RESUMO
Human-adapted bacterial pathogens use a mechanism called phase variation to randomly switch the expression of individual genes to generate a phenotypically diverse population to adapt to challenges within and between human hosts. There are increasing reports of restriction-modification systems that exhibit phase-variable expression. The outcome of phase variation of these systems is global changes in DNA methylation. Analysis of phase-variable Type I and Type III restriction-modification systems in multiple human-adapted bacterial pathogens has demonstrated that global changes in methylation regulate the expression of multiple genes. These systems are called phasevarions (phase-variable regulons). Phasevarion switching alters virulence phenotypes and facilitates evasion of host immune responses. This review describes the characteristics of phasevarions and implications for pathogenesis and immune evasion. We present and discuss examples of phasevarion systems in the major human pathogens Haemophilus influenzae, Neisseria meningitidis, Neisseria gonorrhoeae, Helicobacter pylori, Moraxella catarrhalis, and Streptococcus pneumoniae.
Assuntos
Bactérias/genética , Bactérias/patogenicidade , Epigênese Genética , Regulação Bacteriana da Expressão Gênica , Interações Hospedeiro-Patógeno , Evasão da Resposta Imune , Metilação de DNA , Enzimas de Restrição-Modificação do DNA/genética , Enzimas de Restrição-Modificação do DNA/metabolismo , Humanos , Regulon , VirulênciaRESUMO
Kingella kingae is a common etiological agent of pediatric osteoarticular infections. While current research has expanded our understanding of K. kingae pathogenesis, there is a paucity of knowledge about host-pathogen interactions and virulence gene regulation. Many host-adapted bacterial pathogens contain phase variable DNA methyltransferases (mod genes), which can control expression of a regulon of genes (phasevarion) through differential methylation of the genome. Here, we identify a phase variable type III mod gene in K. kingae, suggesting that phasevarions operate in this pathogen. Phylogenetic studies revealed that there are two active modK alleles in K. kingae Proteomic analysis of secreted and surface-associated proteins, quantitative PCR, and a heat shock assay comparing the wild-type modK1 ON (i.e., in frame for expression) strain to a modK1 OFF (i.e., out of frame) strain revealed three virulence-associated genes under ModK1 control. These include the K. kingae toxin rtxA and the heat shock genes groEL and dnaK Cytokine expression analysis showed that the interleukin-8 (IL-8), IL-1ß, and tumor necrosis factor responses of THP-1 macrophages were lower in the modK1 ON strain than in the modK1::kan mutant. This suggests that the ModK1 phasevarion influences the host inflammatory response and provides the first evidence of this phase variable epigenetic mechanism of gene regulation in K. kingae.
Assuntos
Metilases de Modificação do DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , Interações Hospedeiro-Patógeno , Kingella kingae/crescimento & desenvolvimento , Metilases de Modificação do DNA/genética , Perfilação da Expressão Gênica , Humanos , Kingella kingae/enzimologia , Kingella kingae/genética , Filogenia , Proteoma/análise , Proteômica , Reação em Cadeia da Polimerase em Tempo Real , Regulon , Células THP-1/microbiologia , Virulência , Fatores de Virulência/biossínteseRESUMO
Several human-adapted bacterial pathogens use a phasevarion (ie, a phase-variable regulon) to rapidly and reversibly regulate the expression of many genes, which include known virulence factors, yet the influence of phasevarion-mediated regulation in pathogenesis remains poorly understood. Here we examine the impact of the nontypeable Haemophilus influenzae (NTHI) ModA2 phasevarion on pathogenesis and disease severity in a chinchilla model of experimental otitis media. Chinchillas were challenged with NTHI variant populations that were either inoculated ON and remained ON, inoculated OFF and shifted ON, or inoculated OFF and remained OFF, within the middle ear. We show that populations that shift from OFF to ON within the middle ear induce significantly greater disease severity than populations that are unable to shift. These observations support the importance of phasevarion switching in NTHI pathogenesis and the necessity to considered phasevarion regulation when developing methods to treat and prevent infection.
