Hegde Shivanand, Hegde Shrilakshmi, Zimmermann Martina, Flöck Martina, Spergser Joachim, 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, Austria.
Clinic for Ruminants, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine, Vienna, Austria.
Infect Immun. 2015 Jul;83(7):2751-61. doi: 10.1128/IAI.00403-15. Epub 2015 Apr 27.
Mycoplasmas possess complex pathogenicity determinants that are largely unknown at the molecular level. Mycoplasma agalactiae serves as a useful model to study the molecular basis of mycoplasma pathogenicity. The generation and in vivo screening of a transposon mutant library of M. agalactiae were employed to unravel its host colonization factors. Tn4001mod mutants were sequenced using a novel sequencing method, and functionally heterogeneous pools containing 15 to 19 selected mutants were screened simultaneously through two successive cycles of sheep intramammary infections. A PCR-based negative selection method was employed to identify mutants that failed to colonize the udders and draining lymph nodes in the animals. A total of 14 different mutants found to be absent from ≥ 95% of samples were identified and subsequently verified via a second round of stringent confirmatory screening where 100% absence was considered attenuation. Using this criterion, seven mutants with insertions in genes MAG1050, MAG2540, MAG3390, uhpT, eutD, adhT, and MAG4460 were not recovered from any of the infected animals. Among the attenuated mutants, many contain disruptions in hypothetical genes, implying their previously unknown role in M. agalactiae pathogenicity. These data indicate the putative role of functionally different genes, including hypothetical ones, in the pathogenesis of M. agalactiae. Defining the precise functions of the identified genes is anticipated to increase our understanding of M. agalactiae infections and to develop successful intervention strategies against it.
支原体具有复杂的致病决定因素，在分子水平上大多未知。无乳支原体是研究支原体致病性分子基础的有用模型。利用无乳支原体转座子突变体文库的构建及体内筛选来揭示其宿主定植因子。使用一种新型测序方法对Tn4001mod突变体进行测序，并通过两个连续的绵羊乳房内感染周期同时筛选包含15至19个选定突变体的功能异质库。采用基于PCR的阴性选择方法来鉴定未能在动物乳房和引流淋巴结中定植的突变体。共鉴定出14个不同的突变体，这些突变体在≥95%的样本中均未出现，随后通过第二轮严格的验证性筛选进行验证，其中100%未出现被视为减毒。使用该标准，在MAG1050、MAG2540、MAG3390、uhpT、eutD、adhT和MAG4460基因中插入的7个突变体在任何感染动物中均未检出。在减毒突变体中，许多在假定基因中存在破坏，这意味着它们在无乳支原体致病性中具有先前未知的作用。这些数据表明功能不同的基因，包括假定基因，在无乳支原体发病机制中的推定作用。确定已鉴定基因的确切功能有望增进我们对无乳支原体感染的理解，并制定成功的干预策略。
