Novel type of pilus associated with a Shiga-toxigenic <i>E. coli</i> hybrid pathovar conveys aggregative adherence and bacterial virulence

A large German outbreak in 2011 was caused by a locus of enterocyte effacement (LEE)-negative enterohemorrhagic <i>E. coli</i> (EHEC) strain of the serotype O104:H4. This strain harbors markers that are characteristic of both EHEC and enteroaggregative <i>E. coli</i> (EAEC), including aggregative adhesion fimbriae (AAF) genes. Such rare EHEC/EAEC hybrids are highly pathogenic due to their possession of a combination of genes promoting severe toxicity and aggregative adhesion. We previously identified novel EHEC/EAEC hybrids and observed that one strain exhibited aggregative adherence but had no AAF genes. In this study, a genome sequence analysis showed that this strain belongs to the genoserotype O23:H8, MLST ST26, and harbors a 5.2 Mb chromosome and three plasmids. One plasmid carries some EAEC marker genes, such as <i>aatA</i> and genes with limited protein homology (11–61%) to those encoding the bundle-forming pilus (BFP) of enteropathogenic <i>E. coli</i>. Due to significant protein homology distance to known pili, we designated these as aggregate-forming pili (AFP)-encoding genes and the respective plasmid as pAFP. The <i>afp</i> operon was arranged similarly to the operon of BFP genes but contained an additional gene, <i>afpA2</i>, which is homologous to <i>afpA</i>. The deletion of the <i>afp</i> operon, <i>afpA</i>, or a nearby gene (<i>afpR</i>) encoding an AraC-like regulator, but not <i>afpA2</i>, led to a loss of pilin production, piliation, bacterial autoaggregation, and importantly, a &gt;80% reduction in adhesion and cytotoxicity toward epithelial cells. Gene sets similar to the <i>afp</i> operon were identified in a variety of <i>aatA</i>-positive but AAF-negative intestinal pathogenic <i>E. coli</i>. In summary, we characterized widely distributed and novel fimbriae that are essential for aggregative adherence and cytotoxicity in a LEE-negative Shiga-toxigenic hybrid.