| Congresso Brasileiro de Microbiologia 2023 | Resumo: 992-1 | ||||
Resumo:Zinc (Zn) is a transition metal necessary as a micronutrient for all organisms, due to its importance as an enzyme cofactor. Taking into account that during infection hosts limit zinc availability, bacteria have many mechanisms for zinc uptake. In contrast, excess of Zn can have cytotoxic effects due to protein mismetallation. Thus, maintenance of Zn homeostasis is critical for bacterial survival. Previously, we demonstrated that the ABC-type transporter ZnuABC is required for Zn acquisition and virulence in Chromobacterium violaceum, a Gram-negative, opportunistic bacterium that cause deadly human infections. Here, we intend to identify the global repertoire of genes regulated by the zinc uptake regulator Zur, and characterize the small hypothetical protein CV_RS15040 in C. violaceum. The analysis of the transcriptome was performed comparing RNA sequencing (RNA-seq) of the wild-type strain (WT) with that of the null mutant Δzur. We found that few genes were upregulated in Δzur, including CV_RS15025 (znuC), CV_RS15030 (znuB), CV_RS15035 (znuA), CV_RS15040 (hypothetical protein), CV_RS15045 (metallochaperone), and CV_RS05670 (P-type ATPase). The upregulation of selected genes in Δzur was validated by RT-qPCR. These data indicates that Zur acts as a repressor of a small regulon in C. violaceum. The Zur protein was purified and tested in an EMSA assay with a probe of the zur promoter. Zur binding was detected when Zn was included in the reaction and disrupted in the presence of EDTA, indicating that holo-Zur represses directly its own operon. Among the genes regulated by Zur, we selected the small protein CV_RS15040 to be characterized. We generated a null mutant (ΔCV_RS15040) and a complement strain for this gene. Growth curves performed in LB depleted for Zn with 1 mM EDTA indicated that the ΔCV_RS15040 mutant showed reduced growth compared with WT and complemented strain. We tested the role of CV_RS15040 in C. violaceum virulence using an acute intraperitoneal infection assay in mice. Interesting, the ΔCV_RS15040 was strongly attenuated for virulence when compared with WT and complemented strain. Given that the ΔCV_RS15040 showed the same phenotypes previously observed for a ΔznuABC mutant, we speculate that CV_RS15040 could act as an auxiliary protein of the ZnuABC transporter required for zinc acquisition. Altogether, our data revealed that Zur regulates few genes in C. violaceum, most of them involved in Zn uptake, including the small protein CV_RS15040. Our data revealing strong virulence attenuation of ΔCV_RS15040 suggest that targeting zinc acquisition is a powerful strategy to help in the combat of opportunistic pathogens. Palavras-chave: RNA-seq, ZnuABC, Bacterial Virulence, Zinc Homeostasis, Chromobacterium violaceum Agência de fomento:FAPESP, FAEPA, CNPq, CAPES | |||||