RESUMO
Genome-wide CRISPR screens enable systematic interrogation of gene function. However, guide RNA libraries are costly to synthesize, and their limited diversity compromises the sensitivity of CRISPR screens. Using the Streptococcus pyogenes CRISPR-Cas adaptation machinery, we developed CRISPR adaptation-mediated library manufacturing (CALM), which turns bacterial cells into "factories" for generating hundreds of thousands of crRNAs covering 95% of all targetable genomic sites. With an average gene targeted by more than 100 distinct crRNAs, these highly comprehensive CRISPRi libraries produced varying degrees of transcriptional repression critical for uncovering novel antibiotic resistance determinants. Furthermore, by iterating CRISPR adaptation, we rapidly generated dual-crRNA libraries representing more than 100,000 dual-gene perturbations. The polarized nature of spacer adaptation revealed the historical contingency in the stepwise acquisition of genetic perturbations leading to increasing antibiotic resistance. CALM circumvents the expense, labor, and time required for synthesis and cloning of gRNAs, allowing generation of CRISPRi libraries in wild-type bacteria refractory to routine genetic manipulation.
Assuntos
Sistemas CRISPR-Cas/genética , Genoma Bacteriano/genética , Biblioteca Genômica , Staphylococcus aureus/genética , Escherichia coli/genética , Humanos , RNA Bacteriano/genética , RNA Guia de Cinetoplastídeos/genética , Streptococcus pyogenes/genéticaRESUMO
Multidrug resistance is a global threat as the clinically available potent antibiotic drugs are becoming exceedingly scarce. For example, increasing drug resistance among gram-positive bacteria is responsible for approximately one-third of nosocomial infections. As ribosomes are a major target for these drugs, they may serve as suitable objects for novel development of next-generation antibiotics. Three-dimensional structures of ribosomal particles from Staphylococcus aureus obtained by X-ray crystallography have shed light on fine details of drug binding sites and have revealed unique structural motifs specific for this pathogenic strain, which may be used for the design of novel degradable pathogen-specific, and hence, environmentally friendly drugs.
Assuntos
Antibacterianos/síntese química , Proteínas de Bactérias/química , Desenho de Fármacos , Ribossomos/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Infecção Hospitalar/tratamento farmacológico , Infecção Hospitalar/microbiologia , Cristalografia por Raios X , Deinococcus/efeitos dos fármacos , Deinococcus/genética , Deinococcus/metabolismo , Farmacorresistência Bacteriana Múltipla , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Humanos , Modelos Moleculares , Ribossomos/metabolismo , Ribossomos/ultraestrutura , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Thermus thermophilus/efeitos dos fármacos , Thermus thermophilus/genética , Thermus thermophilus/metabolismoRESUMO
Iron is essential for the survival of most bacteria but presents a significant challenge given its limited bioavailability. Furthermore, the toxicity of iron combined with the need to maintain physiological iron levels within a narrow concentration range requires sophisticated systems to sense, regulate, and transport iron. Most bacteria have evolved mechanisms to chelate and transport ferric iron (Fe3+) via siderophore receptor systems, and pathogenic bacteria have further lowered this barrier by employing mechanisms to utilize the host's hemoproteins. Once internalized, heme is cleaved by both oxidative and nonoxidative mechanisms to release iron. Heme, itself a lipophilic and toxic molecule, presents a significant challenge for transport into the cell. As such, pathogenic bacteria have evolved sophisticated cell surface signaling and transport systems to obtain heme from the host. In this review, we summarize the structure and function of the heme-sensing and transport systems of pathogenic bacteria and the potential of these systems as antimicrobial targets.
Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Membrana Celular/efeitos dos fármacos , Heme/antagonistas & inibidores , Ferro/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Receptores de Superfície Celular/antagonistas & inibidores , Staphylococcus aureus/efeitos dos fármacos , Antibacterianos/síntese química , Antibacterianos/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Transporte Biológico/efeitos dos fármacos , Membrana Celular/metabolismo , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Expressão Gênica , Heme/metabolismo , Metaloporfirinas/síntese química , Metaloporfirinas/farmacologia , Modelos Moleculares , Conformação Proteica , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/metabolismo , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Sideróforos/antagonistas & inibidores , Sideróforos/biossíntese , Staphylococcus aureus/genética , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/metabolismoRESUMO
Prokaryotic type III CRISPR-Cas systems provide immunity against viruses and plasmids using CRISPR-associated Rossman fold (CARF) protein effectors1-5. Recognition of transcripts of these invaders with sequences that are complementary to CRISPR RNA guides leads to the production of cyclic oligoadenylate second messengers, which bind CARF domains and trigger the activity of an effector domain6,7. Whereas most effectors degrade host and invader nucleic acids, some are predicted to contain transmembrane helices without an enzymatic function. Whether and how these CARF-transmembrane helix fusion proteins facilitate the type III CRISPR-Cas immune response remains unknown. Here we investigate the role of cyclic oligoadenylate-activated membrane protein 1 (Cam1) during type III CRISPR immunity. Structural and biochemical analyses reveal that the CARF domains of a Cam1 dimer bind cyclic tetra-adenylate second messengers. In vivo, Cam1 localizes to the membrane, is predicted to form a tetrameric transmembrane pore, and provides defence against viral infection through the induction of membrane depolarization and growth arrest. These results reveal that CRISPR immunity does not always operate through the degradation of nucleic acids, but is instead mediated via a wider range of cellular responses.
Assuntos
Bacteriófagos , Sistemas CRISPR-Cas , Potenciais da Membrana , Staphylococcus aureus , Bacteriófagos/imunologia , Bacteriófagos/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Sistemas CRISPR-Cas/imunologia , Nucleotídeos Cíclicos/metabolismo , RNA Guia de Sistemas CRISPR-Cas , Sistemas do Segundo Mensageiro , Staphylococcus aureus/citologia , Staphylococcus aureus/genética , Staphylococcus aureus/imunologia , Staphylococcus aureus/virologiaRESUMO
Antibiotic resistance is a key medical concern, with antibiotic use likely being an important cause. However, here we describe an alternative route to clinically relevant antibiotic resistance that occurs solely due to competitive interactions among bacterial cells. We consistently observe that isolates of Methicillin-resistant Staphylococcus aureus diversify spontaneously into two distinct, sequentially arising strains. The first evolved strain outgrows the parent strain via secretion of surfactants and a toxic bacteriocin. The second is resistant to the bacteriocin. Importantly, this second strain is also resistant to intermediate levels of vancomycin. This so-called VISA (vancomycin-intermediate S. aureus) phenotype is seen in many hard-to-treat clinical isolates. This strain diversification also occurs during in vivo infection in a mouse model, which is consistent with the fact that both coevolved phenotypes resemble strains commonly found in clinic. Our study shows how competition between coevolving bacterial strains can generate antibiotic resistance and recapitulate key clinical phenotypes.
Assuntos
Staphylococcus aureus Resistente à Meticilina/classificação , Staphylococcus aureus Resistente à Meticilina/genética , Infecções Estafilocócicas/microbiologia , Sequência de Aminoácidos , Animais , Antibacterianos/farmacologia , Bacteriocinas/genética , Bacteriocinas/metabolismo , Biofilmes/efeitos dos fármacos , Evolução Biológica , Feminino , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/fisiologia , Camundongos Endogâmicos BALB C , Fenômenos Microbiológicos , Dados de Sequência Molecular , Pigmentação , Alinhamento de Sequência , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/classificação , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Staphylococcus aureus/fisiologia , Vancomicina/farmacologiaRESUMO
Bacillus subtilis structural maintenance of chromosomes (SMC) complexes are topologically loaded at centromeric sites adjacent to the replication origin by the partitioning protein ParB. These ring-shaped ATPases then translocate down the left and right chromosome arms while tethering them together. Here, we show that the site-specific recombinase XerD, which resolves chromosome dimers, is required to unload SMC tethers when they reach the terminus. We identify XerD-specific binding sites in the terminus region and show that they dictate the site of unloading in a manner that depends on XerD but not its catalytic residue, its partner protein XerC, or the recombination site dif. Finally, we provide evidence that ParB and XerD homologs perform similar functions in Staphylococcus aureus. Thus, two broadly conserved factors that act at the origin and terminus have second functions in loading and unloading SMC complexes that travel between them.
Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/metabolismo , Cromossomos Bacterianos/metabolismo , Integrases/metabolismo , Staphylococcus aureus/enzimologia , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Cromossomos Bacterianos/genética , DNA Primase/genética , DNA Primase/metabolismo , Integrases/genética , Staphylococcus aureus/genéticaRESUMO
Efflux of antibacterial compounds is a major mechanism for developing antimicrobial resistance. In the Gram-positive pathogen Staphylococcus aureus, QacA, a 14 transmembrane helix containing major facilitator superfamily antiporter, mediates proton-coupled efflux of mono and divalent cationic antibacterial compounds. In this study, we report the cryo-EM structure of QacA, with a single mutation D411N that improves homogeneity and retains efflux activity against divalent cationic compounds like dequalinium and chlorhexidine. The structure of substrate-free QacA, complexed to two single-domain camelid antibodies, was elucidated to a resolution of 3.6 Å. The structure displays an outward-open conformation with an extracellular helical hairpin loop (EL7) between transmembrane helices 13 and 14, which is conserved in a subset of DHA2 transporters. Removal of the EL7 hairpin loop or disrupting the interface formed between EL7 and EL1 compromises efflux activity. Chimeric constructs of QacA with a helical hairpin and EL1 grafted from other DHA2 members, LfrA and SmvA, restore activity in the EL7 deleted QacA revealing the allosteric and vital role of EL7 hairpin in antibacterial efflux in QacA and related members.
Assuntos
Infecções Estafilocócicas , Staphylococcus aureus , Humanos , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Microscopia Crioeletrônica , Proteínas de Bactérias/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/química , Antibacterianos/farmacologiaRESUMO
Unregulated cell cycle progression may have lethal consequences and therefore, bacteria have various mechanisms in place for the precise spatiotemporal control of cell cycle events. We have uncovered a new link between chromosome replication/segregation and splitting of the division septum. We show that the DNA translocase domain-containing divisome protein FtsK regulates cellular levels of a peptidoglycan hydrolase Sle1, which is involved in cell separation in the bacterial pathogen Staphylococcus aureus. FtsK interacts with a chaperone (trigger factor, TF) and establishes a FtsK-dependent TF concentration gradient that is higher in the septal region. Trigger factor binds Sle1 and promotes its preferential export at the septal region, while also preventing Sle1 degradation by the ClpXP proteolytic machinery. Upon conditions that lead to paused septum synthesis, such as DNA damage or impaired DNA replication/segregation, TF gradient is dissipated and Sle1 levels are reduced, thus halting premature septum splitting.
