尿路致病性大肠埃希菌乳酸脱氢酶促进巨噬细胞乳酸化增强致病性的机制

Lactate dehydrogenase from uropathogenic Escherichia coli enhances pathogenicity via promoting macrophage lactylation

摘要:

尿路致病性大肠埃希菌(uropathogenic Escherichia coli, UPEC)是引发尿路感染(urinary tract infection, UTI)最常见的病原菌，能够定殖于尿路上皮细胞，可引发严重的败血症、肾衰竭，对人类健康构成威胁。乳酸化是巨噬细胞抗UPEC感染的一种重要蛋白质翻译后修饰方式。UPEC CFT073菌株的DNA序列中包含具有催化乳酸生成功能的乳酸脱氢酶ldhA基因，但其调控巨噬细胞乳酸化的具体机制尚未明确。目的 探讨ldhA基因产物LdhA调控巨噬细胞乳酸化进而影响UPEC致病性的作用机制。方法 利用生物信息学在线工具预测ldhA基因产物LdhA的功能结构域和跨膜区；通过分子克隆技术体外表达重组蛋白rLdhA；使用乳酸脱氢酶活性检测试剂盒测定rLdhA的乳酸脱氢酶活性；采用Western blotting检测rLdhA进入巨噬细胞的方式及其对巨噬细胞乳酸化的影响；运用ELISA检测rLdhA对巨噬细胞炎症因子的影响；利用全自动微生物分析系统检测LdhA对UPEC CFT073耐药性的影响；分别用产生LdhA的野生型UPEC CFT073菌株(CFT073^wt)、敲除ldhA基因的CFT073菌株(CFT073^ΔldhA)，或经rLdhA及乳酸脱氢酶抑制剂预处理的rLdhA感染或尾静脉注射小鼠，观察并测定LdhA对小鼠致病性的影响。结果 LdhA蛋白含有乳酸脱氢酶功能域且能够外分泌。成功构建了ldhA基因体外表达系统，表达并提纯了重组蛋白rLdhA。rLdhA具有乳酸脱氢酶功能，能通过网格蛋白介导的内吞作用进入巨噬细胞，并剂量依赖性地促进巨噬细胞乳酸化水平。rLdhA显著抑制脂多糖(lipopolysaccharide, LPS)诱导的巨噬细胞炎症因子生成。LdhA能够显著影响UPEC CFT073的耐药性，并促进UPEC对小鼠的致病性。结论 LdhA通过促进巨噬细胞的乳酸化水平抑制炎症因子的产生，进而增强UPEC的致病性，其具体的分子调控机制尚需进一步研究。本研究为进一步阐明UPEC的致病性提供了新的科学依据。

Abstract:

Uropathogenic Escherichia coli (UPEC) is the leading cause of urinary tract infections (UTIs). It can adhere to and colonize uroepithelial cells, disseminate systemically, and induce severe sepsis and subsequent renal failure, posing a substantial threat to global public health. Emerging evidence indicates that lactylation, a key post-translational modification (PTM) in macrophages, plays a crucial role in the host defense against UPEC infection. Notably, the UPEC CFT073 strain harbors ldhA, which encodes lactate dehydrogenase (LDH), an enzyme critical for lactate biosynthesis. However, the mechanism by which LdhA (the ldhA-encoded LDH) regulates macrophage lactylation during UPEC infection remains elusive.Objective To elucidate how LdhA modulates macrophage lactylation and thereby impacts UPEC pathogenicity.Methods Online bioinformatics tools were used to predict the functional domains and transmembrane regions of LdhA. The recombinant protein rLdhA was generated via molecular cloning, and its LDH activity was measured by a commercial LDH activity assay kit. Western blotting was performed to assess the cellular entry of rLdhA into macrophages and its regulatory effect on macrophage lactylation. Enzyme-linked immunosorbent assay (ELISA) was employed to measure the secretion of inflammatory cytokines in macrophages treated with rLdhA. The drug resistance profile of UPEC CFT073 (wild-type and ldhA-knockout strains) was analyzed via an automated microbial identification system. To evaluate the role of LdhA in UPEC pathogenicity, we treated mice with the wild-type UPEC CFT073 (CFT073^wt), ldhA-deficient mutant (CFT073^ΔldhA), or rLdhA (with or without pretreatment with an LDH inhibitor) through intraperitoneal injection or tail vein injection, and then observed and quantified pathogenic phenotypes.Results LdhA harbored a LDH domain and was secreted extracellularly. We successfully established an expression system for ldhA and achieved efficient expression and purification of rLdhA. Functional assays confirmed that rLdhA exhibited LDH activity and can enter macrophages via clathrin-mediated endocytosis, subsequently enhancing macrophage lactylation in a dose-dependent manner. Additionally, rLdhA significantly inhibited lipopolysaccharide (LPS)-induced inflammatory cytokine production in macrophages. Furthermore, LdhA was found to substantially modulate the drug resistance profile of UPEC CFT073. In vivo studies demonstrated that LdhA promoted the pathogenicity of UPEC in a mouse infection model.Conclusion Collectively, our findings demonstrate that LdhA enhances UPEC pathogenicity by upregulating macrophage lactylation and suppressing the production of proinflammatory cytokines. However, the underlying molecular mechanisms mediating this regulatory cascade remain to be fully elucidated and warrant further exploration. This study offers a new theoretical basis for deciphering the pathogenic mechanisms of UPEC infections.