Philips Institute of Oral and Craniofacial Molecular Biology, Department of Microbiology and Immunology, Virginia Commonwealth University, Richmond, Virginia, USA.
Infect Immun. 2012 Sep;80(9):3319-31. doi: 10.1128/IAI.00561-12. Epub 2012 Jul 9.
Although the Gram-negative, anaerobic periodontopathogen Porphyromonas gingivalis must withstand nitrosative stress, which is particularly high in the oral cavity, the mechanisms allowing for protection against such stress are not known in this organism. In this study, microarray analysis of P. gingivalis transcriptional response to nitrite and nitric oxide showed drastic upregulation of the PG0893 gene coding for hybrid cluster protein (Hcp), which is a putative hydroxylamine reductase. Although regulation of hcp has been shown to be OxyR dependent in Escherichia coli, here we show that in P. gingivalis its expression is dependent on the Fnr-like regulator designated HcpR. Growth of the isogenic mutant V2807, containing an ermF-ermAM insertion within the hcpR (PG1053) gene, was significantly reduced in the presence of nitrite (P < 0.002) and nitric oxide-generating nitrosoglutathione (GSNO) (P < 0.001), compared to that of the wild-type W83 strain. Furthermore, the upregulation of PG0893 (hcp) was abrogated in V2807 exposed to nitrosative stress. In addition, recombinant HcpR bound DNA containing the hcp promoter sequence, and the binding was hemin dependent. Finally, V2807 was not able to survive with host cells, demonstrating that HcpR plays an important role in P. gingivalis virulence. This work gives insight into the molecular mechanisms of protection against nitrosative stress in P. gingivalis and shows that the regulatory mechanisms differ from those in E. coli.
虽然革兰氏阴性、厌氧牙周病原体牙龈卟啉单胞菌必须承受口腔中特别高的硝化应激，但该生物体中允许其免受此类应激的机制尚不清楚。在这项研究中，牙龈卟啉单胞菌对亚硝酸盐和一氧化氮的转录反应的微阵列分析显示，编码混合簇蛋白（Hcp）的 PG0893 基因的表达急剧上调，Hcp 是一种假定的羟胺还原酶。尽管已经表明大肠杆菌中的 hcp 调节依赖于 OxyR，但在这里我们表明，在牙龈卟啉单胞菌中，其表达依赖于指定为 HcpR 的 Fnr 样调节剂。含有 ermF-ermAM 插入物的同源突变体 V2807 在 hcpR（PG1053）基因内的生长在亚硝酸盐（P <0.002）和产生亚硝酰谷胱甘肽（GSNO）的情况下（P <0.001）与野生型 W83 菌株相比，明显减少。此外，在暴露于硝化应激下的 V2807 中，PG0893（hcp）的上调被阻断。此外，重组 HcpR 结合含有 hcp 启动子序列的 DNA，并且结合依赖于血红素。最后，V2807 无法在宿主细胞中存活，这表明 HcpR 在牙龈卟啉单胞菌的毒力中起着重要作用。这项工作深入了解了牙龈卟啉单胞菌对抗硝化应激的分子机制，并表明调节机制与大肠杆菌中的不同。