Assuntos
Proteínas de Escherichia coli , Infecções Estafilocócicas , Humanos , Segregação de Cromossomos , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Proteínas de Membrana/metabolismo , Divisão Celular , Proteínas de Escherichia coli/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cromossomos Bacterianos/genéticaRESUMO
Lipoproteins of the opportunistic pathogen Staphylococcus aureus play a crucial role in various cellular processes and host interactions. Consisting of a protein and a lipid moiety, they support nutrient acquisition and anchor the protein to the bacterial membrane. Recently, we identified several processed and secreted small linear peptides that derive from the secretion signal sequence of S. aureus lipoproteins. Here, we show, for the first time, that the protein moiety of the S. aureus lipoprotein CamS has a biological role that is distinct from its associated linear peptide staph-cAM373. The small peptide was shown to be involved in interspecies horizontal gene transfer, the primary mechanism for the dissemination of antibiotic resistance among bacteria. We provide evidence that the CamS protein moiety is a potent repressor of cytotoxins, such as α-toxin and leukocidins. The CamS-mediated suppression of toxin transcription was reflected by altered disease severity in in vivo infection models involving skin and soft tissue, as well as bloodstream infections. Collectively, we have uncovered the role of the protein moiety of the staphylococcal lipoprotein CamS as a previously uncharacterized repressor of S. aureus toxin production, which consequently regulates virulence and disease outcomes. Notably, the camS gene is conserved in S. aureus, and we also demonstrated the muted transcriptional response of cytotoxins in 2 different S. aureus lineages. Our findings provide the first evidence of distinct biological functions of the protein moiety and its associated linear peptide for a specific lipoprotein. Therefore, lipoproteins in S. aureus consist of 3 functional components: a lipid moiety, a protein moiety, and a small linear peptide, with putative different biological roles that might not only determine the outcome of host-pathogen interactions but also drive the acquisition of antibiotic resistance determinants.
Assuntos
Infecções Estafilocócicas , Staphylococcus aureus , Humanos , Staphylococcus aureus/genética , Lipoproteínas/genética , Interações Hospedeiro-Patógeno , Moléculas de Adesão Celular , Citotoxinas , PeptídeosRESUMO
Heteroresistance (HR) is an enigmatic phenotype where, in a main population of susceptible cells, small subpopulations of resistant cells exist. This is a cause for concern, as this small subpopulation is difficult to detect by standard antibiotic susceptibility tests, and upon antibiotic exposure the resistant subpopulation may increase in frequency and potentially lead to treatment complications or failure. Here, we determined the prevalence and mechanisms of HR for 40 clinical Staphylococcus aureus isolates, against 6 clinically important antibiotics: daptomycin, gentamicin, linezolid, oxacillin, teicoplanin, and vancomycin. High frequencies of HR were observed for gentamicin (69.2%), oxacillin (27%), daptomycin (25.6%), and teicoplanin (15.4%) while none of the isolates showed HR toward linezolid or vancomycin. Point mutations in various chromosomal core genes, including those involved in membrane and peptidoglycan/teichoic acid biosynthesis and transport, tRNA charging, menaquinone and chorismite biosynthesis and cyclic-di-AMP biosynthesis, were the mechanisms responsible for generating the resistant subpopulations. This finding is in contrast to gram-negative bacteria, where increased copy number of bona fide resistance genes via tandem gene amplification is the most prevalent mechanism. This difference can be explained by the observation that S. aureus has a low content of resistance genes and absence of the repeat sequences that allow tandem gene amplification of these genes as compared to gram-negative species.
Assuntos
Daptomicina , Infecções Estafilocócicas , Humanos , Staphylococcus aureus/genética , Vancomicina , Linezolida/uso terapêutico , Teicoplanina/uso terapêutico , Prevalência , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Infecções Estafilocócicas/genética , Infecções Estafilocócicas/tratamento farmacológico , Oxacilina/uso terapêutico , Mutação , GentamicinasRESUMO
Phage G1 gp67 is a 23 kDa protein that binds to the Staphylococcus aureus (Sau) RNA polymerase (RNAP) σ(A) subunit and blocks cell growth by inhibiting transcription. We show that gp67 has little to no effect on transcription from most promoters but is a potent inhibitor of ribosomal RNA transcription. A 2.0-Å-resolution crystal structure of the complex between gp67 and Sau σ(A) domain 4 (σ(A)(4)) explains how gp67 joins the RNAP promoter complex through σ(A)(4) without significantly affecting σ(A)(4) function. Our results indicate that gp67 forms a complex with RNAP at most, if not all, σ(A)-dependent promoters, but selectively inhibits promoters that depend on an interaction between upstream DNA and the RNAP α-subunit C-terminal domain (αCTD). Thus, we reveal a promoter-specific transcription inhibition mechanism by which gp67 interacts with the RNAP promoter complex through one subunit (σ(A)), and selectively affects the function of another subunit (αCTD) depending on promoter usage.
