Mutational analysis of the major periplasmic loops of Shigella flexneri Wzy: identification of the residues affecting O antigen modal chain length control, and Wzz-dependent polymerization activity

The O antigen (Oag) component of LPS is a major Shigella flexneri virulence determinant. Oag is polymerized by Wzy(Sf), and its modal chain length is determined by Wzz(Sf) and Wzz(pHS2). Site-directed mutagenesis was performed on wzy(Sf) in pWaldo-wzysrTEV-GFP to alter Arg residues in Wzy(Sf)'s two large periplasmic loops (PLs) (PL3 and PL5). Analysis of the LPS profiles conferred by mutated Wzy(Sf) proteins in the wzy(Sf) deficient (Delta wzy) strain identified residues that affect Wzy(Sf) activity. The importance of the guanidium group of the Arg residues was investigated by altering the Arg residues to Lys and Glu, which generated Wzy(Sf) mutants conferring altered LPS Oag modal chain lengths. The dependence of these Wzy(Sf) mutants on Wzz(Sf) was investigated by expressing them in a wzy(Sf) and wzz(Sf) deficient (Delta wzy Delta wzz) strain. Comparison of the LPS profiles identified a role for the Arg residues in the association of Wzy(Sf) and Wzz(Sf) during Oag polymerization. Colicin E2 and bacteriophage Sf6c susceptibility supported this conclusion. Comparison of the expression levels of different mutant Wzy(Sf)-GFPs with the wild-type Wzy(Sf)-GFP showed that certain Arg residues affected production levels of Wzy(Sf) in a Wzz(Sf)-dependent manner. To our knowledge, this is the first report of S. flexneri Wzy(Sf) mutants having an effect on LPS Oag modal chain length, and identified functionally significant Arg residues in Wzy(Sf).