Deletion of glutaredoxin promotes oxidative tolerance and intracellular infection in <i>Listeria monocytogenes</i>

Thiol-disulfide glutaredoxin systems of bacterial cytoplasm favor reducing conditions for the correct disulfide bonding of functional proteins, and therefore were employed by bacteria to defend against oxidative stress. <i>Listeria monocytogenes</i> has been shown to encode a putative glutaredoxin, Grx (encoded by <i>lmo2344</i>), while the underlying roles remain unknown. Here we suggest an unexpected role of <i>L. monocytogenes</i> Grx in oxidative tolerance and intracellular infection. The recombinant Grx was able to efficiently catalyze the thiol-disulfide oxidoreduction of insulin in the presence of DTT as an election donor. Unexpectedly, the deletion of <i>grx</i> resulted in a remarkably increased tolerance and survival ability of this bacteria when exposed to various oxidizing agents, including diamide, and copper and cadmium ions. Furthermore, loss of <i>grx</i> significantly promoted bacterial invasion and proliferation in human epithelial Caco-2 cells and murine macrophages, as well as a notably increasing invasion but not cell-to-cell spread in the murine fibroblasts L929 cells. More importantly, <i>L. monocytogenes</i> lacking the glutaredoxin exhibited more efficient proliferation and recovery in the spleens and livers of the infected mice, and hence became more virulent by upregulating the virulence factors, InlA and InlB. In summary, we here for the first time demonstrated that <i>L. monocytogenes</i> glutaredoxin plays a counterintuitive role in bacterial oxidative resistance and intracellular infection, which is the first report to provide valuable evidence for the role of glutaredoxins in bacterial infection, and more importantly suggests a favorable model to illustrate the functional diversity of bacterial Grx systems during environmental adaption and host infection.

细菌胞质中的巯基-二硫键谷氧还蛋白（glutaredoxin）系统偏好还原环境，以保障功能性蛋白正确形成二硫键，因此细菌借此抵御氧化应激。已有研究表明单核细胞增生李斯特菌（Listeria monocytogenes）可编码一种推定谷氧还蛋白Grx（由lmo2344基因编码），但其具体功能尚未明确。本研究揭示了单核细胞增生李斯特菌Grx在氧化耐受与胞内感染中未曾预料到的功能。重组Grx可在二硫苏糖醇（DTT, dithiothreitol）作为电子供体的条件下，高效催化胰岛素的巯基-二硫键氧化还原反应。出乎意料的是，敲除grx基因后，该菌在暴露于包括双酰胺（diamide）、铜离子与镉离子在内的多种氧化剂时，其耐受能力与存活率显著提升。此外，grx基因缺失可显著促进细菌在人上皮Caco-2细胞与小鼠巨噬细胞中的侵袭与增殖，同时也能显著增强其在小鼠成纤维细胞L929中的侵袭能力，但无法促进细胞间扩散。更为重要的是，缺失谷氧还蛋白的单核细胞增生李斯特菌在感染小鼠的脾脏与肝脏中增殖与恢复能力更强，并通过上调毒力因子InlA与InlB的表达，表现出更强的致病性。综上，本研究首次证实单核细胞增生李斯特菌的谷氧还蛋白在细菌氧化抵抗与胞内感染中发挥与直觉相悖的作用；这是首个为谷氧还蛋白在细菌感染中的功能提供有效证据的研究，更为重要的是，本研究提出了一个理想模型，用以阐释细菌谷氧还蛋白系统在环境适应与宿主感染过程中的功能多样性。