MarR family transcription factors and antibiotic resistance in Chromobacterium violaceum

Author(s)

Kelly Cristina Martins Barroso

• Document type: Master's Dissertation
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC)
• Defense date: 2017-11-09

Examining board members

José Freire da Silva Neto; Rodrigo da Silva Galhardo; Rafael Silva Rocha

• Advisor: José Freire da Silva Neto

Abstract

Antibiotic resistance is a global public health problem with serious consequences for the treatment of various bacterial infections. MarR family transcription factors have been described controlling antibiotic resistance and several other processes in bacteria. In this work, we studied mechanisms of antibiotic resistance in Chromobacterium violaceum, an environmental Gramnegative bacterium that can act as a human opportunistic pathogen. The strategy involved a screening of an available collection of thirteen C. violaceum mutant strains of MarR family transcription factors, by susceptibility testing for 24 antibiotics. These assays revealed that only the ?emrR mutant showed increased resistance to the antibiotic nalidixic acid in relation to the wild-type strain. This increased resistance of the ?emrR mutant to nalidixic acid was reversed in a complemented strain of this mutant, as verified by viability, disk diffusion, and minimal inhibitory concentration (MIC) assays. The phenotype of decreased violacein production of this mutant, observed in a liquid medium, was also complemented. In addition, it was performed the isolation of spontaneous mutants of C. violaceum resistant to nalidixic acid with a point mutation in emrR. DNA microarray assays showed that EmrR represses a few dozen of genes, including the emrCAB operon, which encodes the EmrCAB efflux pump. Northern blot assays confirmed that EmrR represses the emrCAB operon and that the expression of this pump is induced by salicylate, but not other compounds, such as nalidixic acid or ethidium bromide. Electrophoretic mobility shift assays (EMSA) showed that the purified EmrR protein binds directly to the promoter regions of emrR, emrCAB and several other genes of the EmrR regulon, to exert a direct negative regulation of these genes. A ?emrCAB null mutant strain was obtained, but the absence of this efflux pump did not make C. violaceum more susceptible to nalidixic acid, suggesting that it is important only under conditions in which it is induced. These inducing conditions may include 10 oxidative stress since antioxidant enzymes are part of the EmrR regulon and the EmrR protein has formed covalent dimers in the presence of oxidizing agents in vitro. Therefore, our data reveal that point mutations or molecules such as salicylate abolish the repressive activity of the EmrR transcription factor on the emrCAB operon, causing overexpression of the EmrCAB efflux pump and increasing the resistance to nalidixic acid in C. violaceum. (AU)

• FAPESP's process: 15/12605-0 - MarR family transcription factors and antibiotic resistance in Chromobacterium violaceum
• Grantee: Kelly Cristina Martins Barroso
• Support Opportunities: Scholarships in Brazil - Master