Coiled-coil regions play a role in the function of the Shigella flexneri O-antigen chain length regulator WzzpHS2.
Regulation of the length of the O-antigen (Oag) chain attached to LPS in Shigella flexneri is important for virulence and is dependent on the inner-membrane protein Wzz. A lack of high-resolution structural data for Wzz has hampered efforts so far to correlate mutations affecting function of Wzz with structure and describe a mechanism for chain length regulation. Here we have used secondary structure prediction to show that the periplasmic domain of the Wzz(pHS2) protein has three regions of significant coiled-coil (CC) potential, two of which lie within an extended helical region. We describe here the first site-directed mutagenesis study to investigate the role of individual predicted CC regions (CCRs) in Wzz function and oligomerization. We found that CCRs 2 and 3 are necessary for wild-type Oag chain length regulation by Wzz(pHS2). The in vivo cross-linking profile of mutants affected in the three CCRs was not altered, indicating that individually each CCR is not required for oligomerization. Interestingly, the CCR3 mutation resulted in a temperature-sensitive phenotype and an inhibitory effect on Oag polymerization. Analysis of Wzz(pHS2) and the mutant constructs in a S. flexneri degP mutant showed that DegP did not affect the function of wild-type Wzz(pHS2) but its presence influenced the phenotype of the Wzz(pHS2) CCR3 mutant. Additionally, the phenotype of the Wzz(pHS2) CCR3 mutant was suppressed by a cis mutation near the putative cytoplasmic C-terminus of Wzz(pHS2).
Purins
Leanne
L
Australian Bacterial Pathogenesis Program, Discipline of Microbiology and Immunology, School of Molecular and Biomedical Science, University of Adelaide, Adelaide, SA 5005, Australia.
Van Den Bosch
Luisa
L
Australian Bacterial Pathogenesis Program, Discipline of Microbiology and Immunology, School of Molecular and Biomedical Science, University of Adelaide, Adelaide, SA 5005, Australia.
Richardson
Vanessa
V
Australian Bacterial Pathogenesis Program, Discipline of Microbiology and Immunology, School of Molecular and Biomedical Science, University of Adelaide, Adelaide, SA 5005, Australia.
Morona
Renato
R
Australian Bacterial Pathogenesis Program, Discipline of Microbiology and Immunology, School of Molecular and Biomedical Science, University of Adelaide, Adelaide, SA 5005, Australia.
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D001483:Base Sequence; D017353:Gene Deletion; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D006358:Hot Temperature; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019081:O Antigens / Q000378:metabolism*; D033903:Periplasmic Proteins / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism; D012763:Shigella flexneri / Q000276:immunology*; D013489:Suppression, Genetic
D001426:Bacterial Proteins; D006360:Heat-Shock Proteins; D019081:O Antigens; D033903:Periplasmic Proteins; C075969:rol protein, Bacteria; C061682:DegP protease; D012697:Serine Endopeptidases