Assuntos
Inibidores do Crescimento/metabolismo , Myoviridae/metabolismo , Regiões Promotoras Genéticas , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/virologia , Proteínas Virais/metabolismo , Sequência de Bases , Cristalografia por Raios X , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , RNA Bacteriano/genética , RNA Ribossômico/genética , Fator sigma/metabolismo , Staphylococcus aureus/genética , Transcrição GênicaRESUMO
Horizontal gene transfer and mutation are the two major drivers of microbial evolution that enable bacteria to adapt to fluctuating environmental stressors1. Clustered, regularly interspaced, short palindromic repeats (CRISPR) systems use RNA-guided nucleases to direct sequence-specific destruction of the genomes of mobile genetic elements that mediate horizontal gene transfer, such as conjugative plasmids2 and bacteriophages3, thus limiting the extent to which bacteria can evolve by this mechanism. A subset of CRISPR systems also exhibit non-specific degradation of DNA4,5; however, whether and how this feature affects the host has not yet been examined. Here we show that the non-specific DNase activity of the staphylococcal type III-A CRISPR-Cas system increases mutations in the host and accelerates the generation of antibiotic resistance in Staphylococcus aureus and Staphylococcus epidermidis. These mutations require the induction of the SOS response to DNA damage and display a distinct pattern. Our results demonstrate that by differentially affecting both mechanisms that generate genetic diversity, type III-A CRISPR systems can modulate the evolution of the bacterial host.
Assuntos
Sistemas CRISPR-Cas/genética , Sistemas CRISPR-Cas/imunologia , Mutagênese , Mutação , Staphylococcus/genética , Antibacterianos/farmacologia , Bacteriófagos/classificação , Bacteriófagos/fisiologia , Proteínas Associadas a CRISPR/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Desoxirribonucleases/metabolismo , Resistência Microbiana a Medicamentos/efeitos dos fármacos , Resposta SOS em Genética/efeitos dos fármacos , Staphylococcus/efeitos dos fármacos , Staphylococcus/imunologia , Staphylococcus/virologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Staphylococcus aureus/virologia , Staphylococcus epidermidis/efeitos dos fármacos , Staphylococcus epidermidis/genética , Staphylococcus epidermidis/virologia , Fatores de TempoRESUMO
Adaptive immune systems must accurately distinguish between self and non-self in order to defend against invading pathogens while avoiding autoimmunity. Type III CRISPR-Cas systems employ guide RNA to recognize complementary RNA targets, which triggers the degradation of both the invader's transcripts and their template DNA. These systems can broadly eliminate foreign targets with multiple mutations but circumvent damage to the host genome. To explore the molecular basis for these features, we use single-molecule fluorescence microscopy to study the interaction between a type III-A ribonucleoprotein complex and various RNA substrates. We find that Cas10-the DNase effector of the complex-displays rapid conformational fluctuations on foreign RNA targets, but is locked in a static configuration on self RNA. Target mutations differentially modulate Cas10 dynamics and tune the CRISPR interference activity in vivo. These findings highlight the central role of the internal dynamics of CRISPR-Cas complexes in self versus non-self discrimination and target specificity.
