Crawford Matthew A, Tapscott Timothy, Fitzsimmons Liam F, Liu Lin, Reyes Aníbal M, Libby Stephen J, Trujillo Madia, Fang Ferric C, Radi Rafael, Vázquez-Torres Andrés
Department of Immunology and Microbiology, University of Colorado School of Medicine, Aurora, Colorado, USA.
Molecular Biology Program, University of Colorado School of Medicine, Aurora, Colorado, USA.
mBio. 2016 Apr 19;7(2):e02161-15. doi: 10.1128/mBio.02161-15.
The four-cysteine zinc finger motif of the bacterial RNA polymerase regulator DksA is essential for protein structure, canonical control of the stringent response to nutritional limitation, and thiol-based sensing of oxidative and nitrosative stress. This interdependent relationship has limited our understanding of DksA-mediated functions in bacterial pathogenesis. Here, we have addressed this challenge by complementing ΔdksA Salmonella with Pseudomonas aeruginosa dksA paralogues that encode proteins differing in cysteine and zinc content. We find that four-cysteine, zinc-bound (C4) and two-cysteine, zinc-free (C2) DksA proteins are able to mediate appropriate stringent control in Salmonella and that thiol-based sensing of reactive species is conserved among C2 and C4 orthologues. However, variations in cysteine and zinc content determine the threshold at which individual DksA proteins sense and respond to reactive species. In particular, zinc acts as an antioxidant, dampening cysteine reactivity and raising the threshold of posttranslational thiol modification with reactive species. Consequently, C2 DksA triggers transcriptional responses in Salmonella at levels of oxidative or nitrosative stress normally tolerated by Salmonella expressing C4 orthologues. Inappropriate transcriptional regulation by C2 DksA increases the susceptibility of Salmonella to the antimicrobial effects of hydrogen peroxide and nitric oxide, and attenuates virulence in macrophages and mice. Our findings suggest that the redox-active sensory function of DksA proteins is finely tuned to optimize bacterial fitness according to the levels of oxidative and nitrosative stress encountered by bacterial species in their natural and host environments.
In order to cause disease, pathogenic bacteria must rapidly sense and respond to antimicrobial pressures encountered within the host. Prominent among these stresses, and of particular relevance to intracellular pathogens such as Salmonella, are nutritional restriction and the enzymatic generation of reactive oxygen and nitrogen species. The conserved transcriptional regulator DksA controls adaptive responses to nutritional limitation, as well as to oxidative and nitrosative stress. Here, we demonstrate that each of these functions contributes to bacterial pathogenesis. Our observations highlight the importance of metabolic adaptation in bacterial pathogenesis and show the mechanism by which DksA orthologues are optimized to sense the levels of oxidative and nitrosative stress encountered in their natural habitats. An improved understanding of the conserved processes used by bacteria to sense, respond to, and limit host defense will inform the development of novel strategies to treat infections caused by pathogenic, potentially multidrug-resistant bacteria.
细菌RNA聚合酶调节因子DksA的四半胱氨酸锌指基序对于蛋白质结构、对营养限制的严格反应的典型控制以及基于硫醇的氧化和亚硝化应激感应至关重要。这种相互依存的关系限制了我们对DksA介导的细菌致病性功能的理解。在这里，我们通过用铜绿假单胞菌dksA旁系同源物补充ΔdksA沙门氏菌来应对这一挑战，这些旁系同源物编码的蛋白质在半胱氨酸和锌含量上有所不同。我们发现，四半胱氨酸、锌结合型（C4）和二半胱氨酸、无锌型（C2）DksA蛋白能够在沙门氏菌中介导适当的严格控制，并且基于硫醇的活性物质感应在C2和C4直系同源物中是保守的。然而，半胱氨酸和锌含量的变化决定了单个DksA蛋白感应和响应活性物质的阈值。特别是，锌作为一种抗氧化剂，减弱半胱氨酸的反应性并提高活性物质对翻译后硫醇修饰的阈值。因此，C2 DksA在氧化或亚硝化应激水平下触发沙门氏菌中的转录反应，而这些应激水平通常是表达C4直系同源物的沙门氏菌所能耐受的。C2 DksA的不适当转录调控增加了沙门氏菌对过氧化氢和一氧化氮抗菌作用的敏感性，并减弱了其在巨噬细胞和小鼠中的毒力。我们的研究结果表明，DksA蛋白的氧化还原活性传感功能经过精细调节，以根据细菌在其自然和宿主环境中遇到的氧化和亚硝化应激水平优化细菌适应性。
为了引发疾病，病原菌必须迅速感知并应对在宿主体内遇到的抗菌压力。这些压力中突出的，且与沙门氏菌等细胞内病原体特别相关的是营养限制以及活性氧和氮物种的酶促生成。保守的转录调节因子DksA控制对营养限制以及氧化和亚硝化应激的适应性反应。在这里，我们证明这些功能中的每一个都有助于细菌致病。我们的观察结果突出了代谢适应在细菌致病中的重要性，并展示了DksA直系同源物如何被优化以感知其在自然栖息地中遇到的氧化和亚硝化应激水平的机制。对细菌用于感知、响应和限制宿主防御的保守过程的更好理解将为开发治疗由致病性、可能多重耐药的细菌引起的感染的新策略提供信息。
