Hegde Shivanand, Rosengarten Renate, Chopra-Dewasthaly Rohini
Division of Clinical Microbiology and Infection Biology, Institute of Bacteriology, Mycology and Hygiene, Department of Pathobiology, University of Veterinary Medicine Vienna, Vienna, Austria.
PLoS One. 2015 Mar 23;10(3):e0119706. doi: 10.1371/journal.pone.0119706. eCollection 2015.
The utilization of available substrates, the metabolic potential and the growth rates of bacteria can play significant roles in their pathogenicity. This study concentrates on Mycoplasma agalactiae, which causes significant economic losses through its contribution to contagious agalactia in small ruminants by as yet unknown mechanisms. This lack of knowledge is primarily due to its fastidious growth requirements and the scarcity of genetic tools available for its manipulation and analysis. Transposon mutagenesis of M. agalactiae type strain PG2 resulted in several disruptions throughout the genome. A mutant defective in growth in vitro was found to have a transposon insertion in the pdhB gene, which encodes a component of the pyruvate dehydrogenase complex. This growth difference was quite significant during the actively dividing logarithmic phase but a gradual recovery was observed as the cells approached stationary phase. The mutant also exhibited a different and smaller colony morphology compared to the wild type strain PG2. For complementation, pdhAB was cloned downstream of a strong vpma promoter and upstream of a lacZ reporter gene in a newly constructed complementation vector. When transformed with this vector the pdhB mutant recovered its normal growth and colony morphology. Interestingly, the pdhB mutant also had significantly reduced invasiveness in HeLa cells, as revealed by double immunofluorescence staining. This deficiency was recovered in the complemented strain, which had invasiveness comparable to that of PG2. Taken together, these data indicate that pyruvate dehydrogenase might be an important player in infection with and colonization by M. agalactiae.
可用底物的利用、细菌的代谢潜力和生长速率在其致病性中可能发挥重要作用。本研究聚焦于无乳支原体，它通过尚不明确的机制导致小反刍动物发生传染性无乳症，从而造成重大经济损失。这种认知上的不足主要归因于其苛刻的生长要求以及可用于其操作和分析的遗传工具的匮乏。对无乳支原体PG2型菌株进行转座子诱变导致整个基因组出现多处破坏。发现一株在体外生长有缺陷的突变体在pdhB基因中有转座子插入，该基因编码丙酮酸脱氢酶复合体的一个组分。这种生长差异在活跃分裂的对数期非常显著，但随着细胞接近稳定期，观察到其逐渐恢复。与野生型菌株PG2相比，该突变体还表现出不同且更小的菌落形态。为了进行互补，将pdhAB克隆到新构建的互补载体中一个强vpma启动子的下游和lacZ报告基因的上游。用该载体转化时，pdhB突变体恢复了其正常生长和菌落形态。有趣的是，双重免疫荧光染色显示，pdhB突变体在HeLa细胞中的侵袭性也显著降低。在互补菌株中这种缺陷得以恢复，其侵袭性与PG2相当。综上所述，这些数据表明丙酮酸脱氢酶可能在无乳支原体感染和定殖过程中发挥重要作用。
