Purins Leanne, Van Den Bosch Luisa, Richardson Vanessa, Morona Renato
Australian Bacterial Pathogenesis Program, Discipline of Microbiology and Immunology, School of Molecular and Biomedical Science, University of Adelaide, Adelaide, SA 5005, Australia.
Microbiology (Reading). 2008 Apr;154(Pt 4):1104-1116. doi: 10.1099/mic.0.2007/014225-0.
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).
弗氏志贺氏菌中与脂多糖（LPS）相连的O抗原（Oag）链长度的调控对其毒力很重要，且依赖于内膜蛋白Wzz。迄今为止，由于缺乏Wzz的高分辨率结构数据，阻碍了将影响Wzz功能的突变与结构相关联并描述链长度调控机制的研究。在此，我们利用二级结构预测表明，Wzz(pHS2)蛋白的周质结构域有三个具有显著卷曲螺旋（CC）潜力的区域，其中两个位于一个延伸的螺旋区域内。我们在此描述了首个定点诱变研究，以探究各个预测的CC区域（CCRs）在Wzz功能和寡聚化中的作用。我们发现CCRs 2和3对于Wzz(pHS2)调控野生型Oag链长度是必需的。影响三个CCRs的突变体的体内交联图谱未改变，表明单个CCR对于寡聚化并非必需。有趣的是，CCR3突变导致温度敏感表型以及对Oag聚合的抑制作用。在弗氏志贺氏菌degP突变体中对Wzz(pHS2)和突变体构建体的分析表明，DegP不影响野生型Wzz(pHS2)的功能，但它的存在影响了Wzz(pHS2) CCR3突变体的表型。此外，Wzz(pHS2) CCR3突变体的表型被Wzz(pHS2)假定的胞质C末端附近的顺式突变所抑制。
