The involvement of Mycobacterium type III-A CRISPR-Cas system in oxidative stress and intracellular fitness

Previous studies suggest that type I and type II CRISPR-Cas systems were employed to evade host immunity by targeting interference of bacteria’s own genes. Although Mycobacterium bovis (M. bovis) and Mycobacterium tuberculosis (M. tuberculosis) possess integrated type III-A CRISPR-Cas system, its role in mycobacteria remains obscure. Here, we observed that seven cas genes (csm2~6, cas10, cas6) were significantly upregulated under oxidative stress treatment. To explore the function of type III-A CRSIRP-Cas system, Total CRISPR system (TCR) mutant strain was generated in M. bovis Bacille Calmette-Guérin (BCG). Deletion of TCR results in increased sensitivity in response to H2O2 and reduced cell envelope integrity. By using RNA-Seq, 590 differential expression genes were found in mutant for TCR, of which 220 genes were upregulated and 370 genes were downregulated, indicating an important role of TCR in controlling gene expression in mycobacteria. Consistently, disrupting TCR results in poor intracellular survival in vivo and in vitro. Moreover, we showed for the first time that TCR contributed to the regulation of regulatory T cell population, supporting a role of TCR in modulating host immunity. These observations demonstrate that type III-A CRISPR-Cas system as an important factor for intracellular survival and host immunoregulation in mycobacteria, may be a potential target for therapy. For RNA-seq: Examination of 2 strains (Wild type VS ΔTCR).

既往研究表明，I型和II型CRISPR-Cas系统（CRISPR-Cas）可通过靶向干扰细菌自身基因，实现宿主免疫逃逸。尽管牛分枝杆菌（Mycobacterium bovis, M. bovis）与结核分枝杆菌（Mycobacterium tuberculosis, M. tuberculosis）均携带整合型III-A型CRISPR-Cas系统，但该系统在分枝杆菌中的功能仍有待阐明。本研究观察到，7个cas基因（csm2~6、cas10、cas6）在氧化应激处理下显著上调。为探究III-A型CRISPR-Cas系统的功能，我们在牛分枝杆菌卡介苗（Bacille Calmette-Guérin, BCG）中构建了全CRISPR系统（Total CRISPR system, TCR）敲除突变株。敲除TCR可使菌株对过氧化氢（H₂O₂）的敏感性升高，同时降低细胞包膜完整性。通过RNA测序（RNA-Seq）分析，我们在TCR突变株中鉴定出590个差异表达基因，其中220个基因上调、370个基因下调，提示TCR在分枝杆菌的基因表达调控中发挥重要作用。与之相符的是，破坏TCR会导致菌株在体内外的胞内生存能力受损。此外，我们首次证实TCR可调控调节性T细胞（regulatory T cell）群体，进一步支持了TCR在调节宿主免疫中的功能。上述研究结果表明，III-A型CRISPR-Cas系统作为分枝杆菌胞内生存与宿主免疫调控的关键因子，有望成为潜在治疗靶点。RNA测序相关说明：本次研究共检测2组菌株（野生型菌株 vs TCR敲除突变株）。