The Expression of the fim Operon Is Crucial for the Survival of Streptococcus parasanguinis FW213 within Macrophages but Not Acid Tolerance

The acquisition of transition metal ions is essential for the viability and in some cases the expression of virulence genes in bacteria. The fimCBA operon of Streptococcus parasanguinis FW213 encodes a Mn2+/Fe2+-specific ATP-binding cassette transporter. FimA, a lipoprotein in the system, is essential for the development of endocarditis, presumably by binding to fibrin monolayers on the damaged heart tissue. Recent sequence analysis revealed that Spaf_0344 was homologous to Streptococcus gordonii scaR, encoding a metalloregulatory protein for the Sca Mn2+-specific transporter. Based on the homology, Spaf_0344 was designated fimR. By using various fim promoter (pfim) derivatives fused with a promoterless chloramphenicol acetyltransferase gene, the functions of the cis-elements of pfim were analyzed in the wild-type and fimR-deficient hosts. The result indicated that FimR represses the expression of pfim and the palindromic sequences 5′ to fimC are involved in repression of pfim. A direct interaction between FimR and the palindromic sequences was further confirmed by in vitro electrophoresis gel mobility shift assay and in vivo chromatin immunoprecipitation assay (ChIP)-quantitative real-time PCR (qPCR). The result of the ChIP-qPCR analysis also indicated that FimR is activated by Mn2+ and, to a lesser degree, Fe2+. Functional analysis indicated that the expression of FimA in S. parasanguinis was critical for wild-type levels of survival against oxidative stress and within phagocytes, but not for acid tolerance. Taken together, in addition to acting as an adhesin (FimA), the expression of the fim operon is critical for the pathogenic capacity of S. parasanguinis.过渡金属离子的获取对于细菌的生存至关重要，在部分场景下还对细菌毒力基因的表达具有关键调控作用。副血链球菌（Streptococcus parasanguinis）FW213的fimCBA操纵子编码一种Mn²+/Fe²+特异性ATP结合盒（ATP-binding cassette）转运蛋白。该系统中的脂蛋白FimA是心内膜炎发生的必需因子，推测其通过结合受损心脏组织表面的纤维蛋白单层来介导致病过程。近期的序列分析显示，Spaf_0344与戈登链球菌（Streptococcus gordonii）的scaR基因同源，后者编码一种针对Sca型Mn²+特异性转运蛋白的金属调控蛋白。基于该同源性，Spaf_0344被命名为fimR。研究人员将一系列fim启动子（pfim）衍生物与无启动子氯霉素乙酰转移酶基因进行融合，以此在野生型菌株与fimR缺陷宿主中分析pfim的顺式作用元件功能。结果表明，FimR可抑制pfim的转录，且fimC基因上游的回文序列参与了pfim的转录抑制过程。通过体外电泳迁移率变动分析（electrophoresis gel mobility shift assay）与体内染色质免疫沉淀（chromatin immunoprecipitation, ChIP）-实时定量PCR（quantitative real-time PCR, qPCR）实验，研究团队进一步证实了FimR与该回文序列之间存在直接相互作用。ChIP-qPCR分析结果还显示，FimR可被Mn²+激活，而Fe²+对其的激活效应相对较弱。功能分析结果显示，副血链球菌中FimA的表达对于菌株在氧化应激条件下以及吞噬细胞内维持野生型水平的存活能力至关重要，但与菌株的耐酸性无关。综上，除了作为黏附素FimA发挥功能之外，fim操纵子的表达对于副血链球菌的致病能力同样具有核心意义。