RESUMO
Persister cells are a subpopulation of transiently antibiotic-tolerant bacteria associated with chronic infection and antibiotic treatment failure. Toxin-antitoxin systems have been linked to persister cell formation but the molecular mechanisms leading to bacterial persistence are mostly unknown. Here, we show that SprF1, a type I antitoxin, associates with translating ribosomes from the major human pathogen Staphylococcus aureus to reduce the pathogen's overall protein synthesis during growth. Under hyperosmotic stress, SprF1 levels increase due to enhanced stability, accumulate on polysomes and attenuate protein synthesis. Using an internal 6-nucleotide sequence on its 5'-end, SprF1 binds ribosomes and interferes with initiator transfer RNA binding, thus reducing translation initiation. An excess of messenger RNA displaces the ribosome-bound antitoxin, freeing the ribosomes for new translation cycles; however, this RNA antitoxin can also displace ribosome-bound mRNA. This translation attenuation mechanism, mediated by an RNA antitoxin, promotes antibiotic persister cell formation. The untranslated SprF1 is a dual-function RNA antitoxin that represses toxin expression by its 3'-end and fine-tunes overall bacterial translation via its 5'-end. These findings demonstrate a general function for a bacterial RNA antitoxin beyond protection from toxicity. They also highlight an RNA-guided molecular process that influences antibiotic persister cell formation.
Assuntos
Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Staphylococcus aureus/metabolismo , Staphylococcus aureus/patogenicidade , Sistemas Toxina-Antitoxina/fisiologia , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana , Polirribossomos/metabolismo , Staphylococcus aureus/genéticaRESUMO
In Staphylococcus aureus, the transcription factor CodY modulates the expression of hundreds of genes, including most virulence factors, in response to the availability of key nutrients like GTP and branched-chain amino acids. Despite numerous studies examining how CodY controls gene expression directly or indirectly, virtually nothing is known about the extent to which CodY exerts its effect through small regulatory RNAs (sRNAs). Herein, we report the first set of sRNAs under the control of CodY. We reveal that staphylococcal sRNA RsaD is overexpressed >20-fold in a CodY-deficient strain in three S. aureus clinical isolates and in S. epidermidis. We validated the CodY-dependent regulation of rsaD and demonstrated that CodY directly represses rsaD expression by binding the promoter. Using a combination of molecular techniques, we show that RsaD posttranscriptionally regulates alsS (acetolactate synthase) mRNA and enzyme levels. We further show that RsaD redirects carbon overflow metabolism, contributing to stationary phase cell death during exposure to weak acid stress. Taken together, our data delineate a role for CodY in controlling sRNA expression in a major human pathogen and indicate that RsaD may integrate nutrient depletion and other signals to mount a response to physiological stress experienced by S. aureus in diverse environments.
Assuntos
Proteínas de Bactérias/genética , Pequeno RNA não Traduzido/genética , Proteínas Repressoras/genética , Staphylococcus aureus , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Regiões Promotoras Genéticas , Proteínas Repressoras/metabolismo , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Estresse Fisiológico/genética , Fatores de Transcrição/metabolismo , Transcriptoma/genéticaRESUMO
Bacterial type I toxin-antitoxin (TA) systems are widespread, and consist of a stable toxic peptide whose expression is monitored by a labile RNA antitoxin. We characterized Staphylococcus aureus SprA2/SprA2AS module, which shares nucleotide similarities with the SprA1/SprA1AS TA system. We demonstrated that SprA2/SprA2AS encodes a functional type I TA system, with the cis-encoded SprA2AS antitoxin acting in trans to prevent ribosomal loading onto SprA2 RNA. We proved that both TA systems are distinct, with no cross-regulation between the antitoxins in vitro or in vivo. SprA2 expresses PepA2, a toxic peptide which internally triggers bacterial death. Conversely, although PepA2 does not affect bacteria when it is present in the extracellular medium, it is highly toxic to other host cells such as polymorphonuclear neutrophils and erythrocytes. Finally, we showed that SprA2AS expression is lowered during osmotic shock and stringent response, which indicates that the system responds to specific triggers. Therefore, the SprA2/SprA2AS module is not redundant with SprA1/SprA1AS, and its PepA2 peptide exhibits an original dual mode of action against bacteria and host cells. This suggests an altruistic behavior for S. aureus in which clones producing PepA2 in vivo shall die as they induce cytotoxicity, thereby promoting the success of the community.
Assuntos
Proteínas de Bactérias/genética , Infecções Estafilocócicas/genética , Staphylococcus aureus/genética , Sistemas Toxina-Antitoxina/genética , Sequência de Aminoácidos/genética , Regulação Bacteriana da Expressão Gênica/genética , Interações Hospedeiro-Patógeno/genética , Humanos , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/patogenicidade , Staphylococcus aureus/fisiologiaRESUMO
Staphylococcus aureus pathogenesis is directed by regulatory proteins and RNAs. We report the case of an RNA attenuating virulence and host uptake, possibly to sustain commensalism. A S. aureus sRNA, SprC (srn_3610), reduced virulence and bacterial loads in a mouse infection model. S. aureus deleted for sprC became more virulent and increased bacterial dissemination in colonized animals. Conversely, inducing SprC expression lowered virulence and the bacterial load. Without sprC, S. aureus phagocytosis by monocytes and macrophages was higher, whereas bacteria were internalized at lower yields when SprC expression was stimulated. Without sprC, higher internalization led to a greater number of extracellular bacteria, facilitating colonization. SprC expression decreased after phagocytosis, concurring with the facilitated growth of bacteria lacking the sRNA in the presence of an oxidant. The major staphylococcal autolysin facilitates S. aureus uptake by human phagocytes. ATL proved to be negatively regulated by SprC. The SprC domains involved in pairing with atl mRNA were analyzed. The addition of ATL reduced phagocytosis of bacteria lacking sprC with no effects on wild-type bacterial uptake, implying that SprC influences phagocytosis, at least in part, by controlling ATL. Since the control of SprC on ATL was modest, other factors must contribute to atl regulation.
Assuntos
Fagocitose , RNA Bacteriano/metabolismo , Pequeno RNA não Traduzido/metabolismo , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidade , Animais , Linhagem Celular , Feminino , Regulação Bacteriana da Expressão Gênica , Humanos , Camundongos , N-Acetil-Muramil-L-Alanina Amidase/genética , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Estresse Oxidativo , Fagócitos/microbiologia , RNA Mensageiro/química , Ribossomos/metabolismo , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/metabolismo , VirulênciaRESUMO
Tight recognition of codon-anticodon pairings by the ribosome ensures the accuracy and fidelity of protein synthesis. In eubacteria, translational surveillance and ribosome rescue are performed by the 'tmRNA-SmpB' system (transfer messenger RNA-small protein B). Remarkably, entry and accommodation of aminoacylated-tmRNA into stalled ribosomes occur without a codon-anticodon interaction but in the presence of SmpB. Here, we show that within a stalled ribosome, SmpB interacts with the three universally conserved bases G530, A1492 and A1493 that form the 30S subunit decoding centre, in which canonical codon-anticodon pairing occurs. The footprints at positions A1492 and A1493 of a small decoding centre, as well as on a set of conserved SmpB amino acids, were identified by nuclear magnetic resonance. Mutants at these residues display the same growth defects as for DeltasmpB strains. The SmpB protein has functional and structural similarities with initiation factor 1, and is proposed to be a functional mimic of the pairing between a codon and an anticodon.