Assuntos
Autoimunidade , Proteínas de Bactérias/imunologia , Proteínas Associadas a CRISPR/imunologia , Sistemas CRISPR-Cas/imunologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/imunologia , RNA Bacteriano/imunologia , Tolerância a Antígenos Próprios , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/imunologia , Cinética , Microscopia de Fluorescência , Mutação , Conformação de Ácido Nucleico , Conformação Proteica , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Transdução de Sinais , Imagem Individual de Molécula/métodos , Staphylococcus aureus/enzimologia , Staphylococcus aureus/genética , Staphylococcus aureus/imunologia , Staphylococcus epidermidis/enzimologia , Staphylococcus epidermidis/genética , Staphylococcus epidermidis/imunologia , Relação Estrutura-AtividadeRESUMO
Bacterial infections are often polymicrobial. Pseudomonas aeruginosa and Staphylococcus aureus cause chronic co-infections, which are more problematic than mono-species infections. Understanding the mechanisms of their interactions is crucial for treating co-infections. Staphyloxanthin (STX), a yellow pigment synthesized by the S. aureus crt operon, promotes S. aureus resistance to oxidative stress and neutrophil-mediated killing. We found that STX production by S. aureus, either as surface-grown macrocolonies or planktonic cultures, was elevated when exposed to the P. aeruginosa exoproduct, 2-heptyl-4-hydroxyquinoline N-oxide (HQNO). This was observed with both mucoid and non-mucoid P. aeruginosa strains. The induction phenotype was found in a majority of P. aeruginosa and S. aureus clinical isolates examined. When subjected to hydrogen peroxide or human neutrophils, P. aeruginosa survival was significantly higher when mixed with wild-type (WT) S. aureus, compared to P. aeruginosa alone or with an S. aureus crt mutant deficient in STX production. In a murine wound model, co-infection with WT S. aureus, but not the STX-deficient mutant, enhanced P. aeruginosa burden and disease compared to mono-infection. In conclusion, we identified a role for P. aeruginosa HQNO mediating polymicrobial interactions with S. aureus by inducing STX production, which consequently promotes resistance to the innate immune effectors H2O2 and neutrophils. These results further our understanding of how different bacterial species cooperatively cause co-infections.
Assuntos
Coinfecção , Infecções Estafilocócicas , Humanos , Animais , Camundongos , Staphylococcus aureus/genética , Peróxido de Hidrogênio/farmacologia , Neutrófilos , Infecções Estafilocócicas/microbiologia , Pseudomonas aeruginosa/genética , Fatores Biológicos , BiofilmesRESUMO
Cellular processes require precise and specific gene regulation, in which continuous mRNA degradation is a major element. The mRNA degradation mechanisms should be able to degrade a wide range of different RNA substrates with high efficiency, but should at the same time be limited, to avoid killing the cell by elimination of all cellular RNA. RNase Y is a major endoribonuclease found in most Firmicutes, including Bacillus subtilis and Staphylococcus aureus. However, the molecular interactions that direct RNase Y to cleave the correct RNA molecules at the correct position remain unknown. In this work we have identified transcripts that are homologs in S. aureus and B. subtilis, and are RNase Y targets in both bacteria. Two such transcript pairs were used as models to show a functional overlap between the S. aureus and the B. subtilis RNase Y, which highlighted the importance of the nucleotide sequence of the RNA molecule itself in the RNase Y targeting process. Cleavage efficiency is driven by the primary nucleotide sequence immediately downstream of the cleavage site and base-pairing in a secondary structure a few nucleotides downstream. Cleavage positioning is roughly localised by the downstream secondary structure and fine-tuned by the nucleotide immediately upstream of the cleavage. The identified elements were sufficient for RNase Y-dependent cleavage, since the sequence elements from one of the model transcripts were able to convert an exogenous non-target transcript into a target for RNase Y.
