Molecular characterisation of Shigella flexneri outer membrane protease IcsP.

Abstract

Shigella is a genus of Gram-negative bacteria responsible for bacillary dysentery in humans. Shigella flexneri type 2a in particular is responsible for the majority of incidents in developing countries. The S. flexneri protease IcsP, is a member of the Omptin family of outer membrane (OM) proteases which cleaves IcsA, a polarly localised OM protein required for Shigella virulence. Mutations in icsP have been shown to effect the observed distribution of IcsA, however the significance of IcsP in Shigella virulence is incompletely understood. In this study, aspects of IcsP biology were investigated. S. flexneri 2457T and M90T icsP mutants were constructed to investigate the role of IcsP in Shigella intercellular spread, and it was found that icsP in both S. flexneri backgrounds did not appear to be essential for cell-tocell spread in human cervical cancer HeLa cells, but enhanced cell-to-cell spread in monkey kidney CV-1 cells (as determined by plaque assays). Complementation with icsP returned the mutant phenotype to wild-type. The results suggest IcsP does play a role in Shigella intercellular spread. The 2457T icsP mutant was subsequently complemented with an altered icsP gene encoding a haemagglutinin epitope tagged IcsP (IcsPHA) to determine the distribution of IcsP on the cell surface. In both S. flexneri and E. coli K-12 possessing smooth and rough lipopolysaccharide (LPS), the distribution of IcsPHA was found to be punctate across the cell surface. Deconvolution analysis revealed that IcsP distribution was punctate and banded in both LPS backgrounds. A smooth LPS E. coli K-12 yfdI mutant strain expressing IcsPHA was also constructed, and experiments involving treatment of this strain with bacteriophage Sf6 tail spike protein suggested that LPS O antigen chains masked IcsP in smooth LPS strains. During these studies, double-labelling of IcsPHA and LPS in a S. flexneri 5a M90T strain revealed a helical distribution of LPS in this strain. Overall, the results suggest IcsP has a punctate, banded distribution across the cell surface. The effect of virK and rmlD mutations on IcsP was then investigated by constructing a virK, rmlD and virK/rmlD double mutant in S. flexneri 2457T. Western immunoblotting showed no change in IcsP expression levels in either the virK, rmlD or virK/rmlD mutants compared to wild-type. Surprisingly, the virK mutant showed no change in IcsA expression levels by Western immunoblotting and plaque assays (using HeLa and CV-1 cells) suggested that virK was not essential for Shigella intercellular spread (contradicting the published data on this gene). No effect was also observed on IcsP expression level or on IcsP’s ability to cleave IcsA into culture supernatants. Finally alternative substrates for the protease activity of IcsP were investigated against known Omptin substrates (plasminogen, α2-antiplasmin, complement, protamine and colicins). However, IcsP appeared to have no effect on these substrates as determined by proteolytic cleavage assays and antimicrobial assay. Interestingly, Plg cleavage by rough LPS S. flexneri, and α2AP cleavage by both smooth and rough LPS S. flexneri, was observed.