Signaling and regulatory pathways of beta-lactamases in Chromobacterium violaceum

Author(s)

Luís Gustavo Laranjeiro Alves

• Document type: Master's Dissertation
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC)
• Defense date: 2024-02-07

Examining board members

José Freire da Silva Neto; Rodrigo da Silva Galhardo; Eliana Guedes Stehling

• Advisor: José Freire da Silva Neto

Abstract

One of the biggest public health problems is the increase in antibiotic resistance in pathogenic bacteria. Worldwide, there is a high prevalence of resistance to beta-lactam antibiotics mediated by beta-lactamases enzymes. By producing two chromosomal beta-lactamases, AmpC and CphA, the environmental opportunistic bacterium Chromobacterium violaceum is intrinsically resistant to several beta-lactam antibiotics. However, little is known about the signaling and regulation pathways of these enzymes in this bacterium. In this study, we isolated spontaneous mutants resistant to the antibiotic ceftazidime to elucidate how these beta-lactamases are regulated. By antibiogram, we confirmed resistance to several other beta-lactams in 13 of these spontaneous mutants. In 12 of them, there was overexpression of the ampC and cphA genes, as verified by beta-galactosidase assays. DNA sequencing of the three paralogous genes of the C. violaceum AmpD amidase, an enzyme involved in peptidoglycan recycling, revealed mutations of different types in AmpD1 (CV_0566) in most spontaneous mutants, but not in AmpD2 or AmpD3. Single and combined null mutants of the three-amidase genes were obtained. Overexpression of both beta-lactamases and increased resistance to beta-lactams were observed only in mutants with ampD1 deletion. When introduced into ampD1 null or spontaneous mutants, the ampD1 gene rescued the phenotypes of these mutants. The AmpD1 amidase from C. violaceum has a unique architecture among bacterial amidases with the presence of an N-terminal acetyltransferase domain. Three spontaneous mutants were chosen for whole genome sequencing (WGS). The results revealed mutations in the genes CV_1820 (RNAse E), CV_2060 (RseB, a negative regulator of sigma E) and CV_3538 (IspG, isoprenoid synthesis enzyme), suggesting new pathways potentially involved in resistance to beta-lactam antibiotics. The mutation identified by WGS in the intergenic region of the CV_1922 transcription factor actually turned out to be an artifact resulting from the excision of the CV_1923 gene, one of the five copies of transposases found in C. violaceum. Alternatively, a DNA affinity pull-down assay allowed us to isolate different proteins that could be candidates for transcriptional regulators of both beta- lactamases. In conclusion, our data indicate that the occurrence of mutations in the AmpD1 amidase is a common mechanism that leads to the overexpression of the beta-lactamases AmpC and CphA and the high resistance of C. violaceum to several beta-lactam antibiotics. (AU)

• FAPESP's process: 21/01911-3 - Signaling and regulatory pathways of beta-lactamases in Chromobacterium violaceum
• Grantee: Luís Gustavo Laranjeiro Alves
• Support Opportunities: Scholarships in Brazil - Master