Molecular analysis of the psa permease complex of Streptococcus pneumoniae
Molecular analysis of the psa permease complex of Streptococcus pneumoniae
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DOI：
10.1111/j.1365-2958.2004.04164.x
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发表时间：
2004-08-01
影响因子：
3.6
通讯作者：
Paton, JC
中科院分区：
文献类型：
--
作者：
McAllister, LJ;Tseng, HJ;Paton, JC
肺炎链球菌的psaBCA基因座编码推定的ABC Mn 2 +-通透酶复合物。操纵子的下游是psaD，它可以共转录并编码巯基过氧化物酶。以前，关于psa基因座突变的表型影响一直存在不一致，突变体结构的差异和极性效应的可能性使其解决复杂化。在这里，我们构建了未标记的，在框内缺失突变体DeltapsaB，DeltapsaC，DeltapsaA，DeltapsaD，DeltapsaBC，DeltapsaBCA和DeltapsaBCAD在S。pneumoniae D39的基因座中的每一个基因的作用，在Mn 2+摄取，氧化应激的易感性，毒力，鼻咽定植和链形态。还研究了所有突变体的生长和转化对Mn 2+的需求。电感耦合等离子体质谱（ICP-MS）分析提供了第一个直接证据，证明PsaBCA确实是一种Mn 2+转运蛋白。然而，这项研究并没有证实以前的报告，该位点发挥作用，胆碱结合蛋白的生产或链形态。我们还证实了Psa通透酶在系统毒力和对超氧化物和过氧化氢的抗性中的重要性，并首次证明了其在鼻咽定植中的作用。进一步的证据支持Mn ~（2+）补充对DeltapsaB、DeltapsaC、DeltapsaA、DeltapsaBC、DeltapsaBCA和DeltapsaBCAD突变体生长和转化的需要。然而，DeltapsaD突变体的转化以及生长并不依赖于Mn 2+的补充。我们还表明，除了对过氧化氢的敏感性，DeltapsaD突变体表现出与野生型基本相似的表型。用PsaD抗血清进行的Western印迹分析表明，删除PsaD上游的任何基因都不影响其表达。然而，我们发现，删除psaB导致PsaA相对于D39中的表达降低，而删除psaB和psaC两者导致至少野生型水平的PsaA。
The psaBCA locus of Streptococcus pneumoniae encodes a putative ABC Mn2+-permease complex. Downstream of the operon is psaD, which may be co-transcribed and encodes a thiol peroxidase. Previously, there has been discordance concerning the phenotypic impact of mutations in the psa locus, resolution of which has been complicated by differences in mutant construction and the possibility of polar effects. Here, we constructed unmarked, in frame deletion mutants DeltapsaB, DeltapsaC, DeltapsaA, DeltapsaD, DeltapsaBC, DeltapsaBCA and DeltapsaBCAD in S. pneumoniae D39 to examine the role of each gene within the locus in Mn2+ uptake, susceptibility to oxidative stress, virulence, nasopharyngeal colonization and chain morphology. The requirement for Mn2+ for growth and transformation was also investigated for all mutants. Inductively coupled plasma mass spectrometry (ICP-MS) analysis provided the first direct evidence that PsaBCA is indeed a Mn2+ transporter. However, this study did not substantiate previous reports that the locus plays a role in choline-binding protein pro-duction or chain morphology. We also confirmed the importance of the Psa permease in systemic virulence and resistance to superoxide and hydrogen peroxide, as well as demonstrating a role in nasopharyngeal colonization for the first time. Further evi-dence is provided to support the requirement for Mn2+ supplementation for growth and transformation of DeltapsaB, DeltapsaC, DeltapsaA, DeltapsaBC, DeltapsaBCA and DeltapsaBCAD mutants. However, transformation, as well as growth, of the DeltapsaD mutant was not dependent upon Mn2+ supplementation. We also show that, apart from sensitivity to hydrogen peroxide, the DeltapsaD mutant exhibited essentially similar phenotypes to those of the wild type. Western blot analysis with a PsaD antiserum showed that deleting any of the genes upstream of psaD did not affect its expression. However, we found that deleting psaB resulted in decreased expression of PsaA relative to that in D39, whereas deleting both psaB and psaC resulted in at least wild-type levels of PsaA.