Hha toxin regulates diverse phenotypes of uropathogenic Proteus mirabilis including virulence

Background: To disclose Proteus mirabilis copper detoxification mechanisms, we performed Tn5-mutagenesis and an hha gene-disrupted mutant was isolated, exhibiting increased copper sensitivity. We investigated the role of Hha in P. mirabilis, focusing on the virulence aspects. Methods: Assays of copper susceptibility, swarming/swimming, biofilm formation, persister cell formation, killing by macrophages, adhesion, invasion, urothelial cell cytokine expression, wax moth virulence, were performed. The invertible element assay, TEM, EMSA and SDM were used to examine MR/P fimbria expression, flagellar production, Hha-flhDC promoter binding and the importance of Hha 13th amino acid cysteine (Hha C13), respectively. RT-PCR was conducted to demonstrate the tomB-hha operon. Hha-related gene expression and regulation were elucidated by RT-qPCR and transcriptional reporter assay. Results: We showed P. mirabilis hha and tomB form an operon and TomB antagonized Hha toxicity. The Hha was involved in copper susceptibility, swimming/swarming, biofilm formation and persister cell formation. The hha mutant had lower adhesion/invasion abilities, triggered higher cytokine expression of urothelial cells and was more sensitive to macrophage killing and attenuated in wax moth. P. mirabilis Hha C13 is crucial in toxicity and Hha-TomB interaction. Deletion of hha reduced relA/rpoS expression. CpxR controlled hha promoter activity. Finally, copper was the signal to induce cpxR/hha expression. Conclusion: We disclosed that P. mirabilis Hha of the toxin-antitoxin system (TA) participates in virulence and its regulation by copper-sensing CpxR. This work provides Hha as a potential drug target for combating P. mirabilis urinary tract infections and opens new perspectives for studying TA aiming at controlling bacterial virulence.