METHODS: We tested growth of KPC-Kp isolates in human serum. In serial isolates from a single patient, we performed whole genome sequencing and tested for complement resistance and binding by mixing study, direct enzyme-linked immunosorbent assay, flow cytometry, and electron microscopy. We utilized an isogenic deletion mutant in phagocytosis assays and an acute lung infection model.
RESULTS: We found serum resistance in 16 of 59 (27%) KPC-Kp clinical bloodstream isolates. In 5 genetically related bloodstream isolates from a single patient, we noted a loss-of-function mutation in the capsule biosynthesis gene, wcaJ. Disruption of wcaJ was associated with decreased polysaccharide capsule, resistance to complement-mediated killing, and surprisingly, increased binding of complement proteins. Furthermore, an isogenic wcaJ deletion mutant exhibited increased opsonophagocytosis in vitro and impaired in vivo control in the lung after airspace macrophage depletion in mice.
CONCLUSIONS: Loss of function in wcaJ led to increased complement resistance, complement binding, and opsonophagocytosis, which may promote KPC-Kp persistence by enabling coexistence of increased bloodstream fitness and reduced tissue virulence.
方法:我们测试了KPC-Kp分离株在人血清中的生长。在单个患者的连续分离株中,我们进行了全基因组测序,并通过混合研究测试了补体抗性和结合,直接酶联免疫吸附测定,流式细胞术,和电子显微镜。我们在吞噬作用测定和急性肺部感染模型中使用了等基因缺失突变体。
结果:我们在59例KPC-Kp临床血流分离株中发现了16例(27%)的血清耐药性。在一名患者的5种遗传相关血流分离物中,我们注意到胶囊生物合成基因的功能丧失突变,wcaj.wcaJ的破坏与多糖胶囊减少有关,对补体介导的杀伤的抗性,令人惊讶的是,补体蛋白的结合增加。此外,等基因wcaJ缺失突变体在小鼠空域巨噬细胞消耗后,在体外表现出肺中的调理吞噬作用增加和体内控制受损。
结论:wcaJ功能丧失导致补体抵抗增加,补体结合,和调理吞噬作用,这可能会促进KPC-Kp的持久性,使血液适应性增加和组织毒力降低共存。