IpsA, a novel transcriptional regulator required for inositol derived lipid formation in Corynebacteria and Mycobacteria. Corynebacterium glutamicum ATCC 13032

The development of new drugs against tuberculosis and diphtheria is focused on disrupting the biogenesis of the cell wall, the unique architecture of which confers resistance against current therapies. The enzymatic pathways involved in the synthesis of the cell wall by these pathogens are well understood but the underlying regulatory mechanisms are largely unknown. Here, we characterize IpsA, a LacI-type transcriptional regulator conserved among Mycobacteria and Corynebacteria that plays a role in the regulation of cell wall biogenesis. IpsA triggers myo-inositol formation by activating ino1, which encodes inositol phosphate synthase. An IpsA deletion mutant of Corynebacterium glutamicum cultured on glucose displayed significantly impaired growth and presented an elongated cell morphology. Analysis of the polar lipid fraction of the cell wall revealed the absence of inositol-derived lipids. The phenotype of the C. glutamicum ΔipsA mutant was complemented by homologues from Corynebacterium diphtheriae (dip1969) and Mycobacterium tuberculosis (rv3575), indicating the conserved function of IpsA in the pathogenic species. Additional targets of IpsA with putative functions in cell wall biogenesis were identified and IpsA was shown to bind to a conserved palindromic motif within the corresponding promoter regions. myo-inositol was identified as an effector of IpsA, causing the dissociation of the IpsA-DNA complex in vitro. This characterization of IpsA function and of its regulon sheds light on the complex transcriptional control of cell wall biogenesis in the mycolata taxon and generates novel targets for drug development. Overall design: To identify genes which were regulated by IpsA (Cg2910), we performed DNA microarray analyses of ATCC 13032 ΔipsA against wild type. For this purpose RNA was isolated from cells cultivated in CGXII minimal medium with 2% glucose (w v-1) and harvested in the exponential growth phase at an OD600 of 1. Three biological replicates were performed.

针对结核病与白喉的新药研发，核心方向为破坏病原菌细胞壁的生物发生过程：其独特的结构赋予了病原体对抗现有治疗方案的耐药性。目前学界已明确此类病原体合成细胞壁所涉及的酶促通路，但其中潜在的调控机制仍未被充分阐释。本研究对IpsA开展了系统表征：该蛋白属于LacI型转录调控因子（LacI-type transcriptional regulator），在分枝杆菌属（Mycobacteria）与棒杆菌属（Corynebacteria）中保守存在，参与细胞壁生物发生的调控过程。IpsA通过激活编码肌醇磷酸合酶的ino1基因，触发myo-肌醇（myo-inositol）的合成。在葡萄糖培养基中培养的谷氨酸棒杆菌（Corynebacterium glutamicum）IpsA缺失突变株，其生长能力显著受损，且呈现细胞形态拉长的表型。对该突变株细胞壁的极性脂组分进行分析后发现，其中不存在肌醇衍生脂质。谷氨酸棒杆菌ΔipsA突变株的表型可被白喉棒杆菌（Corynebacterium diphtheriae）的同源基因dip1969以及结核分枝杆菌（Mycobacterium tuberculosis）的同源基因rv3575互补，这表明IpsA在致病物种中具有保守的功能。本研究还鉴定出IpsA的其他潜在靶基因，这些靶基因推测参与细胞壁生物发生过程；同时证实IpsA可结合对应启动子区域内的保守回文基序。myo-肌醇（myo-inositol）被鉴定为IpsA的效应分子，可在体外诱导IpsA-DNA复合物发生解离。本研究对IpsA功能及其调控子（regulon）的系统表征，阐明了霉菌酸类细菌类群（mycolata）中细胞壁生物发生的复杂转录调控机制，同时为新药研发提供了全新的靶标。整体实验设计：为鉴定受IpsA（Cg2910）调控的基因，我们以野生型ATCC 13032为对照，对ΔipsA突变株开展DNA微阵列分析。具体步骤为：从以2%葡萄糖（w/v）添加至CGXII最小培养基中培养的细胞内分离RNA，于指数生长期（OD600为1）时收集样本。实验共设置3次生物学重复。