Characterisation of Shigella flexneri polysaccharide co-polymerase (PCP) protein Wzz: analysis of structure, function and protein interaction.

Abstract

In Shigella flexneri, Wzz[subscript]SF determines the lipopolysaccharides (LPS) O antigen (Oag) modal chain length of 11-17 repeat units (RUs). Wzz[subscript]SF has two transmembrane regions, the N-terminal TM1 and the C-terminal TM2, and previous studies have shown that the TM2 region is important in Wzz function. The mechanism of Oag modal chain length regulation has not been revealed. Previous studies have probed the structure-function relationship of Wzz[subscript]SF and have suggested that function is a result of the overall structure and not one particular region. Genetic evidence suggests that Wzz may form a complex with other Oag processing proteins, possibly with the Wzy polymerase. This thesis describes in-frame linker mutagenesis of Wzz[subscript]SF, and five classes of Wzz insertion (Wzz[subscript]i) mutants with 5 amino acid insertions were identified: Class I (non-functional), Class II (conferred very short (VS) Oag chain length, 2-10 RUs), Class III (8-14 RUs), Class IV (11-19 RUs), and Class V (16-25 RUs). The susceptibility of strains expressing Wzzi mutants to colicin E2 was investigated, and a correlation between random/VS LPS Oag modal chain length, and susceptibility to colicin E2 was found. Chemical cross-linking analyses were conducted to assess Wzz oligomerisation and showed that high molecular weight proteins were easily detected in wild-type Wzz[subscript]SF and mutants from Classes V, but not easily detected in Classes II and III, and only Wzz[subscript]SF, Wzz[subscript]i Class IV and Class V dimers were detected after heating to 100ºC and in the presence of SDS, suggesting wild-type/longer LPS Oag modal chain length may be dependent on dimer stability. The involvement of the TM2 region in Wzz:Wzz interactions was also investigated. S. typhimurium Wzz (Wzz[subscript]ST) confers an LPS Oag modal chain length of Long-type (L-type) 19-30 RUs, and Wzz[subscript]ST and Wzz[subscript]SF have identical residues in the conserved residues of their TM2 regions. Wzz[subscript]ST expression in Wzz deficient S. flexneri strain confers an L-type LPS Oag modal chain length, however co-expression of Wzz[subscript]ST with Wzz[subscript]SF resulted in mono-modal LPS Oag modal chain length. A previously constructed Wzz mutant which had two G305A/G311A substitutions in the TM2 region confers a VS LPS Oag modal chain length (3-8 RUs), however co-expression of Wzz[subscript]G305A/G311A with wild-type Wzz[subscript]SF resulted in LPS with a bimodal Oag chain length distribution (both wild-type 11-17 RUs, and VS-type 3-8 RUs). Strains expressing His[subscript]6-Wzz[subscript]SF and FLAG-tagged version of Wzz[subscript]ST, Wzz[subscript]SF and Wzz[subscript]G305A/G311A were used in co-purification assays. Purified His[subscript]6-Wzz[subscript]SF and had FLAGWzz[subscript]ST was in the eluted fraction, demonstrating that FLAG-Wzz[subscript]ST interacted with His[subscript]6-Wzz[subscript]SF. However when His[subscript]6-Wzz[subscript]SF was purified from a strain co-expressing FLAGWzz[subscript]G305A/G311A, very low amounts of the latter were detected in the elution fraction, indicating that His[subscript]6-Wzz[subscript]SF interacted very poorly with FLAG-Wzz[subscript]G305A/G311A. These data implicate residues G305 and G311 in Wzz:Wzz interactions. A fusion of the red fluorescent protein mCherry to Wzz[subscript]SF was performed and showed the localisation of this protein was in the periphery regions of the cell, as determined by epifluorescence microscopy. Wzy was tagged with StrepTag-II at its amino terminal end; the resulting StrepII-Wzy protein was able to complement a wzy mutation, however the smooth LPS produced lacked any Oag modal chain length control. Two GFP⁺-tagged Wzy fusion proteins were constructed, pGFP⁺-StrepII-Wzy, (with the strepII linker region between gfp+ and wzy), and pGFP⁺-Wzy (strepII excised). GFP⁺-StrepII-Wzy had near wild-type Wzy functionality, and the complemented wzy mutant exhibited the wild-type trait of resistance to the lethal action of colicin E2, but GFP⁺-StrepII-Wzy could not be detected by epifluorescence microscopy, and was poorly detectable by Western immunoblotting. GFP⁺-Wzy was partially functional and the complemented wzy mutant was susceptible to colicin E2, like the StrepII-Wzy complemented wzy mutant. GFP⁺-Wzy could be detected by both Western immunoblotting and epi-fluorescence microscopy. These results implicate the N-terminal region of Wzy in Oag modal chain length determination.