Roy F, Vanterpool E, Fletcher H M
Department of Biochemistry and Microbiology, School of Medicine, Loma Linda University, Loma Linda, CA 92350, USA.
Microbiology (Reading). 2006 Nov;152(Pt 11):3391-3398. doi: 10.1099/mic.0.29147-0.
In several micro-organisms, HtrA, a serine periplasmic protease, is considered an important virulence factor that plays a regulatory role in oxidative and temperature stress. The authors have previously shown that the vimA gene product is an important virulence regulator in Porphyromonas gingivalis. Further, purified recombinant VimA physically interacted with the major gingipains and the HtrA from P. gingivalis. To further evaluate a role for HtrA in the pathogenicity of this organism, a 1.5 kb fragment containing the htrA gene was PCR-amplified from the chromosomal DNA of P. gingivalis W83. This gene was insertionally inactivated using the ermF-ermAM antibiotic-resistance cassette and used to create an htrA-deficient mutant by allelic exchange. In one randomly chosen isogenic mutant designated P. gingivalis FLL203, there was increased sensitivity to hydrogen peroxide. Growth of this mutant at an elevated temperature was more inhibited compared to the wild-type. Further, in contrast to the wild-type, there was a significant decrease in Arg-gingipain activity after heat shock in FLL203. However, the gingipain activity in the mutant returned to normal levels after a further 30 min incubation at room temperature. Collectively, these data suggest that HtrA may play a similar role in oxidative and temperature stress in P. gingivalis as observed in other organisms.
在几种微生物中,HtrA(一种丝氨酸周质蛋白酶)被认为是一种重要的毒力因子,在氧化应激和温度应激中发挥调节作用。作者之前已经表明,牙龈卟啉单胞菌中的vimA基因产物是一种重要的毒力调节因子。此外,纯化的重组VimA与牙龈卟啉单胞菌的主要牙龈蛋白酶和HtrA发生物理相互作用。为了进一步评估HtrA在该生物体致病性中的作用,从牙龈卟啉单胞菌W83的染色体DNA中通过PCR扩增出一个包含htrA基因的1.5 kb片段。使用ermF-ermAM抗生素抗性盒对该基因进行插入失活,并通过等位基因交换创建一个htrA缺陷突变体。在一个随机选择的同基因突变体牙龈卟啉单胞菌FLL203中,对过氧化氢的敏感性增加。与野生型相比,该突变体在高温下的生长受到更明显的抑制。此外,与野生型相比,FLL203在热休克后Arg-牙龈蛋白酶活性显著降低。然而,在室温下再孵育30分钟后,突变体中的牙龈蛋白酶活性恢复到正常水平。总体而言,这些数据表明,HtrA在牙龈卟啉单胞菌的氧化应激和温度应激中可能发挥与其他生物体中类似的作用。