Table_1_The Lack of the TetR-Like Repressor Gene BCG_2177c (Rv2160A) May Help Mycobacteria Overcome Intracellular Redox Stress and Survive Longer Inside Macrophages When Surrounded by a Lipid Environment.docx

Mycobacteria, like other microorganisms, survive under different environmental variations by expressing an efficient adaptive response, oriented by regulatory elements, such as transcriptional repressors of the TetR family. These repressors in mycobacteria also appear to be related to cholesterol metabolism. In this study, we have evaluated the effect of a fatty acid (oleic–palmitic–stearic)/cholesterol mixture on some phenotypic and genotypic characteristics of a tetR-mutant strain (BCG_2177c mutated gene) of M. bovis BCG, a homologous of Rv2160A of M. tuberculosis. In order to accomplish this, we have analyzed the global gene expression of this strain by RNA-seq and evaluated its neutral-lipid storage capacity and potential to infect macrophages. We have also determined the macrophage response by measuring some pro- and anti-inflammatory cytokine expressions. In comparison with wild-type microorganisms, we showed that the mutation in the BCG_2177c gene did not affect the growth of M. bovis BCG in the presence of lipids but it probably modified the structure/composition of its cell envelope. Compared to with dextrose, an overexpression of the transcriptome of the wild-type and mutant strains was observed when these mycobacteria were cultured in lipids, mainly at the exponential phase. Twelve putative intracellular redox balance maintenance genes and four others coding for putative transcriptional factors (including WhiB6 and three TetR-like) were the main elements repeatedly overexpressed when cultured in the presence of lipids. These genes belonged to the central part of what we called the “genetic lipid signature” for M. bovis BCG. We have also found that all these mycobacteria genotypic changes affected the outcome of BCG-infected macrophages, being the mutant strain most adapted to persist longer inside the host. This high persistence result was also confirmed when mutant-infected macrophages showed overexpression of the anti-inflammatory cytokine TGF-β versus pro-inflammatory cytokines. In summary, the lack of this TetR-like repressor expression, within a lipid environment, may help mycobacteria overcome intracellular redox stress and survive longer inside their host.

分枝杆菌(Mycobacteria)与其他微生物类似，可通过高效的适应性应答应对不同环境变化，该应答由调控元件介导，例如TetR家族转录阻遏蛋白（TetR family transcriptional repressors）。而分枝杆菌中的这类阻遏蛋白，似乎还与胆固醇代谢相关。本研究评估了脂肪酸（油酸-棕榈酸-硬脂酸）/胆固醇混合物对牛分枝杆菌卡介苗（M. bovis BCG）tetR突变菌株（BCG_2177c基因突变株，该基因对应结核分枝杆菌（M. tuberculosis）的Rv2160A同源基因）的部分表型与基因型特征的影响。为完成该研究，我们通过RNA测序（RNA-seq）分析了该菌株的全局基因表达情况，并评估了其中性脂质储存能力以及感染巨噬细胞的潜能。同时我们还通过检测部分促炎与抗炎细胞因子的表达水平，测定了巨噬细胞的应答状态。与野生型菌株相比，我们发现BCG_2177c基因的突变并未影响牛分枝杆菌卡介苗在脂质环境下的生长，但可能改变了其细胞壁的结构与组成。与葡萄糖培养基相比，当分枝杆菌在脂质培养基中培养时（尤其是在指数生长期），野生型与突变菌株的转录组均出现上调表达。在脂质环境下培养时，共有12个假定的胞内氧化还原平衡维持基因，以及4个假定转录因子编码基因（包括WhiB6与3个TetR类蛋白）被反复显著上调表达。这些基因构成了我们所称的牛分枝杆菌卡介苗‘脂质遗传特征’的核心部分。我们还发现，所有上述分枝杆菌的基因型变化均会影响卡介苗感染巨噬细胞后的感染结局，其中突变菌株更适应在宿主细胞内长期存活。该高持久性结果也得到了验证：突变菌株感染的巨噬细胞中，抗炎细胞因子转化生长因子β（TGF-β）的表达水平高于促炎细胞因子。总而言之，在脂质环境中缺失此类TetR类阻遏蛋白的表达，或许可帮助分枝杆菌抵御胞内氧化还原应激，并在宿主内存活更久。