Previato-Mello, M. | bioRxiv Science

bioRxiv2024

Mutations in ampD cause hyperproduction of AmpC and CphA beta-lactamases and high resistance to beta-lactam antibiotics in Chromobacterium violaceum

Bacterial resistance to beta-lactam antibiotics mediated by beta-lactamase enzymes is widespread worldwide. Chromobacterium violaceum, an environmental Gram-negative bacteria pathogen, is intrinsically resistant to some beta-lactam antibiotics. In this work, we found that mutations in an ampD gene, encoding a peptidoglycan-recycling amidase, cause hyperproduction of two chromosomal beta-lactamases (AmpC and CphA), conferring high beta-lactam resistance in C. violaceum. Susceptibility tests using {Delta}ampC, {Delta}cphA, and{Delta} ampC{Delta}cphA mutant strains revealed specific susceptibility profiles to penicillin, cephalosporin, and carbapenem beta-lactams, suggesting that AmpC is a broad-spectrum beta-lactamase (penicillinase and cephalosporinase), while CphA is a narrow-spectrum metallo-carbapenemase. Beta-galactosidase assays indicate that the expression of ampC and cphA increased in response to beta-lactams. We isolated C. violaceum spontaneous mutants resistant to the antibiotic ceftazidime and found that most mutants were also resistant to several other beta-lactams and overexpressed ampC and cphA. DNA sequencing of the three paralog genes encoding the C. violaceum AmpD amidases revealed mutations of different types in AmpD1 (CV_0566) in most of the spontaneous mutants, but no mutation was found in AmpD2 or AmpD3. Analysis of single and combined null amidase mutants revealed overexpression of both beta-lactamases and increased resistance to beta-lactams only in mutants with deleted ampD1. When introduced into ampD1 null or spontaneous mutants, the ampD1 gene rescued the antibiotic-related phenotypes. The AmpD1 amidase from C. violaceum has a unique architecture with an N-terminal acetyltransferase domain. Our work offers new insights into the mechanisms of beta-lactamase-mediated antibiotic resistance and open perspectives to improve the treatment of C. violaceum infections.

microbiology↗

bioRxiv2022

A quorum sensing-regulated type VI secretion system containing multiple nonredundant VgrG proteins is required for interbacterial competition in Chromobacterium violaceum

The environmental pathogenic bacterium Chromobacterium violaceum kills Gram-positive bacteria delivering violacein packed into outer membrane vesicles, but nothing is known about its contact-dependent competition mechanisms. In this work, we demonstrate that C. violaceum utilizes a type VI secretion system (T6SS) containing multiple VgrG proteins primarily for interbacterial competition. The single T6SS of C. violaceum contains six vgrG genes, which are located in the main T6SS cluster and four vgrG islands. Using T6SS-core component null mutant strains, western blot, fluorescence microscopy, and competition assays, we show that the C. violaceum T6SS is active and required for competition against Gram-negative bacteria such as Pseudomonas aeruginosa but dispensable for C. violaceum infection in mice. Characterization of single and multiple vgrG mutants revealed that, despite having high sequence similarity, the six VgrGs show little functional redundancy, with VgrG3 showing a major role in T6SS function. Our coimmunoprecipitation data support a model of VgrG3 assembling heterotrimers with the other VgrGs. Moreover, we determined that the promoter activities of T6SS genes increased at high cell density, but the produced Hcp protein was not secreted under such condition. This T6SS growth-phase-dependent regulation was dependent on CviR but not on CviI, the components of a C. violaceum quorum sensing (QS) system. Indeed, a {Delta}cviR but not a {Delta}cviI mutant was completely defective in Hcp secretion, T6SS activity, and interbacterial competition. Overall, our data reveal that C. violaceum relies on a QS-regulated T6SS to outcompete other bacteria and expand our knowledge about the redundancy of multiple VgrGs. IMPORTANCEThe type VI secretion system (T6SS) is a contractile nanomachine used by many Gram-negative bacteria to inject toxic effectors into adjacent cells. The delivered effectors are bound to the components of a puncturing apparatus containing the protein VgrG. The T6SS has been implicated in pathogenesis and, more commonly, in competition among bacteria. Chromobacterium violaceum is an environmental bacterium that causes deadly infections in humans. In this work, we characterized the single T6SS of C. violaceum ATCC 12472, including its six VgrG proteins, regarding its function and regulation. This previously undescribed C. violaceum T6SS is active, regulated by QS, and required for interbacterial competition instead of acute infection in mice. Among the VgrGs, VgrG3, encoded outside of the main T6SS cluster, showed a major contribution to T6SS function. These results shed light on a key contact-dependent killing mechanism used by C. violaceum to antagonize other bacteria.

microbiology↗