Abstract
Shigella flexneri can synthesize polysaccharide chains having complex sugars and a regulated number of repeating units. S. flexneri lipopolysaccharide O antigen (Oag) is synthesized by the Wzy-dependent pathway, which is the most common pathway used in bacteria for polysaccharide synthesis. The inner membrane protein WzyB polymerizes the Oag repeat units into chains, while the polysaccharide copolymerases WzzB and WzzpHS2 determine the average number of repeat units or "the modal length," termed short type and very long type. Our data show for the first time a direct interaction between WzyB and WzzpHS2, with and without the use of the chemical cross-linker dithiobis (succinimidyl propionate) (DSP). Additionally, mutations generated via random and site-directed mutagenesis identify a region of WzyB that caused diminished function and significantly decreased very long Oag chain polymerization, and that affected the aforementioned interaction. These results provide insight into the mechanisms underlying the regulation of Oag biosynthesis. IMPORTANCE Complex polysaccharide chains are synthesized by bacteria, usually at a regulated number of repeating units, which has broad implications for bacterial pathogenesis. One example is the O antigen (Oag) component of lipopolysaccharide that is predominantly synthesized by the Wzy-dependent pathway. Our findings show for the first time a direct physical interaction between WzyB and WzzpHS2. Additionally, a set of Wzy mutant constructs were generated, revealing a proposed active site/switch region involved in the activity of WzyB and the physical interaction with WzzpHS2. Combined, these findings further understanding of the Wzy-dependent pathway. The identification of a novel interaction with the polysaccharide copolymerase WzzpHS2 and the region of WzyB that is involved in this aforementioned interaction and its impact on WzyB Oag synthesis activity have significant implication for the prevention/treatment of bacterial diseases and discovery of novel biotechnologies.