Genetic mechanisms of Coxiella burnetii lipopolysaccharide phase variation

Coxiella burnetii is an intracellular pathogen that causes human Q fever, a disease that normally presents as a severe flu-like illness. Due to high infectivity and disease severity, the pathogen is considered a risk group 3 organism. Full-length lipopolysaccharide (LPS) is required for full virulence and disease by C. burnetii and is the only virulence factor currently defined by infection of an immunocompetent animal. Transition of virulent phase I bacteria with smooth LPS, to avirulent phase II bacteria with rough LPS, occurs during in vitro passage. Semi-rough intermediate forms are also observed. Here, the genetic basis of LPS phase conversion was investigated to obtain a more complete understanding of C. burnetii pathogenesis. Whole genome sequencing of strains producing intermediate and/or phase II LPS identified several common mutations in predicted LPS biosynthesis genes. After passage in broth culture for 30 weeks, phase I strains from different genomic groups exhibited similar phase transition kinetics and elevation of mutations in LPS biosynthesis genes. Targeted mutagenesis and genetic complementation using a new C. burnetii nutritional selection system based on lysine auxotrophy confirmed that six of the mutated genes were necessary for production of phase I LPS. Disruption of two of these genes in a C. burnetii phase I strain resulted in production of phase II LPS, suggesting inhibition of the encoded enzymes could represent a new therapeutic strategy for treatment of Q fever. Additionally, targeted mutagenesis of genes encoding LPS biosynthesis enzymes can now be used to construct new phase II strains from different genomic groups for use in pathogen-host studies at a risk group 2 level.贝氏柯克斯体（Coxiella burnetii）是一种胞内病原体，可引发人类Q热（Q fever），该病通常表现为重症流感样疾病。由于其感染性强、致病严重，该病原体被归为三级风险微生物。完整长度的脂多糖（lipopolysaccharide, LPS）是贝氏柯克斯体实现完全毒力与致病能力的必需因子，也是目前经免疫健全动物感染实验证实的唯一毒力因子。具有光滑型LPS的强毒I相细菌，在体外传代过程中会转变为携带粗糙型LPS的弱毒II相细菌，同时也可观察到半粗糙的中间过渡型菌株。本研究针对脂多糖相转换的遗传基础展开探究，以期更全面地解析贝氏柯克斯体的致病机制。对产生中间型和/或II相LPS的菌株进行全基因组测序，在预测的脂多糖生物合成基因中发现了多处共通突变。在肉汤培养基中传代培养30周后，不同基因组群的I相菌株均表现出相似的相转换动力学特征，且脂多糖生物合成基因的突变丰度显著升高。依托基于赖氨酸营养缺陷型构建的新型贝氏柯克斯体营养筛选系统，开展靶向诱变与基因互补实验，证实其中6个突变基因对于I相LPS的产生必不可少。在I相贝氏柯克斯体菌株中敲除其中两个基因即可使其产生II相LPS，这提示抑制上述基因编码的酶类或可成为治疗Q热的全新治疗策略。此外，靶向诱变脂多糖生物合成相关基因的技术手段，如今可用于构建不同基因组群的新型II相菌株，用于二级风险水平下的病原体-宿主交互研究。