Department of Microbiology & Immunology, University of Michigan, Ann Arbor, Michigan, United States of America.
Department of Pathology, University of Michigan, Ann Arbor, Michigan, United States of America.
PLoS Pathog. 2021 Mar 15;17(3):e1009376. doi: 10.1371/journal.ppat.1009376. eCollection 2021 Mar.
Hypervirulent K. pneumoniae (hvKp) is a distinct pathotype that causes invasive community-acquired infections in healthy individuals. Hypermucoviscosity (hmv) is a major phenotype associated with hvKp characterized by copious capsule production and poor sedimentation. Dissecting the individual functions of CPS production and hmv in hvKp has been hindered by the conflation of these two properties. Although hmv requires capsular polysaccharide (CPS) biosynthesis, other cellular factors may also be required and some fitness phenotypes ascribed to CPS may be distinctly attributed to hmv. To address this challenge, we systematically identified genes that impact capsule and hmv. We generated a condensed, ordered transposon library in hypervirulent strain KPPR1, then evaluated the CPS production and hmv phenotypes of the 3,733 transposon mutants, representing 72% of all open reading frames in the genome. We employed forward and reverse genetic screens to evaluate effects of novel and known genes on CPS biosynthesis and hmv. These screens expand our understanding of core genes that coordinate CPS biosynthesis and hmv, as well as identify central metabolism genes that distinctly impact CPS biosynthesis or hmv, specifically those related to purine metabolism, pyruvate metabolism and the TCA cycle. Six representative mutants, with varying effect on CPS biosynthesis and hmv, were evaluated for their impact on CPS thickness, serum resistance, host cell association, and fitness in a murine model of disseminating pneumonia. Altogether, these data demonstrate that hmv requires both CPS biosynthesis and other cellular factors, and that hmv and CPS may serve distinct functions during pathogenesis. The integration of hmv and CPS to the metabolic status of the cell suggests that hvKp may require certain nutrients to specifically cause deep tissue infections.
高毒力肺炎克雷伯菌（hvKp）是一种独特的病原体，可导致健康个体发生侵袭性社区获得性感染。高黏液性（hmv）是与 hvKp 相关的主要表型，其特征为大量荚膜产生和不良沉降。由于这两种特性的混淆，解析 CPS 产生和 hmv 在 hvKp 中的个体功能受到了阻碍。尽管 hmv 需要荚膜多糖（CPS）生物合成，但其他细胞因子也可能是必需的，并且一些归因于 CPS 的适应性表型可能明显归因于 hmv。为了解决这一挑战，我们系统地鉴定了影响荚膜和 hmv 的基因。我们在高毒力菌株 KPPR1 中生成了一个浓缩的、有序的转座子文库，然后评估了 3733 个转座子突变体的 CPS 产生和 hmv 表型，这些突变体代表了基因组中所有开放阅读框的 72%。我们采用正向和反向遗传筛选来评估新型和已知基因对 CPS 生物合成和 hmv 的影响。这些筛选扩大了我们对协调 CPS 生物合成和 hmv 的核心基因的理解，同时确定了对 CPS 生物合成或 hmv 具有明显影响的中心代谢基因，特别是与嘌呤代谢、丙酮酸代谢和 TCA 循环相关的基因。我们评估了具有不同 CPS 生物合成和 hmv 效应的六个代表性突变体对 CPS 厚度、血清抗性、宿主细胞关联和在弥漫性肺炎小鼠模型中的适应性的影响。总的来说，这些数据表明 hmv 需要 CPS 生物合成和其他细胞因子，并且 hmv 和 CPS 在发病机制中可能具有不同的功能。将 hmv 和 CPS 与细胞的代谢状态整合在一起表明，hvKp 可能需要某些营养物质来特异性地引起深部组织感染。