Assuntos
Variação Antigênica , Antígenos de Bactérias/imunologia , Infecções por Haemophilus/microbiologia , Infecções por Haemophilus/patologia , Haemophilus influenzae/patogenicidade , Otite Média/microbiologia , Otite Média/patologia , Animais , Antígenos de Bactérias/genética , Chinchila , Estudos de Coortes , Modelos Animais de Doenças , Índice de Gravidade de DoençaRESUMO
UNLABELLED: Klebsiella pneumoniae is an important cause of nosocomial infections, primarily through the formation of surface-associated biofilms to promote microbial colonization on host tissues. Expression of type 3 fimbriae by K. pneumoniae facilitates surface adherence, a process strongly activated by the cyclic di-GMP (c-di-GMP)-dependent transcriptional activator MrkH. In this study, we demonstrated the critical importance of MrkH in facilitating K. pneumoniae attachment on a variety of medically relevant materials and demonstrated the mechanism by which bacteria activate expression of type 3 fimbriae to colonize these materials. Sequence analysis revealed a putative MrkH recognition DNA sequence ("MrkH box"; TATCAA) located in the regulatory region of the mrkHI operon. Mutational analysis, electrophoretic mobility shift assay, and quantitative PCR experiments demonstrated that MrkH binds to the cognate DNA sequence to autoregulate mrkHI expression in a c-di-GMP-dependent manner. A half-turn deletion, but not a full-turn deletion, between the MrkH box and the -35 promoter element rendered MrkH ineffective in activating mrkHI expression, implying that a direct interaction between MrkH and RNA polymerase exists. In vivo analyses showed that residues L260, R265, N268, C269, E273, and I275 in the C-terminal domain of the RNA polymerase α subunit are involved in the positive control of mrkHI expression by MrkH and revealed the regions of MrkH required for DNA binding and transcriptional activation. Taken together, the data suggest a model whereby c-di-GMP-dependent MrkH recruits RNA polymerase to the mrkHI promoter to autoactivate mrkH expression. Increased MrkH production subsequently drives mrkABCDF expression when activated by c-di-GMP, leading to biosynthesis of type 3 fimbriae and biofilm formation. IMPORTANCE: Bacterial biofilms can cause persistent infections that are refractory to antimicrobial treatments. This study investigated how a commonly encountered hospital-acquired pathogen, Klebsiella pneumoniae, controls the expression of MrkH, the principal regulator of type 3 fimbriae and biofilm formation. We discovered a regulatory circuit whereby MrkH acts as a c-di-GMP-dependent transcriptional activator of both the gene cluster of type 3 fimbriae and the mrkHI operon. In this positive-feedback loop, whereby MrkH activates its own production, K. pneumoniae has evolved a mechanism to ensure rapid MrkH production, expression of type 3 fimbriae, and subsequent biofilm formation under favorable conditions. Deciphering the molecular mechanisms of biofilm formation by bacterial pathogens is important for the development of innovative treatment strategies for biofilm infections.
