RESUMO
Toxin-antitoxin (TA) systems are ubiquitous genetic elements in prokaryotes, but their biological importance is poorly understood. Mycobacterium smegmatis contains eight putative TA systems. Previously, seven TAs have been studied, with five of them being verified as functional. Here, we show that Ms0251-0252 is a novel TA system in that expression of the toxin Ms0251 leads to growth inhibition that can be rescued by the antitoxin Ms0252. To investigate the functional roles of TA systems in M. smegmatis, we deleted the eight putative TA loci and assayed the mutants for resistance to various stresses. Deletion of all eight TA loci resulted in decreased survival under starvation conditions and altered fitness when exposed to environmental stresses. Furthermore, we showed that deletion of the eight TA loci decreased resistance to phage infection in Sauton medium compared with the results using 7H10 medium, suggesting that TA systems might have different contributions depending on the nutrient environment. Furthermore, we found that MazEF specifically played a dominant role in resistance to phage infection. Finally, transcriptome analysis revealed that MazEF overexpression led to differential expression of multiple genes, including those related to iron acquisition. Altogether, we demonstrate that TA systems coordinately function to allow M. smegmatis to adapt to changing environmental conditions. IMPORTANCE Toxin-antitoxin (TA) systems are mechanisms for rapid adaptation of bacteria to environmental changes. Mycobacterium smegmatis, a model bacterium for studying Mycobacterium tuberculosis, encodes eight putative TA systems. Here, we constructed an M. smegmatis mutant with deletions of all eight TA-encoding genes and evaluated the resistance of these mutants to environmental stresses. Our results showed that different TA systems have overlapping and, in some cases, opposing functions in adaptation to various stresses. We suggest that complementary TA modules may function together to regulate the bacterial stress response, enabling adaptation to changing environments. Together, this study provides key insights into the roles of TA systems in resistance to various environmental stresses, drug tolerance, and defense against phage infection.
Assuntos
Antitoxinas , Toxinas Bacterianas , Mycobacterium tuberculosis , Sistemas Toxina-Antitoxina , Mycobacterium smegmatis/metabolismo , Sistemas Toxina-Antitoxina/genética , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Mycobacterium tuberculosis/genética , Antitoxinas/genética , Antitoxinas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismoRESUMO
Although not evolved to function in eukaryotes, prokaryotic toxin Kid induces apoptosis in human cells, and this is avoided by coexpression of its neutralizing antitoxin, Kis. Inspired by the way Kid becomes active in bacterial cells we had previously engineered a synthetic toxin-antitoxin system bearing a Kis protein variant that is selectively degraded in cells expressing viral oncoprotein E6, thus achieving highly selective killing of cancer cells transformed by human papillomavirus. Here we aimed to broaden the type of oncogenic insults, and therefore of cancer cells, that can be targeted using this approach. We show that appropriate linkage of the kis gene to a single, fully complementary, target site for an oncogenic human microRNA enables the construction of a synthetic toxin-antitoxin pair that selectively kills cancer cells overexpressing that particular microRNA. Importantly, the resulting system spares nontargeted cells from collateral damage, even when they overexpress highly homologous, though nontargeted, microRNAs.
Assuntos
Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , MicroRNAs/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Western Blotting , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Doxiciclina/farmacologia , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/genética , Células HEK293 , Humanos , MicroRNAs/genética , Sistemas Toxina-Antitoxina/genética , Sistemas Toxina-Antitoxina/fisiologiaRESUMO
The molecular mechanisms of tolerance and persistence associated with several compounds in Acinetobacter baumannii clinical isolates are unknown. Using transcriptomic and phenotypic studies, we found a link between mechanisms of bacterial tolerance to chlorhexidine and the development of persistence in the presence of imipenem in an A. baumannii strain belonging to clinical clone ST-2 (OXA-24 ß-lactamase and AbkAB toxin-antitoxin [TA] system carried in a plasmid). Interestingly, the strain A. baumannii ATCC 17978 (AbkAB TA system from plasmid) showed persistence in the presence of imipenem and chlorhexidine.