| Congresso Brasileiro de Microbiologia 2023 | Resumo: 763-1 | ||||
Resumo:Heme, an iron-containing protoporphyrin, is an essential molecule to almost every organism. It acts as a cofactor of enzymes like cytochromes, catalases, and peroxidases that are involved in crucial cell processes. This abundant iron source is in the center of the battle between pathogens and hosts for metals during infection, a process known as nutritional immunity. Heme is also highly reactive and its synthesis, uptake, and efflux must be tightly regulated to avoid oxidative stress. Previously, we demonstrated that Chromobacterium violaceum, a Gram-negative environmental bacterium that causes deadly opportunistic infections, acquires and uses exogenous heme as an iron source via the ChuRSTUV system. Moreover, we showed that this system has a role in C. violaceum virulence, and its expression is regulated by ChuP, a HemP/HmuP family regulator, possibly in a post-transcriptional manner. Here, we confirm that ChuP acts as a post-transcriptional regulator of genes involved in heme uptake not only under iron deficiency but also under heme excess in C. violaceum. Using RNA-seq and RT-qPCR, we demonstrate that ChuP regulates the expression of a small regulon comprising 9 genes (chuPRSTUV operon, vbuA, CV_1141, and CV_1142) under iron deficiency. In silico analyses revealed HPREs (HmuP-responsive elements) that form secondary structures in the 5’-UTR or intergenic regions of these genes. Employing translational fusions to GFP, we show that the ChuP regulator and these HPREs are both required for chuR and vbuA expression as verified by flow cytometry and fluorescence microscopy. Moreover, mutations in highly conserved amino acid residues of ChuP as well deletion of HPRE-chuR from the genome abolished heme uptake in nutritional assays. We also discovered that the post-transcriptional regulatory mechanism mediated by ChuP stabilizes chuR and vbuA transcripts as determined by mRNA decay assays. Additionally, we demonstrate that besides its role in the uptake of the siderophore viobactin, the TonB-dependent receptor VbuA is also involved in heme acquisition in C. violaceum. Employing an RNA-seq analysis under heme excess, we identified more than 100 differentially expressed genes that are involved in different metabolic processes, such as energy production, cell wall maintenance, and lipid and amino acid transport/metabolism. Interestingly, genes that belong to the chu operon had a different pattern of expression under this condition, with an increase in chuPR and a decrease in chuSTUV transcripts. Together, these data suggests that ChuP is a heme-sensor post-transcriptional regulator acting over the mRNA stability of its gene targets according to heme levels in C. violaceum. Palavras-chave: iron homeostasis, heme homeostasis, siderophore, post-transcriptional regulation, Chromobacterium violaceum Agência de fomento:Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) | |||||