ORCID Profile
0000-0002-5441-6285
Current Organisation
University of Tasmania
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Publisher: American Society for Microbiology
Date: 2015
DOI: 10.1128/JB.01885-14
Abstract: The O-antigen (Oag) component of lipopolysaccharide (LPS) is a major virulence determinant of Shigella flexneri and is synthesized by the O-antigen polymerase, Wzy Sf . Oag chain length is regulated by chromosomally encoded Wzz Sf and pHS-2 plasmid-encoded Wzz pHS2 . To identify functionally important amino acid residues in Wzy Sf , random mutagenesis was performed on the wzy Sf gene in a pWaldo-TEV-GFP plasmid, followed by screening with colicin E2. Analysis of the LPS conferred by mutated Wzy Sf proteins in the wzy Sf -deficient (Δ wzy ) strain identified 4 different mutant classes, with mutations found in periplasmic loop 1 (PL1), PL2, PL3, and PL6, transmembrane region 2 (TM2), TM4, TM5, TM7, TM8, and TM9, and cytoplasmic loop 1 (CL1) and CL5. The association of Wzy Sf and Wzz Sf was investigated by transforming these mutated wzy Sf plasmids into a wzy Sf - and wzz Sf -deficient (Δ wzy Δwzz ) strain. Comparison of the LPS profiles in the Δ wzy and Δ wzy Δwzz backgrounds identified Wzy Sf mutants whose polymerization activities were Wzz Sf dependent. Colicin E2 and bacteriophage Sf6c sensitivities were consistent with the LPS profiles. Analysis of the expression levels of the Wzy Sf -GFP mutants in the Δ wzy and Δ wzy Δwzz backgrounds identified a role for Wzz Sf in Wzy Sf stability. Hence, in addition to its role in regulating Oag modal chain length, Wzz Sf also affects Wzy Sf activity and stability.
Publisher: Microbiology Society
Date: 09-2015
DOI: 10.1099/MIC.0.000132
Abstract: The O antigen (Oag) component of Shigella flexneri lipopolysaccharide (LPS) is important for virulence and a protective antigen. It is synthesized by the Wzy-dependent mechanism. S. flexneri Wzy has 12 transmembrane segments and two large periplasmic loops. The modal chain length of the Oag is determined by Wzz. Experimental evidence supports multi-protein interactions in the Wzy-dependent pathway. However, evidence for direct interaction of Wzy with the other proteins of the Wzy-dependent pathway is limited. Initially, we purified Wzy-GFP-His8 and detected the presence of a dimeric form. In vivo cross-linking was then performed in an S. flexneri wzy mutant strain carrying plasmids encoding Wzy-GFP-His8 and untagged Wzz. Following solubilization with n-dodecyl-β-D-maltopyranoside (DDM) and affinity purification of Wzy-GFP-His8, Western immunoblotting with Wzz antibody detected co-purification of Wzz this was supported by MS analysis. To the best of our knowledge, this is the first reported isolation of a complex between Wzy and Wzz. Wzy mutants (WzyR164A, WzyV92M, WzyY137H, and WzyR250K) whose properties are affected by Wzz were able to form complexes with Wzz. Their mutational alterations did not affect the interaction of Wzy with Wzz. Thus, the interaction may involve many regions of Wzy.
Publisher: Microbiology Society
Date: 04-2015
DOI: 10.1099/MIC.0.000042
Abstract: 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.
No related grants have been discovered for Pratiti Nath.