Wang Ge, Maier Robert J
Department of Microbiology, University of Georgia, Athens, Georgia 30602, USA.
Infect Immun. 2004 Mar;72(3):1391-6. doi: 10.1128/IAI.72.3.1391-1396.2004.
Oxidative stress resistance is one of the key properties that enable pathogenic bacteria to survive the toxic reactive oxygen species released by the host. In a previous study characterizing oxidative stress resistance mutants of Helicobacter pylori, a novel potential antioxidant protein (MdaB) was identified by the observation that the expression of this protein was significantly upregulated to compensate for the loss of other major antioxidant components. In this study, we characterized an H. pylori mdaB mutant and the MdaB protein. While the wild-type strain can tolerate 10% oxygen for growth, the growth of the mdaB mutant was significantly inhibited by this oxygen condition. The mdaB mutant is also more sensitive to H(2)O(2), organic hydroperoxides, and the superoxide-generating agent paraquat. Although the wild-type strain can survive more than 10 h of air exposure, exposure of the mutant strain to air for 8 h resulted in recovery of no viable cells. The oxidative stress sensitivity of the mdaB mutant resulted in a deficiency in the ability to colonize mouse stomachs. H. pylori was recovered from 10 of 11 mouse stomachs inoculated with the wild-type strain, with about 5,000 to 45,000 CFU/g of stomach. However, only 3 of 12 mice that were inoculated with the mdaB mutant strain were found to harbor any H. pylori, and these 3 contained less than 2,000 CFU/g of stomach. A His-tagged MdaB protein was purified and characterized. It was shown to be a flavoprotein that catalyzes two-electron transfer from NAD(P)H to quinones. It reduces both ubiquinones and menaquinones with similar efficiencies and preferably uses NADPH as an electron donor. We propose that the physiological function of the H. pylori MdaB protein is that of an NADPH quinone reductase that plays an important role in managing oxidative stress and contributes to successful colonization of the host.
抗氧化应激是使病原菌能够在宿主释放的有毒活性氧中存活的关键特性之一。在之前一项对幽门螺杆菌抗氧化应激突变体进行表征的研究中，通过观察到一种新型潜在抗氧化蛋白（MdaB）的表达显著上调以补偿其他主要抗氧化成分的缺失，从而鉴定出了该蛋白。在本研究中，我们对幽门螺杆菌mdaB突变体和MdaB蛋白进行了表征。野生型菌株能够耐受10%的氧气用于生长，而mdaB突变体的生长在此氧气条件下受到显著抑制。mdaB突变体对过氧化氢、有机氢过氧化物和超氧化物产生剂百草枯也更敏感。虽然野生型菌株能够在空气暴露超过10小时后存活，但突变株暴露于空气中8小时后未检测到存活细胞。mdaB突变体的氧化应激敏感性导致其在小鼠胃中定殖的能力存在缺陷。接种野生型菌株的11只小鼠中有10只的胃中检测到幽门螺杆菌，每克胃组织中约有5000至45000 CFU。然而，接种mdaB突变株的12只小鼠中只有3只被发现携带幽门螺杆菌，且这3只小鼠每克胃组织中的细菌含量少于2000 CFU。一种带有His标签的MdaB蛋白被纯化并进行了表征。结果表明它是一种黄素蛋白，催化从NAD(P)H到醌的双电子转移。它以相似的效率还原泛醌和甲萘醌，并且优先使用NADPH作为电子供体。我们提出幽门螺杆菌MdaB蛋白的生理功能是作为一种NADPH醌还原酶，在应对氧化应激中起重要作用，并有助于在宿主体内成功定殖。
