Quach Darin, van Sorge Nina M, Kristian Sascha A, Bryan Joshua D, Shelver Daniel W, Doran Kelly S
Department of Biology and Center for Microbial Sciences, San Diego State University, CA 92182, USA.
J Bacteriol. 2009 Apr;191(7):2023-32. doi: 10.1128/JB.01216-08. Epub 2008 Dec 29.
Group B Streptococcus (GBS) is major cause of invasive disease in newborn infants and the leading cause of neonatal meningitis. To gain access to the central nervous system (CNS), GBS must not only subvert host defenses in the bloodstream but also invade and survive within brain microvascular endothelial cells (BMEC), the principal cell layer composing the blood-brain barrier (BBB). While several GBS determinants that contribute to the invasion of BMEC have been identified, little is known about the GBS factors that are required for intracellular survival and ultimate disease progression. In this study we sought to identify these factors by screening a random GBS mutant library in an in vitro survival assay. One mutant was identified which contained a disruption in a two-component regulatory system homologous to CiaR/CiaH, which is present in other streptococcal pathogens. Deletion of the putative response regulator, ciaR, in GBS resulted in a significant decrease in intracellular survival within neutrophils, murine macrophages, and human BMEC, which was linked to increased susceptibility to killing by antimicrobial peptides, lysozyme, and reactive oxygen species. Furthermore, competition experiments with mice showed that wild-type GBS had a significant survival advantage over the GBS DeltaciaR mutant in the bloodstream and brain. Microarray analysis comparing gene expression between wild-type and DeltaciaR mutant GBS bacteria revealed several CiaR-regulated genes that may contribute to stress tolerance and the subversion of host defenses by GBS. Our results identify the GBS CiaR response regulator as a crucial factor in GBS intracellular survival and invasive disease pathogenesis.
B族链球菌（GBS）是新生儿侵袭性疾病的主要病因，也是新生儿脑膜炎的首要病因。为了进入中枢神经系统（CNS），GBS不仅必须破坏血液中的宿主防御机制，还必须侵入脑微血管内皮细胞（BMEC）并在其中存活，BMEC是构成血脑屏障（BBB）的主要细胞层。虽然已经确定了几种有助于GBS侵入BMEC的决定因素，但对于GBS在细胞内存活和最终疾病进展所需的因素却知之甚少。在本研究中，我们试图通过在体外存活试验中筛选随机GBS突变体文库来鉴定这些因素。我们鉴定出一个突变体，其在与其他链球菌病原体中存在的CiaR/CiaH同源的双组分调节系统中存在破坏。在GBS中缺失假定的反应调节因子ciaR，导致其在中性粒细胞、小鼠巨噬细胞和人BMEC内的细胞内存活率显著降低，这与对抗菌肽、溶菌酶和活性氧的杀伤敏感性增加有关。此外，与小鼠的竞争实验表明，野生型GBS在血液和大脑中比GBS DeltaciaR突变体具有显著的生存优势。比较野生型和DeltaciaR突变体GBS细菌基因表达的微阵列分析揭示了几个可能有助于GBS应激耐受和破坏宿主防御的CiaR调节基因。我们的结果确定GBS CiaR反应调节因子是GBS细胞内存活和侵袭性疾病发病机制中的关键因素。
