分枝杆菌aceE基因影响细胞壁合成代谢的机制研究

The integrity of the cell wall structure has important biological significance on the in vivo survival for mycobacteria. However, the metabolic mechanisms of the cell wall biosynthesis in mycobacteria have not been fully explored. aceE is encoding the E1 components of pyruvate dehydrogenase complex (PDH). The PDH catalyses the conversion of pyruvate to acetyl-CoA, which feeds into the tricarboxylic acid cycle, one of the main pathways of cellular respiration and biosynthesis in organisms. But aceE gene in Mycobacteria was rarely reported. Only is Tian in 2005 established the E1 components of PDH in Mycobacterium tuberculosis (Mtb) is aceE and another study found that aceE is seem to be expressed more readily in the virulent Mtb H37Rv than in the attenuated H37Ra throughout the course of infection. It is not clear that aceE in Mtb exists other functions independently of the PDH. In the process of screening of M.smegmatis transposon libraries, we found that the plaque of aceE-deficent mutants was quiet different from that of the wildtype strain, with a small and smooth morphpology on the solid culture medium. Meanwhile, the mutant strain disperses uniformly, while the WT M. smegamatis is much easy to form agglomerate in Middlebrook 7H9 medium. Let the culture medium stand for 2 ~ 3 more days, a thick deposit of biofilm was formed around the wall of the tube in the culture of WT strain, but absent in the culture of mutant strain. What’s more, a distinct cell wall mycolic acid composition in mutant strain (with higher proportion of short-chain mycolic acid but lower proportion of long-chain mycolic acid) that is significantly different from WT M.smegmatis strain was found, suggesting a potential role of aceE gene in cell wall mycolic acid metabolism. A recent study isolated colony morphotype mutants by screening transposon mutagenesis in M. smegmatis mc26 and also found an aceE gene loss mutant with a severe defect in biofilm pellicle formation which was in consistence with our study. So there are several questions worthy of discussion. Is aceE gene indeed involved in mycobacteria cell wall systhesis? How it works? We will research the change of cell morphology and cell wall construction using electron microscope, analyze the difference compositon of lipids and mycolic acids by TLC, and find the change of upstream and downstream components in aceE metabolic by the transcriptome and metabolomic analysis between the mutant strain and WT strain, to investigate how aceE gene plays a role on the cell wall synthesis, expounding the new function of aceE gene of mycobacteria, providing new target for the treatment of TB disease.

细胞壁合成代谢通路一直是抗结核新药研制的研究热点。然而，关于分枝杆菌细胞壁合成代谢的研究尚未完全清楚。我们的前期工作发现，参与丙酮酸代谢的aceE基因失活后，极大地影响耻垢分枝杆菌在培养基上的生长形态、细菌形态、生物膜的形成及分枝菌酸的构成，表明该基因或其参与的代谢途径与分枝杆菌细胞壁分枝菌酸或脂类合成相关。我们将运用双向薄层层析和高效液相色谱串联质谱技术分析野生型与突变体菌株细胞壁脂类及分枝菌酸组成的差异，转录组及代谢组学分析aceE基因参与代谢通路上下游组份的改变，免疫共沉淀及酶活性分析aceE基因发挥作用的功能，研究aceE基因对分枝杆菌细胞壁合成代谢的影响及其参与该代谢过程的分子机理，明确该基因在维持分枝杆菌细胞壁完整性中的作用，并阐明该基因发挥作用的机制，以增进对分枝杆菌细胞壁合成代谢的认识，为抗结核新药的研制提供新靶标。

背景: 细胞壁结构的完整性对分枝杆菌的体内外生理代谢具有重要的生物学意义。但细胞壁合成的相关机制尚不完全清楚。.方法: 利用转座子文库筛选影响耻垢分枝杆菌细胞壁合成相关基因，对体外生长形态变异的转座失活突变株进行转座失活基因测序鉴定, 确定该突变株为基因组中aceE基因（MSMEG_4323基因）被转座失活的突变株。比较野生型耻垢分枝杆菌（野生株）和转座失活突变株（突变株）在固体和液体培养基中的体外生长形态，电镜观察突变体的细菌形态和细胞壁的结构，生物膜形成试验测定菌株生物膜形成，HPLC-sherlock系统分析细胞壁分支菌酸组成，并用LC-MS对细胞壁的各种脂质进行检测。药物敏感性试验测定野生株和突变株对常规十二种抗结核药物的敏感性，并比较野生株和突变株感染巨噬细胞后的体内生长情况，研究aceE基因对于耻垢分枝杆菌体内外生长代谢的影响及可能的作用机制。.结果: 与野生株相比，突变株在固体培养基上具有明显的形态差异。在7H9液体培养基中，野生株与突变株之间生长速度无明显差异，但突变株生长分散较为均一，不易聚集成团。将静置培养时间延长至5‒7天，可观察到野生株易在试管壁上沿形成一层生物膜，突变株则无类似的生物膜形成。电镜观察发现突变株的细胞表面光滑，且细胞壁厚度与野生株相比要薄。进一步的细胞壁分支菌酸组成分析，发现突变株中短链分支菌酸的组成比例较高，长链分支菌酸的组成比例较低，提示aceE基因的失活可能影响细胞壁分支菌酸碳链的延伸。LC-MS的脂质谱分析，发现突变株中alpha-MA, alpha prime-MA and keto/epoxy/omega-1-MA等分支菌酸合成缺陷。药敏试验结果表明，突变株与野生株对常规十二种抗结核药物的药物敏感程度基本一致。突变株感染巨噬细胞的入侵和生长速度与野生株无明显差异。.结论: aceE基因转座失活导致细胞壁分支菌酸的合成代谢发生异常，从而导致耻垢分枝杆菌固体培养基上的菌落形态异常，影响生物膜的形成，但对耻垢分枝杆菌的抗药性及在巨噬细胞内的生存均无显著影响，表明aceE基因并非影响耻垢分枝杆菌体内外生长的关键基因。