Max Planck Institute for Infection Biology, Berlin, Germany.
PLoS Pathog. 2022 Sep 9;18(9):e1010825. doi: 10.1371/journal.ppat.1010825. eCollection 2022 Sep.
Bacteria from the genus Providencia are ubiquitous Gram-negative opportunistic pathogens, causing "travelers' diarrhea", urinary tract, and other nosocomial infections in humans. Some Providencia strains have also been isolated as natural pathogens of Drosophila melanogaster. Despite clinical relevance and extensive use in Drosophila immunity research, little is known about Providencia virulence mechanisms and the corresponding insect host defenses. To close this knowledge gap, we investigated the virulence factors of a representative Providencia species-P. alcalifaciens which is highly virulent to fruit flies and amenable to genetic manipulations. We generated a P. alcalifaciens transposon mutant library and performed an unbiased forward genetics screen in vivo for attenuated mutants. Our screen uncovered 23 mutants with reduced virulence. The vast majority of them had disrupted genes linked to lipopolysaccharide (LPS) synthesis or modifications. These LPS mutants were sensitive to cationic antimicrobial peptides (AMPs) in vitro and their virulence was restored in Drosophila mutants lacking most AMPs. Thus, LPS-mediated resistance to host AMPs is one of the virulence strategies of P. alcalifaciens. Another subset of P. alcalifaciens attenuated mutants exhibited increased susceptibility to reactive oxygen species (ROS) in vitro and their virulence was rescued by chemical scavenging of ROS in flies prior to infection. Using genetic analysis, we found that the enzyme Duox specifically in hemocytes is the source of bactericidal ROS targeting P. alcalifaciens. Consistently, the virulence of ROS-sensitive P. alcalifaciens mutants was rescued in flies with Duox knockdown in hemocytes. Therefore, these genes function as virulence factors by helping bacteria to counteract the ROS immune response. Our reciprocal analysis of host-pathogen interactions between D. melanogaster and P. alcalifaciens identified that AMPs and hemocyte-derived ROS are the major defense mechanisms against P. alcalifaciens, while the ability of the pathogen to resist these host immune responses is its major virulence mechanism. Thus, our work revealed a host-pathogen conflict mediated by ROS and AMPs.
普罗威登斯菌属的细菌是普遍存在的革兰氏阴性机会性病原体，导致人类“旅行者腹泻”、尿路感染和其他医院获得性感染。一些普罗威登斯菌菌株也被分离为黑腹果蝇的天然病原体。尽管具有临床相关性，并在果蝇免疫研究中广泛应用，但对普罗威登斯菌的毒力机制和相应的昆虫宿主防御机制知之甚少。为了弥补这一知识空白，我们研究了一种代表物种——具有高度毒性的粪产碱普罗威登斯菌（P. alcalifaciens）的毒力因子，该菌对果蝇具有高度毒性，并且易于进行遗传操作。我们生成了粪产碱普罗威登斯菌转座子突变体文库，并在体内进行了无偏正向遗传学筛选，以寻找减毒突变体。我们的筛选发现了 23 个毒力降低的突变体。绝大多数突变体的基因与脂多糖（LPS）合成或修饰有关。这些 LPS 突变体在体外对阳离子抗菌肽（AMPs）敏感，并且在缺乏大多数 AMPs 的果蝇突变体中其毒力得以恢复。因此，LPS 介导的对宿主 AMP 的抗性是粪产碱普罗威登斯菌的一种毒力策略。粪产碱普罗威登斯菌的另一个衰减突变体亚群在体外对活性氧（ROS）的敏感性增加，并且在感染前用化学清除 ROS 可以挽救其毒力。通过遗传分析，我们发现特异性存在于血细胞中的酶 Duox 是针对粪产碱普罗威登斯菌的杀菌 ROS 的来源。一致地，ROS 敏感的粪产碱普罗威登斯菌突变体的毒力在血细胞中 Duox 敲低的果蝇中得到挽救。因此，这些基因通过帮助细菌抵抗 ROS 免疫反应而作为毒力因子发挥作用。我们对黑腹果蝇和粪产碱普罗威登斯菌之间的宿主-病原体相互作用的相互分析表明，AMPs 和血细胞衍生的 ROS 是抵抗粪产碱普罗威登斯菌的主要防御机制，而病原体抵抗这些宿主免疫反应的能力是其主要毒力机制。因此，我们的工作揭示了由 ROS 和 AMPs 介导的宿主-病原体冲突。