Assuntos
Bacillus subtilis , Regulação Bacteriana da Expressão Gênica , Clivagem do RNA , Estabilidade de RNA , RNA Bacteriano , Staphylococcus aureus , Staphylococcus aureus/genética , Staphylococcus aureus/enzimologia , Bacillus subtilis/genética , Bacillus subtilis/enzimologia , Bacillus subtilis/metabolismo , RNA Bacteriano/metabolismo , RNA Bacteriano/genética , Estabilidade de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Endorribonucleases/metabolismo , Endorribonucleases/genética , Conformação de Ácido Nucleico , Sequência de BasesRESUMO
rRNA modifications play crucial roles in fine-tuning the delicate balance between translation speed and accuracy, yet the underlying mechanisms remain elusive. Comparative analyses of the rRNA modifications in taxonomically distant bacteria could help define their general, as well as species-specific, roles. In this study, we identified a new methyltransferase, RlmQ, in Staphylococcus aureus responsible for the Gram-positive specific m7G2601, which is not modified in Escherichia coli (G2574). We also demonstrate the absence of methylation on C1989, equivalent to E. coli C1962, which is methylated at position 5 by the Gram-negative specific RlmI methyltransferase, a paralog of RlmQ. Both modifications (S. aureus m7G2601 and E. coli m5C1962) are situated within the same tRNA accommodation corridor, hinting at a potential shared function in translation. Inactivation of S. aureus rlmQ causes the loss of methylation at G2601 and significantly impacts growth, cytotoxicity, and biofilm formation. These findings unravel the intricate connections between rRNA modifications, translation, and virulence in pathogenic Gram-positive bacteria.
Assuntos
Proteínas de Escherichia coli , Escherichia coli , Staphylococcus aureus/genética , Proteínas de Escherichia coli/genética , RNA , Virulência/genética , RNA Ribossômico 23S/genética , Metiltransferases/genéticaRESUMO
Staphylococcus aureus is an opportunistic pathogen capable of causing many different human diseases. During colonization and infection, S. aureus will encounter a range of hostile environments, including acidic conditions such as those found on the skin and within macrophages. However, little is known about the mechanisms that S. aureus uses to detect and respond to low pH. Here, we employed a transposon sequencing approach to determine on a genome-wide level the genes required or detrimental for growth at low pH. We identified 31 genes that were essential for the growth of S. aureus at pH 4.5 and confirmed the importance of many of them through follow up experiments using mutant strains inactivated for individual genes. Most of the genes identified code for proteins with functions in cell wall assembly and maintenance. These data suggest that the cell wall has a more important role than previously appreciated in promoting bacterial survival when under acid stress. We also identified several novel processes previously not linked to the acid stress response in S. aureus. These include aerobic respiration and histidine transport, the latter by showing that one of the most important genes, SAUSA300_0846, codes for a previously uncharacterized histidine transporter. We further show that under acid stress, the expression of the histidine transporter gene is increased in WT S. aureus. In a S. aureus SAUSA300_0846 mutant strain expression of the histidine biosynthesis genes is induced under acid stress conditions allowing the bacteria to maintain cytosolic histidine levels. This strain is, however, unable to maintain its cytosolic pH to the same extent as a WT strain, revealing an important function specifically for histidine transport in the acid stress response of S. aureus.
Assuntos
Infecções Estafilocócicas , Staphylococcus aureus , Humanos , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Histidina/genética , Histidina/metabolismo , Infecções Estafilocócicas/genética , Infecções Estafilocócicas/microbiologia , Concentração de Íons de Hidrogênio , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismoRESUMO
Staphylococcus aureus is a facultative intracellular pathogen of human macrophages, which facilitates chronic infection. The genotypes, pathways, and mutations influencing that phenotype remain incompletely explored. Here, we used two distinct strategies to ascertain S. aureus gene mutations affecting pathogenesis in macrophages. First, we analyzed isolates collected serially from chronic cystic fibrosis (CF) respiratory infections. We found that S. aureus strains evolved greater macrophage invasion capacity during chronic human infection. Bacterial genome-wide association studies (GWAS) identified 127 candidate genes for which mutation was significantly associated with macrophage pathogenesis in vivo. In parallel, we passaged laboratory S. aureus strains in vitro to select for increased infection of human THP-1 derived macrophages, which identified 15 candidate genes by whole-genome sequencing. Functional validation of candidate genes using isogenic transposon mutant knockouts and CRISPR interference (CRISPRi) knockdowns confirmed virulence contributions from 37 of 39 tested genes (95%) implicated by in vivo studies and 7 of 10 genes (70%) ascertained from in vitro selection, with one gene in common to the two strategies. Validated genes included 17 known virulence factors (39%) and 27 newly identified by our study (61%), some encoding functions not previously associated with macrophage pathogenesis. Most genes (80%) positively impacted macrophage invasion when disrupted, consistent with the phenotype readily arising from loss-of-function mutations in vivo. This work reveals genes and mechanisms that contribute to S. aureus infection of macrophages, highlights differences in mutations underlying convergent phenotypes arising from in vivo and in vitro systems, and supports the relevance of S. aureus macrophage pathogenesis during chronic respiratory infection in CF. Additional studies will be needed to illuminate the exact mechanisms by which implicated mutations affect their phenotypes.
