Hypothiocyanous acid (HOSCN) is an antimicrobial oxidant produced by heme peroxidases from hydrogen peroxide and thiocyanate anions in secretory fluids such as in the human respiratory tract. While some respiratory tract pathogens display tolerance to HOSCN, there might be therapeutic value in targeting bacterial antioxidant systems that protect against HOSCN; however, surprisingly little is known about the bacterial defense mechanisms involved. We hypothesized that tolerant pathogens have a flavoprotein disulfide reductase (FDR) that uses NAD(P)H to directly reduce HOSCN, similar to thioredoxin reductase in mammalian cells. Here, we report the discovery of a previously uncharacterized FDR with HOSCN reductase activity, which we term Har, in Streptococcus pneumoniae, a bacterium previously found to be tolerant of HOSCN. S. pneumoniae generates large amounts of hydrogen peroxide that can be converted to HOSCN in the respiratory tract. Using knockouts, we demonstrate that the HOSCN reductase is dispensable for growth of S. pneumoniae in the presence of lactoperoxidase and thiocyanate. However, we also show bacterial growth in the HOSCN-generating system was completely crippled when deletion of HOSCN reductase activity was combined with disruption of glutathione import or recycling. Our findings identify a new bacterial HOSCN reductase, and demonstrate a role for this protein in combination with glutathione utilization to protect S. pneumoniae from HOSCN.

次硫氰酸 (HOSCN) 是一种抗菌氧化剂，由血红素过氧化物酶从分泌液（如人体呼吸道）中的过氧化氢和硫氰酸根阴离子产生。虽然一些呼吸道病原体表现出对 HOSCN 的耐受性，但靶向保护免受 HOSCN 的细菌抗氧化系统可能具有治疗价值；然而，令人惊讶的是，人们对所涉及的细菌防御机制知之甚少。我们假设耐受病原体具有使用 NAD(P)H 直接还原 HOSCN 的黄素蛋白二硫键还原酶 (FDR)，类似于哺乳动物细胞中的硫氧还蛋白还原酶。在这里，我们报告在肺炎链球菌中发现了一种以前未鉴定的具有 HOSCN 还原酶活性的 FDR，我们称之为 Har ，一种先前发现对 HOSCN 耐受的细菌。肺炎链球菌产生大量过氧化氢，可在呼吸道中转化为 HOSCN。使用基因敲除，我们证明了在乳过氧化物酶和硫氰酸盐存在的情况下， HOSCN 还原酶对于肺炎链球菌的生长是可有可无的。然而，我们还表明，当 HOSCN 还原酶活性的缺失与谷胱甘肽输入或回收的中断相结合时，HOSCN 生成系统中的细菌生长完全瘫痪。我们的研究结果确定了一种新的细菌 HOSCN 还原酶，并证明了这种蛋白质与谷胱甘肽联合利用来保护肺炎链球菌免受 HOSCN 侵害的作用。