Escherichia coli O157:H7 (O157) are noninvasive and weak biofilm producers; however, a subset of O157 are exceptions. O157 ATCC 43895 forms biofilms and invades epithelial cells. Tn5 mutagenesis identified mutation insertions that map within the curli csgB fimbriae locus to be responsible for both phenotypes. Screening of O157 strains for biofilm formation and cell invasion identify a bovine and a clinical isolate with those characteristics. A single base pair A to T transversion, intergenic to the curli divergent operons csgDEFG and csgBAC, is present only in biofilm-producing and invasive strains. Using site-directed mutagenesis, this single base change was introduced into two curli-negative/noninvasive O157 strains and modified strains to form biofilms, produce curli, and gain invasive capability. Transmission electron microscopy (EM) and immuno-EM confirmed curli fibers. EM of bovine epithelial cells (MAC-T) co-cultured with curli-expressing O157 show intracellular bacteria. The role of curli in O157 persistence in cattle was examined by challenging cattle with curli-positive and -negative O157 and comparing carriage. The duration of bovine colonization with the O157 curli-negative mutant was shorter than its curli-positive isogenic parent. These findings definitively demonstrate that a single base pair stably confers biofilm formation, epithelial cell invasion, and persistence in cattle.
Enterohemorrhagic Escherichia coli (EHEC) cause human disease with symptoms ranging from self-limited watery diarrhea to life-threatening hemorrhagic colitis, and hemolytic uremic syndrome . E. coli O157:H7 (O157) is the best studied EHEC serotype and is the predominant strain associated with disease outbreaks in North America, the United Kingdom, and Japan . Cattle and other ruminants carry this pathogen with no apparent symptoms  and are the most common source for human infections . O157 colonize at the bovine recto-anal junction (RAJ) and the bacteria persist in the feces of individual animals from a few days to several months . Attachment to biological surfaces is a first critical step in colonization and is mediated by multiple bacterial factors. Surface-associated factors of O157 contributing to tissue adherence and persistence in the bovine host include O-antigen , fimbriae , adhesins such as intimin , and some autotransporters . There is evidence that the duration of colonization and the bovine immune responses are strain/variant dependent .
Curli fimbriae, comprised of polymerized amyloid protein, are expressed on the surface of many members of the Enterobacteriaceae and other Gammaproteobacteria . Curli binds amyloid-specific dyes, such as Congo red and certain host proteins, including fibronectin, laminin, and plasminogen . During infections, curli complexes with extracellular matrix DNA. In a mouse model for lupus erythematosus autoimmunity, these curli-DNA complexes interact with Toll-like receptors (TLRs) 2 and 9 on dendritic and macrophage cells resulting in the production of autoantibodies . In most non-O157 E. coli, curli is regulated by σs and synthesized at low temperature, in nutrient-deprivation, and/or in stationary phase, conditions that promote biofilm formation . Curli synthesis requires the expression of genes from two divergently transcribed operons, designated csgDEFG and csgBAC. Genes with identified functions include the regulator CsgD, the type VIII secretion machinery components CsgE-G, the curli major subunit CsgA, and the curli nucleation protein CsgB . The intergenic region between csgDEFG and csgBAC is large and contains many putative binding sites for regulatory factors. CsgD is essential for the transcription of the curli operons . Curli promotes biofilm adhesion to abiotic surfaces as well as to mammalian cells . Although both operons are present in all sequenced O157 strains , the majority of O157 (approximately 95%) are curli-negative. This is because the prophage carrying Shiga toxin type-1 inserts into mlrA, a positive regulator of csgD . The few curli-positive O157 strains produce curli constitutively, including at 37 °C, and have acquired a suppressor mutation overriding the normal requirement for mlrA .
O157 is a weak biofilm producer and is considered an extracellular pathogen . However, some strains do not meet this general characterization. In a previous study, we showed that O157 strain 43895, an outbreak isolate from hamburger, produces biofilms at 37 °C, invades epithelial cells, and persists longer in cattle than a biofilm-negative strain . Curli expression has been found in certain O157 strains , but the underlying mechanism has not been fully explored.