Assuntos
Fibrose Cística , Estudo de Associação Genômica Ampla , Macrófagos , Infecções Estafilocócicas , Staphylococcus aureus , Humanos , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidade , Macrófagos/microbiologia , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/genética , Infecções Estafilocócicas/patologia , Fibrose Cística/microbiologia , Mutação , Virulência/genética , Fatores de Virulência/genética , Adaptação FisiológicaRESUMO
Human activities such as agriculturalization and domestication have led to the emergence of many new pathogens via host-switching events between humans, domesticated and wild animals. Staphylococcus aureus is a multi-host opportunistic pathogen with a global healthcare and economic burden. Recently, it was discovered that laboratory and wild rodents can be colonised and infected with S. aureus, but the origins and zoonotic potential of rodent S. aureus is unknown. In order to trace their evolutionary history, we employed a dataset of 1249 S. aureus genome sequences including 393 of isolates from rodents and other small mammals (including newly determined sequences for 305 isolates from 7 countries). Among laboratory mouse populations, we identified multiple widespread rodent-specific S. aureus clones that likely originated in humans. Phylogeographic analysis of the most common murine lineage CC88 suggests that it emerged in the 1980s in laboratory mouse facilities most likely in North America, from where it spread to institutions around the world, via the distribution of mice for research. In contrast, wild rodents (mice, voles, squirrels) were colonized with a unique complement of S. aureus lineages that are widely disseminated across Europe. In order to investigate the molecular basis for S. aureus adaptation to rodent hosts, genome-wide association analysis was carried out revealing a unique complement of bacteriophages associated with a rodent host ecology. Of note, we identified novel prophages and pathogenicity islands in rodent-derived S. aureus that conferred the potential for coagulation of rodent plasma, a key phenotype of abscess formation and persistence. Our findings highlight the remarkable capacity of S. aureus to expand into new host populations, driven by the acquisition of genes promoting survival in new host-species.
Assuntos
Infecções Estafilocócicas , Staphylococcus aureus , Animais , Staphylococcus aureus/genética , Staphylococcus aureus/virologia , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/veterinária , Camundongos , Roedores/microbiologia , Roedores/virologia , Bacteriófagos/genética , Humanos , Filogenia , Genoma Bacteriano , Fagos de Staphylococcus/genéticaRESUMO
Pathogenic bacteria's metabolic adaptation for survival and proliferation within hosts is a crucial aspect of bacterial pathogenesis. Here, we demonstrate that citrate, the first intermediate of the tricarboxylic acid (TCA) cycle, plays a key role as a regulator of gene expression in Staphylococcus aureus. We show that citrate activates the transcriptional regulator CcpE and thus modulates the expression of numerous genes involved in key cellular pathways such as central carbon metabolism, iron uptake and the synthesis and export of virulence factors. Citrate can also suppress the transcriptional regulatory activity of ferric uptake regulator. Moreover, we determined that accumulated intracellular citrate, partly through the activation of CcpE, decreases the pathogenic potential of S. aureus in animal infection models. Therefore, citrate plays a pivotal role in coordinating carbon metabolism, iron homeostasis, and bacterial pathogenicity at the transcriptional level in S. aureus, going beyond its established role as a TCA cycle intermediate.