| Congresso Brasileiro de Microbiologia 2023 | Resumo: 862-1 | ||||
Resumo:Zinc is an essential trace element for all forms of life by playing structural and catalytic functions in many metalloproteins. However, in high concentrations zinc mismetalates non-target proteins, inhibiting their functions and blocking key biological processes. Zinc toxicity can be used by the host as a defense mechanism against pathogens, in which immune cells can accumulate zinc in the phagosome as part of the antimicrobial response. Cadmium, in turn, is highly toxic and has no apparent biological role. Bacteria have many mechanisms for protection against metal intoxication, such as the extrusion of metals by P-type ATPase efflux pumps. In this work, we characterized the role of two divergent genes, CV_1154 and CV_1153, in protection against metal intoxication in the opportunistic pathogenic bacterium Chromobacterium violaceum. These genes encode a putative P-type ATPase and a predicted MerR family transcription factor that we named ZntA and ZntR, respectively. In silico analysis revealed that ZntA has a typical amino acid signature of the P1B-2-subgroup of P-type ATPases. We obtained null ΔzntA and ΔzntR mutant strains by an allelic exchange protocol, and their respective complemented strains, which were confirmed by PCR. Phenotypic analyzes by growth curves, disk diffusion, and CFU assays revealed that both mutant strains, ΔzntA and ΔzntR, were more susceptible to zinc and cadmium intoxication, when compared with the WT strain. These phenotypes were reverted in the complemented strains. We investigated the expression of zntA and zntR in response to zinc and cadmium in the wild-type strain by fusing their promoter regions to the promoterless lacZ gene. Beta-galactosidase assays indicated that the expression of zntA, but not of zntR, increased under conditions of zinc and cadmium excess. Furthermore, Zur had a role in ZntA regulation since the activity of the zntA promoter increased in a Δzur strain. To verify the importance of ZntA in C. violaceum virulence, we performed survival assays in mice. The ΔzntA was as virulent as the wild-type strain, suggesting that ZntA does not have a role in C. violaceum virulence in an acute model of infection. We will investigate the role of ZntA in other infection models, such as macrophages and amoebas. Collectively, our data indicate that the P-type ATPase ZntA and the regulator ZntR are determinants of zinc and cadmium tolerance in C. violaceum, and suggest that ZntR could be a transcriptional activator of zntA. This work sheds new insights into the molecular mechanisms used by C. violaceum to overcome zinc and cadmium toxicity. Palavras-chave: Chromobacterium violaceum, Efflux pump, MerR transcription factor, Metal resistance, P-type ATPase Agência de fomento:FAPESP, FAEPA, CNPq | |||||