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| Author(s): |
Vinicius Marques de Lima
Total Authors: 1
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| Document type: | Master's Dissertation |
| Press: | Ribeirão Preto. |
| Institution: | Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC) |
| Defense date: | 2020-09-15 |
| Examining board members: |
José Freire da Silva Neto;
Aline Maria da Silva;
Alessandra Alves de Souza
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| Advisor: | José Freire da Silva Neto |
| Abstract | |
Iron is an essential metal in several cell processes, including respiration and gene expression, acting as a cofactor of different bacterial proteins. An important iron source in the host is found into the heme group of hemoproteins like hemoglobin. Therefore, bacteria evolved efficient mechanism for iron acquisition, like the production of small chelating molecules known as siderophores and the expression of specialized transporters for the uptake of siderophores and hemoproteins. In this work, we characterized the mechanisms of heme uptake in Chromobacterium violaceum, an opportunistic Gram-negative beta-proteobacterium that occasionally causes severe infections in humans. In silico analyses in the C. violaceum ATCC 12472 genome revealed the genes CV_3895-96-97-RS19290-CV_3898-99, here named chuPRSTUV, as candidates for coding a regulatory protein (ChuP), an outer membrane TonB-dependet receptor (ChuR), a heme degradation enzyme (ChuS), and an inner membrane ABC-type transporter (ChuTUV). Null-mutant strains with deletion of chuP, chuR, chuS, chuTUV, and chuPRSTUV were obtained; a mutant strain with deletion of the chuPRSTUV and cbaCEBA genes, the latter for siderophore synthesis, was also generated. Both the wild-type and mutant strains showed high tolerance to heme toxicity assessed by cell viability. With the exception of chuS, all other genes in the chuPRSTUV operon were required for heme and hemoglobin utilization in stimulation assays. Besides, the DchuP, DchuPRSTUV, and DcbaCEBADchuPRSTUV strains displayed delayed growth under iron deficiency and/or heme sufficiency. Interestingly, the chuP gene demonstrated a role in siderophore activity, displayed by increased halos in CAS assay for the mutants without chuP. Expression assays demonstrated that the chuPRSTUV operon is controlled by a promoter in front of the chuP gene that responds to the presence of iron and heme, being more expressed in a situation where these molecules are limited. The ChuP protein interacted with heme in vitro and its regulatory activity appears to be post-transcriptional, as ChuP showed no DNA-binding activity. In vivo assays with mice revealed that the ChuPRSTUV system plays a role in the C. violaceum virulence, with attenuation of virulence observed in mutants of the chu operon mainly in the absence of siderophores. Therefore, the data obtained in this work revealed that C. violaceum uses hierarchically the mechanisms of iron uptake by siderophores and by heme to establish a successful infection in the host. (AU) | |
| FAPESP's process: | 18/17716-2 - Mechanisms of heme uptake in Chromobacterium violaceum |
| Grantee: | Vinicius Marques de Lima |
| Support Opportunities: | Scholarships in Brazil - Master |