting severe toxicity and aggregative adhesion. Previously, we discovered novel EHEC/EAEC hybrids and one revealed aggregative adherence but no AAF genes. In this study, genome sequence analysis uncovered that this strain belongs to the genoserotype O23:H8, MLST ST26 and further reveals a 5.2 Mb chromosome and three plasmids. One plasmid comprised some EAEC marker genes, such as aatA, and genes with limited protein homology (11-61%) to the bundle-forming pilus (BFP) of enteropathogenic E. coli. Due to significant protein distance to known pili, we designated those as aggregate-forming pili (AFP) and the respective plasmid as pAFP. The afp operon was arranged similar to the BFP genes, but contained an additional gene, afpA2 homologous to afpA. Deletion of the afp operon, afpA, or a nearby located gene of an AraC-like regulator afpR, but not afpA2, led to loss of pilin production, piliation, bacterial autoaggregation, and importantly > 80% reduced adhesion and cytotoxicity towards epithelial cells. Gene sets similar to the afp operon were present in a variety of aatA-positive but AAF-negative intestinal pathogenic E. coli. In conclusion, we identified widely distributed novel fimbriae essential for aggregative adherence and cytotoxicity in a LEE-negative Shiga-toxigenic hybrid.