Department of Molecular Medicine, University of Padua, Padua, Italy.
Institut de Pharmacologie et de Biologie Structurale, IPBS, Université de Toulouse, CNRS, UPS, Toulouse, France.
BMC Microbiol. 2022 Apr 1;22(1):85. doi: 10.1186/s12866-022-02493-2.
Aminoacyl-phosphatidylglycerol (aaPG) synthases are bacterial enzymes that usually catalyze transfer of aminoacyl residues to the plasma membrane phospholipid phosphatidylglycerol (PG). The result is introduction of positive charges onto the cytoplasmic membrane, yielding reduced affinity towards cationic antimicrobial peptides, and increased resistance to acidic environments. Therefore, these enzymes represent an important defense mechanism for many pathogens, including Staphylococcus aureus and Mycobacterium tuberculosis (Mtb), which are known to encode for lysyl-(Lys)-PG synthase MprF and LysX, respectively. Here, we used a combination of bioinformatic, genetic and bacteriological methods to characterize a protein encoded by the Mtb genome, Rv1619, carrying a domain with high similarity to MprF-like domains, suggesting that this protein could be a new aaPG synthase family member. However, unlike homologous domains of MprF and LysX that are positioned in the cytoplasm, we predicted that the MprF-like domain in LysX2 is in the extracytoplasmic region.
Using genetic fusions to the Escherichia coli proteins PhoA and LacZ of LysX2, we confirmed this unique membrane topology, as well as LysX and MprF as benchmarks. Expression of lysX2 in Mycobacterium smegmatis increased cell resistance to human β-defensin 2 and sodium nitrite, enhanced cell viability and delayed biofilm formation in acidic pH environment. Remarkably, MtLysX2 significantly reduced the negative charge on the bacterial surface upon exposure to an acidic environment. Additionally, we found LysX2 orthologues in major human pathogens and in rapid-growing mycobacteria frequently associated with human infections, but not in environmental and non-pathogenic mycobacteria.
Overall, our data suggest that LysX2 is a prototype of a new class within the MprF-like protein family that likely enhances survival of the pathogenic species through its catalytic domain which is exposed to the extracytoplasmic side of the cell membrane and is required to decrease the negative charge on the bacterial surface through a yet uncharacterized mechanism.
氨酰基-磷酰基甘油(aaPG)合酶是一种细菌酶,通常催化将氨酰基残基转移到质膜磷脂酰甘油(PG)上。其结果是在细胞质膜上引入正电荷,降低了对阳离子抗菌肽的亲和力,并增加了对酸性环境的抵抗力。因此,这些酶代表了许多病原体的重要防御机制,包括金黄色葡萄球菌和结核分枝杆菌(Mtb),它们分别编码赖氨酸(Lys)-PG 合酶 MprF 和 LysX。在这里,我们使用生物信息学、遗传和细菌学方法的组合,对 Mtb 基因组编码的一种蛋白进行了表征,该蛋白编码的蛋白含有与 MprF 样结构域高度相似的结构域,表明该蛋白可能是一种新的 aaPG 合酶家族成员。然而,与 MprF 和 LysX 的同源结构域位于细胞质中不同,我们预测 LysX2 中的 MprF 样结构域位于细胞外区。
我们使用 LysX2 与大肠杆菌 PhoA 和 LacZ 蛋白的遗传融合,证实了这种独特的膜拓扑结构,以及 LysX 和 MprF 作为基准。在耻垢分枝杆菌中表达 lysX2 可提高细胞对人β-防御素 2 和亚硝酸钠的抗性,增强细胞在酸性 pH 环境下的活力并延迟生物膜的形成。值得注意的是,Mtl LysX2 在暴露于酸性环境时显著降低了细菌表面的负电荷。此外,我们在主要的人类病原体和与人类感染相关的快速生长分枝杆菌中发现了 LysX2 同源物,但在环境和非致病性分枝杆菌中没有发现。
总体而言,我们的数据表明 LysX2 是 MprF 样蛋白家族中新一类的原型,该蛋白可能通过其暴露在细胞膜细胞外侧面的催化结构域来增强致病性物种的生存能力,并通过一种尚未确定的机制降低细菌表面的负电荷。