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 WzySf, and its modal chain length is determined by WzzSf and WzzpHS2. Site-directed mutagenesis was performed on wzySf in pWaldo-wzySf-TEV-GFP to alter Arg residues in WzySf's two large periplasmic loops (PLs) (PL3 and PL5). Analysis of the LPS profiles conferred by mutated WzySf proteins in the wzySf deficient (Δwzy) strain identified residues that affect WzySf activity. The importance of the guanidium group of the Arg residues was investigated by altering the Arg residues to Lys and Glu, which generated WzySf mutants conferring altered LPS Oag modal chain lengths. The dependence of these WzySf mutants on WzzSf was investigated by expressing them in a wzySf and wzzSf deficient (Δwzy Δwzz) strain. Comparison of the LPS profiles identified a role for the Arg residues in the association of WzySf and WzzSf during Oag polymerization. Colicin E2 and bacteriophage Sf6c susceptibility supported this conclusion. Comparison of the expression levels of different mutant WzySf-GFPs with the wild-type WzySf-GFP showed that certain Arg residues affected production levels of WzySf in a WzzSf-dependent manner. To our knowledge, this is the first report of S. flexneri WzySf mutants having an effect on LPS Oag modal chain length, and identified functionally significant Arg residues in WzySf.