Abstract

Copper, an essential trace element for all lives, is toxic when accumulated in excess. Thus, bacteria evolve copper detoxification systems to maintain copper homeostasis. Copper is also a member of the human innate immunity. For Proteus mirabilis, an important uropathogen often causing catheter-associated urinary tract infections, the approaches to maintain copper homeostasis remain unclear. Using Tn5-mutagenesis, we isolated cueO and cueR mutants which exhibited more sensitive to copper than the wild-type. The cueO gene, encoding a periplasmic multicopper oxidase, belongs to COG category of cell division and chromosome partitioning. The cueR gene encodes a copper-responsive transcriptional factor. Transcriptomic analysis revealed that, cueO expression increased 11-fold under the copper stress. We then investigated the role of cueO in cell differentiation and found that overexpression of cueO decreased swarming motility and swarmer cell length, while cueO mutant had increased swarming motility and cell length compared to wild-type, suggesting that cueO may play a role in cell differentiation in P. mirabilis. In addition, we found cueO mutant showed decreased tolerance to oxidative stress and killing by macrophages and reduced ability to adhere and invade urothelial cells, which correspond to the lower colonization in the mouse urinary tract compared to the wild-type. Mouse colonization assay also revealed lower colonization ability of cueR mutant. We further showed the copper-induced expression of cueO through CueR. Interestingly, we found that aldehydes such as methylglyoxal can decrease cueR mRNA level and uplift bacterial susceptibility to copper. In summary, a copper detoxification system made of CueO and CueR was found in P. mirabilis. Besides copper homeostasis, CueR and CueO also participated in virulence. To our knowledge, this is the first report disclosing the role of P. mirabilis copper detoxification system in virulence.