Identification of virulence determinants for endocarditis in Streptococcus sanguinis by signature-tagged mutagenesis
Identification of virulence determinants for endocarditis in Streptococcus sanguinis by signature-tagged mutagenesis
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DOI：
10.1128/iai.73.9.6064-6074.2005
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发表时间：
2005-09-01
影响因子：
3.1
通讯作者：
Kitten, T
中科院分区：
文献类型：
--
作者：
Paik, S;Senty, L;Kitten, T
血链球菌是一种革兰氏阳性兼性厌氧菌，是人类口腔的正常居民。它也是感染性心内膜炎（一种严重的血管内感染）最常见的病原体之一。为了鉴定感染性心内膜炎的毒力因子，对SK 36株S. sanguinis，其基因组正在测序中。STM允许大规模的创建，在体内筛选，并恢复一系列的突变体与改变的毒力。STM筛选800个突变体，鉴定出38个推定无毒突变体和5个推定超毒突变体。随后对这些突变体的一个子集进行分子分析，鉴定了编码十一烯醇激酶、高丝氨酸激酶、厌氧核糖核苷酸还原酶、腺苷酸琥珀酸裂解酶和一种假设蛋白质的基因。通过竞争指数测定证实了毒力降低2- 150倍。鉴定了一种在基因间区域插入转座子的pupectin高毒力菌株，但在竞争指数测定中未证实毒力增加。除了高丝氨酸激酶突变体外，所有突变体在需氧肉汤培养中均生长至SK 36。该突变体的生长通过向培养基中添加苏氨酸而恢复。在厌氧核糖核苷酸还原酶基因中含有插入或框内缺失的突变体在严格厌氧条件下不能生长。结果表明，细胞壁合成、氨基酸和核酸合成以及在厌氧条件下的生存能力等管家功能是S.血液性心内膜炎
Streptococcus sanguinis is a gram-positive, facultative anaerobe and a normal inhabitant of the human oral cavity. It is also one of the most common agents of infective endocarditis, a serious endovascular infection. To identify virulence factors for infective endocarditis, signature-tagged mutagenesis (STM) was applied to the SK36 strain of S. sanguinis, whose genome is being sequenced. STM allows the large-scale creation, in vivo screening, and recovery of a series of mutants with altered virulence. Screening of 800 mutants by STM identified 38 putative avirulent and 5 putative hypervirulent mutants. Subsequent molecular analysis of a subset of these mutants identified genes encoding undecaprenol kinase, homoserine kinase, anaerobic ribonucleotide reductase, adenylosuccinate lyase, and a hypothetical protein. Virulence reductions ranging from 2-to 150-fold were confirmed by competitive index assays. One putatively hypervirulent strain with a transposon insertion in an intergenic region was identified, though increased virulence was not confirmed in competitive index assays. All mutants grew comparably to SK36 in aerobic broth culture except for the homoserine kinase mutant. Growth of this mutant was restored by the addition of threonine to the medium. Mutants containing an insertion or in-frame deletion in the anaerobic ribonucleotide reductase gene failed to grow under strictly anaerobic conditions. The results suggest that housekeeping functions such as cell wall synthesis, amino acid and nucleic acid synthesis, and the ability to survive under anaerobic conditions are important virulence factors in S. sanguinis endocarditis.