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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 (CFT073wt), ldhA-deficient mutant (CFT073ΔldhA ), 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.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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