Increased biofilm staining after deletion of sepA in enteroaggregative E. coli strains is not due to the lack of SepA

Abstract

Background Enteroaggregative E. coli (EAEC) is associated with acute and chronic diarrhea worldwide. EAEC is thought to form thick biofilms on the intestinal mucosa. Epidemiological data suggest that SepA, a serine protease autotransporter of Enterobacteriaceae (SPATE), is important for EAEC disease. The genes for SepA, and some proteins that are important for EAEC biofilm formation and aggregative adherence, are encoded by the pAA plasmid. Methods We deleted sepA 49-4043 from six EAEC clinical isolates and inserted a chloramphenicol resistance gene (cat) in place of the sepA coding sequence. In vitro biofilm formation was assessed after growth in 96-well plates. Complementation studies were conducted with genetic and protein-based techniques. We moved the pAA plasmid from EAEC strains into a commensal E. coli. Finally, we sequenced the pAA of several strains. Results All of the wild-type EAEC strains secreted similar amounts of SepA as assessed by Western blot analysis. Four of the six mutant EAEC strains exhibited increased biofilm staining when sepA was deleted. Additionally, we found that introduction of pAAΔsepA K261 into a nalidixic acid resistant commensal E. coli strain, (HSNalR) resulted in significantly increased biofilm staining relative to HSNalR (pAAK261). Therefore, pAAΔsepA K261 alone was sufficient to confer the elevated biofilm phenotype onto HSNalR. However, introduction of sepA via a plasmid or on the pAA did not restore biofilm staining to wild-type levels in the ΔsepA mutant strains that showed elevated biofilm staining. Furthermore, the addition of exogenous SepA to the biofilm in vitro did not reduce biofilm staining. Conclusions Taken together, our results suggest that deletion of sepA causes elevated biofilm formation in some EAEC strains, but that the increased biofilm staining is not directly due to the loss of SepA.