Role of long polar fimbriae from EHEC O157:H7 in intestinal colonization and adherence, and as a tool for molecular diagnostics

BOTKIN DJ; LLOYD SJ; GALLI L; RITCHIE JM; BLUMENTRITT CA; WALDOR MK; RIVAS M; TORRES AG

Amsterdam

Simposio; 8th International Symposium on Shiga Toxin (Verocytotoxin) - Producing Escherichia coli Infections; 2012

In addition to a variety of other adhesins, STEC O157:H7 possess two long polar fimbriae (Lpf) structures. The genes encoding Lpf are prevalent among pathogenic E.coli, and Lpf have been implicated in both colonization using different animal models and human intestinal tissue tropism. STEC canalso produce curli, thin aggregative fimbriae, and they have been suggested to be involved in biofilm formation, invasion, and intestinal inflammation. Recently, we identified a putative compensatory link between Lpf and curli with regards to colonization and adherenceof intestinal tissue. Finally, given the wide distribution of lpf genes in pathogenic E. coli, we examined the possibility that lpfA (encoding the putative major subunit of Lpf) alleles can be incorporatedin a diagnostic strategy to differentiate STEC and EPEC strains. The goal of these studies was to examine the role of Lpf and curli using models of intestinal colonization and adherence, and develop a multiplex PCR (mPCR) approach to identify pathogenic E. coli, focusing on lpfA-encoding genes. The infant rabbit model of infection was employed to assess the importance of Lpf in intestinal colonization, while the role of Lpf in adherence was tested using cultured colonicepithelial cells. A mPCR approach was developed and optimizedusing lpfA alleles as well as other virulence-associated genes uniqueto STEC and EPEC strains. Competition experiments between wt EHEC and an isogenic lpfA1/2 double mutant revealed that the wt strain outcompeted the mutant in the ileum, cecum, and mid-colon, indicating a rolefor Lpf in intestinal colonization. However, the lpfA1/2 double mutant exhibited increased adherence to cultured colonic epithelial cells, compared to wt EHEC. Curli fiber production was observed byimmuno gold electron microscopy and western blotting in bacteria cultured with the colonic cell line. However, deletion of csgA (encoding the putative major subunit of curli) did not appear to affect intestinal colonization. Inclusion of lpfA alleles in the mPCR assay permitted differentiation of EHEC1 and EPEC2 groups, as wellas EHEC1 and EHEC2. This methodology resulted in a clinical sensitivity and specificity of 91% and 84%, respectively, when analyzing clinical strains from Argentina. Further work is needed to more fully examine the linkbetween curli and Lpf production and the specific contribution ofeach one during colonization and persistence. The use of lpfA alleles in the multiplex PCR assay allowed us to discriminate different classesof pathogenic E. coli strains, suggesting this can be used for diagnosisof STEC and EPEC infections.