Division of Bacteriology & Mycology, ICAR-Indian Veterinary Research Institute, Izatnagar, 243 122, India.
Division of Biochemistry, ICAR-Indian Veterinary Research Institute, Izatnagar, 243 122, India.
Mol Biol Rep. 2021 Apr;48(4):3195-3203. doi: 10.1007/s11033-021-06381-2. Epub 2021 May 5.
Salmonella Typhimurium survives and replicates inside the oxidative environment of phagocytic cells. Proteins, because of their composition and location, are the foremost targets of host inflammatory response. Among others, Met-residues are highly prone to oxidation. Methionine sulfoxide reductase (Msr), with the help of thioredoxin-thioredoxin reductase, can repair oxidized methionine (Met-SO) residues to Met. There are four methionine sulfoxide reductases localized in the cytosol of S. Typhimurium, MsrA, MsrB, MsrC and BisC. MsrA repairs both protein-bound and free 'S' Met-SO, MsrB repairs protein-bound 'R' Met-SO, MsrC repairs free 'R' Met-SO and BisC repairs free 'S' Met-SO. To assess the role(s) of various Msrs in Salmonella, few studies have been conducted by utilizing ΔmsrA, ΔmsrB, ΔmsrC, ΔmsrAΔmsrB, ΔmsrBΔmsrC and ΔbisC mutant strains of S. Typhimurium. Out of the above-mentioned mutants, ΔmsrA and ΔmsrC were found to play important role in the stress survival of this bacterium; however, the combined roles of these two genes have not been determined. In the current study, we have generated msrAmsrC double gene deletion strain (ΔmsrAΔmsrC) of S. Typhimurium and evaluated the effect of gene deletions on the survival of Salmonella against hypochlorite stress and intramacrophage replication. In in vitro growth curve analysis, ΔmsrAΔmsrC mutant strain showed a longer lag phase during the initial stages of the growth; however, it attained similar growth as the wild type strain of S. Typhimurium after 5 h. The ΔmsrAΔmsrC mutant strain has been highly (~ 3000 folds more) sensitive (p < 0.001) to hypochlorite stress. Further, ΔmsrA and ΔmsrAΔmsrC mutant strains showed more than 8 and 26 folds more susceptibility to poultry macrophages, respectively. Our data suggest that the deletion of both msrA and msrC genes severely affect the oxidative stress survival and intramacrophage proliferation of S. Typhimurium.
鼠伤寒沙门氏菌在吞噬细胞的氧化环境中存活并复制。由于其组成和位置，蛋白质是宿主炎症反应的首要靶标。其中，蛋氨酸残基极易氧化。甲硫氨酸还原酶（Msr）在硫氧还蛋白-硫氧还蛋白还原酶的帮助下，可以将氧化的蛋氨酸（Met-SO）残基修复为 Met。有四种甲硫氨酸还原酶定位于鼠伤寒沙门氏菌的细胞质中，分别是 MsrA、MsrB、MsrC 和 BisC。MsrA 修复结合蛋白和游离的“S”Met-SO，MsrB 修复结合蛋白的“R”Met-SO，MsrC 修复游离的“R”Met-SO，BisC 修复游离的“S”Met-SO。为了评估各种 Msr 在沙门氏菌中的作用，已经利用鼠伤寒沙门氏菌的ΔmsrA、ΔmsrB、ΔmsrC、ΔmsrAΔmsrB、ΔmsrBΔmsrC 和ΔbisC 突变株进行了一些研究。在上述突变体中，ΔmsrA 和 ΔmsrC 被发现对该细菌的应激生存起着重要作用；然而，这两个基因的联合作用尚未确定。在本研究中，我们构建了鼠伤寒沙门氏菌的 msrAmsrC 双基因缺失株（ΔmsrAΔmsrC），并评估了基因缺失对沙门氏菌抵抗次氯酸盐应激和细胞内复制的影响。在体外生长曲线分析中，ΔmsrAΔmsrC 突变株在生长初期表现出较长的迟滞期；然而，在 5 小时后，它的生长与鼠伤寒沙门氏菌的野生型菌株相似。ΔmsrAΔmsrC 突变株对次氯酸盐应激高度敏感（p<0.001），约（~3000 倍）。此外，ΔmsrA 和 ΔmsrAΔmsrC 突变株对禽类巨噬细胞的敏感性分别增加了 8 倍和 26 倍以上。我们的数据表明，msrA 和 msrC 基因的缺失严重影响鼠伤寒沙门氏菌的氧化应激生存和细胞内增殖。
