Griffith Institute for Drug Discovery, Griffith University, Nathan, Queensland, Australia.
Vice-Chancellor's Unit, University of Wollongong, Wollongong, New South Wales, Australia.
PLoS One. 2020 Nov 20;15(11):e0241306. doi: 10.1371/journal.pone.0241306. eCollection 2020.
Identification of bacterial virulence factors is critical for understanding disease pathogenesis, drug discovery and vaccine development. In this study we used two approaches to predict virulence factors of Burkholderia pseudomallei, the Gram-negative bacterium that causes melioidosis. B. pseudomallei is naturally antibiotic resistant and there are no clinically available melioidosis vaccines. To identify B. pseudomallei protein targets for drug discovery and vaccine development, we chose to search for substrates of the B. pseudomallei periplasmic disulfide bond forming protein A (DsbA). DsbA introduces disulfide bonds into extra-cytoplasmic proteins and is essential for virulence in many Gram-negative organism, including B. pseudomallei. The first approach to identify B. pseudomallei DsbA virulence factor substrates was a large-scale genomic analysis of 511 unique B. pseudomallei disease-associated strains. This yielded 4,496 core gene products, of which we hypothesise 263 are DsbA substrates. Manual curation and database screening of the 263 mature proteins yielded 81 associated with disease pathogenesis or virulence. These were screened for structural homologues to predict potential B-cell epitopes. In the second approach, we searched the B. pseudomallei genome for homologues of the more than 90 known DsbA substrates in other bacteria. Using this approach, we identified 15 putative B. pseudomallei DsbA virulence factor substrates, with two of these previously identified in the genomic approach, bringing the total number of putative DsbA virulence factor substrates to 94. The two putative B. pseudomallei virulence factors identified by both methods are homologues of PenI family β-lactamase and a molecular chaperone. These two proteins could serve as high priority targets for future B. pseudomallei virulence factor characterization.
鉴定细菌毒力因子对于了解疾病发病机制、药物发现和疫苗开发至关重要。在这项研究中，我们使用了两种方法来预测伯克霍尔德氏菌的毒力因子，这种革兰氏阴性菌会导致类鼻疽病。伯克霍尔德氏菌天然具有抗药性，目前也没有临床可用的类鼻疽病疫苗。为了确定伯克霍尔德氏菌蛋白作为药物发现和疫苗开发的靶点，我们选择寻找伯克霍尔德氏菌周质中二硫键形成蛋白 A（DsbA）的底物。DsbA 将二硫键引入细胞外蛋白中，对于许多革兰氏阴性菌（包括伯克霍尔德氏菌）的毒力至关重要。鉴定伯克霍尔德氏菌 DsbA 毒力因子底物的第一种方法是对 511 株独特的与疾病相关的伯克霍尔德氏菌菌株进行大规模基因组分析。这产生了 4496 个核心基因产物，我们假设其中 263 个是 DsbA 的底物。对 263 个成熟蛋白进行手动编辑和数据库筛选，得到了 81 个与疾病发病机制或毒力相关的蛋白。对这些蛋白进行结构同源性筛选，以预测潜在的 B 细胞表位。在第二种方法中，我们在伯克霍尔德氏菌基因组中搜索其他细菌中超过 90 种已知的 DsbA 底物的同源物。使用这种方法，我们鉴定了 15 种可能的伯克霍尔德氏菌 DsbA 毒力因子底物，其中两种在基因组方法中已经被鉴定出来，这使得潜在的 DsbA 毒力因子底物总数达到 94 种。这两种通过两种方法鉴定的可能的伯克霍尔德氏菌毒力因子是 PenI 家族β-内酰胺酶和分子伴侣的同源物。这两种蛋白可以作为未来伯克霍尔德氏菌毒力因子特征分析的高优先级目标。
