Mechanisms controlling the regulation of beta-lactamases in Chromobacterium violaceum

• Grant number: 22/07135-8
• Support Opportunities: Scholarships abroad - Research Internship - Master's degree
• Start date: February 01, 2023
• End date: July 31, 2023
• Field of knowledge: Biological Sciences - Genetics - Molecular Genetics and Genetics of Microorganisms
• Principal Investigator: José Freire da Silva Neto
• Grantee: Luís Gustavo Laranjeiro Alves
• Supervisor: Isabel da Silva Henriques
• Host Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Institution abroad: Universidade de Coimbra (UC), Portugal
• Associated to the scholarship: 21/01911-3 - Signaling and regulatory pathways of beta-lactamases in Chromobacterium violaceum, BP.MS

Abstract

The bacterial cell wall is composed of peptidoglycan, a macromolecule that acts to confer rigidity, resistance, and osmotic stability. The cycle of peptidoglycan synthesis, degradation, and recycling is critical for bacterial cell homeostasis. Used on the front lines against different infections, ²-lactam antibiotics prevent peptidoglycan synthesis. Different forms of bacterial resistance to ²-lactam antibiotics have already been reported, the main one being the production of ²-lactamases enzymes that inactivate them by cleavage. The hyperproduction of ²-lactamases can occur by mutations in the peptidoglycan recycling pathway, mainly in ampD, which encodes amidases that act in this elegant recycling process. Previous studies by our group indicate that the environmental opportunistic bacterium Chromobacterium violaceum is intrinsically resistant to beta-lactam antibiotics because it expresses two chromosomal beta-lactamases, AmpC and CphA. In this master's project, we seek to understand how these beta-lactamases are regulated. For this, we isolated spontaneous mutants of C. violaceum in the antibiotic ceftazidime and confirmed by antibiogram that 19 isolates are also resistant to other ²-lactam antibiotics. Most of these mutants overexpress both ²-lactamases (AmpC and CphA). The sequencing of the ampD genes (CV_0566, CV_1309, and CV_3031) in these mutants revealed that 10 of them have mutations in a single ampD gene, CV_0566, while the others have their resistance linked to other components of the genome that are not ampD. In this BEPE proposal, we intend to characterize spontaneous mutants and null mutants in genes of the peptidoglycan recycling pathway in order to understand how the regulation of the expression of ²-lactamases AmpC and CphA of C. violaceum occurs. (AU)