Yadav Archana, Brewer Melissa N, Elshahed Mostafa S, Shaw Edward I
Department of Microbiology and Molecular Genetics. Oklahoma State University. Stillwater, OK.USA.
Biological Sciences. Southeastern Oklahoma State University. Durant, OK. USA.
bioRxiv. 2023 Feb 6:2023.02.06.527305. doi: 10.1101/2023.02.06.527305.
(Cb) is an obligate intracellular pathogen in nature and the causative agent of acute Q fever as well as chronic diseases. In an effort to identify genes and proteins crucial to their normal intracellular growth lifestyle, we applied a "Reverse evolution" approach where the avirulent Nine Mile Phase II strain of Cb was grown for 67 passages in chemically defined ACCM-D media and gene expression patterns and genome integrity from various passages was compared to passage number one following intracellular growth. Transcriptomic analysis identified a marked downregulation of the structural components of the type 4B secretion system (T4BSS), the general secretory (sec) pathway, as well as 14 out of 118 previously identified genes encoding effector proteins. Additional downregulated pathogenicity determinants genes included several chaperones, LPS, and peptidoglycan biosynthesis. A general marked downregulation of central metabolic pathways was also observed, which was balanced by a marked upregulation of genes encoding transporters. This pattern reflected the richness of the media and diminishing anabolic and ATP-generation needs. Finally, genomic sequencing and comparative genomic analysis demonstrated an extremely low level of mutation across passages, despite the observed Cb gene expression changes following acclimation to axenic media.
伯纳特立克次体（Cb）在自然界中是一种专性细胞内病原体，是急性Q热以及慢性疾病的病原体。为了确定对其正常细胞内生长方式至关重要的基因和蛋白质，我们采用了一种“反向进化”方法，即无毒的九英里II期Cb菌株在化学成分明确的ACCM-D培养基中传代培养67次，并将不同传代后的基因表达模式和基因组完整性与细胞内生长后的第一代进行比较。转录组分析发现，4B型分泌系统（T4BSS）的结构成分、一般分泌（sec）途径以及118个先前鉴定的编码效应蛋白的基因中的14个显著下调。其他下调的致病性决定因素基因包括几种伴侣蛋白、脂多糖和肽聚糖生物合成。还观察到中央代谢途径普遍显著下调，这被编码转运蛋白的基因显著上调所平衡。这种模式反映了培养基的丰富性以及合成代谢和ATP生成需求的减少。最后，基因组测序和比较基因组分析表明，尽管在适应无细胞培养基后观察到Cb基因表达发生了变化，但各传代间的突变水平极低。