Assuntos
Aderência Bacteriana , GMP Cíclico/análogos & derivados , Regulação Bacteriana da Expressão Gênica , Homeostase , Klebsiella pneumoniae/fisiologia , Fatores de Transcrição/metabolismo , Sítios de Ligação , Biofilmes/crescimento & desenvolvimento , GMP Cíclico/metabolismo , Análise Mutacional de DNA , DNA Bacteriano/química , DNA Bacteriano/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Fímbrias Bacterianas/fisiologia , Perfilação da Expressão Gênica , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/metabolismo , Óperon , Regiões Promotoras Genéticas , Ligação Proteica , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNA , Fatores de Transcrição/genéticaRESUMO
Enterohemorrhagic Escherichia coli (EHEC) O157:H7 causes bloody diarrhea and hemolytic-uremic syndrome (HUS) and is the most prevalent E. coli serotype associated with food-borne illness worldwide. This pathogen is transmitted via the fecal-oral route and has a low infectious dose that has been estimated to be between 10 and 100 cells. We and others have previously identified three prophage-encoded AraC-like transcriptional regulators, PatE, PsrA, and PsrB in the EHEC O157:H7 EDL933 strain. Our analysis showed that PatE plays an important role in facilitating survival of EHEC under a number of acidic conditions, but the contribution of PsrA and PsrB to acid resistance (AR) was unknown. Here, we investigated the involvement of PsrA and PsrB in the survival of E. coli O157:H7 in acid. Our results showed that PsrB, but not PsrA, enhanced the survival of strain EDL933 under various acidic conditions. Transcriptional analysis using promoter-lacZ reporters and electrophoretic mobility shift assays demonstrated that PsrB activates transcription of the hdeA operon, which encodes a major acid stress chaperone, by interacting with its promoter region. Furthermore, using a mouse model, we showed that expression of PsrB significantly enhanced the ability of strain EDL933 to overcome the acidic barrier of the mouse stomach. Taken together, our results indicate that EDL933 acquired enhanced acid tolerance via horizontally acquired regulatory genes encoding transcriptional regulators that activate its AR machinery.
Assuntos
Ácidos/toxicidade , Escherichia coli O157/efeitos dos fármacos , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Prófagos/genética , Estresse Fisiológico , Fatores de Transcrição/metabolismo , Animais , Fusão Gênica Artificial , DNA Bacteriano/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Escherichia coli O157/genética , Escherichia coli O157/fisiologia , Escherichia coli O157/virologia , Proteínas de Escherichia coli/genética , Perfilação da Expressão Gênica , Genes Reporter , Camundongos Endogâmicos C57BL , Viabilidade Microbiana/efeitos dos fármacos , Ligação Proteica , Fatores de Transcrição/genética , beta-Galactosidase/análise , beta-Galactosidase/genéticaRESUMO
With the aim of discovering small molecule inhibitors of the sporulation process in Clostridioides difficile, we prepared a series of C-7 α-(4-substituted-1H-1,2,3-triazol-1-yl)acetamide analogues of cefotetan, a known inhibitor of the C. difficile sporulation-specific protein target CdSpoVD. These analogues were evaluated using both in vitro binding assays with CdSpoVD and antisporulation assays against C. difficile. Further design concepts were aided utilizing the predicted docking scores (DS) using both AlphaFold (AF) models, and a crystal structure of the CdSpoVD protein (PDB 7RCZ). Despite being 1 order of magnitude more potent as a sporulation inhibitor than cefotetan, in vivo studies on compound 6a in a murine-model of C. difficile infection demonstrated comparable spore shedding capabilities as cefotetan. Importantly, compound 6a had no concerning broad spectrum antibacterial activities, toxicity, or hemolytic activity and thus has potential for further drug development.
Assuntos
Cefamicinas , Clostridioides difficile , Infecções por Clostridium , Animais , Camundongos , Cefamicinas/metabolismo , Clostridioides , Cefotetan/metabolismo , Esporos Bacterianos , Antibacterianos/química , Proteínas de Bactérias/metabolismoRESUMO
NtrYX is a sensor-histidine kinase/response regulator two-component system that has had limited characterization in a small number of Alphaproteobacteria. Phylogenetic analysis of the response regulator NtrX showed that this two-component system is extensively distributed across the bacterial domain, and it is present in a variety of Betaproteobacteria, including the human pathogen Neisseria gonorrhoeae. Microarray analysis revealed that the expression of several components of the respiratory chain was reduced in an N. gonorrhoeae ntrX mutant compared to that in the isogenic wild-type (WT) strain 1291. These included the cytochrome c oxidase subunit (ccoP), nitrite reductase (aniA), and nitric oxide reductase (norB). Enzyme activity assays showed decreased cytochrome oxidase and nitrite reductase activities in the ntrX mutant, consistent with microarray data. N. gonorrhoeae ntrX mutants had reduced capacity to survive inside primary cervical cells compared to the wild type, and although they retained the ability to form a biofilm, they exhibited reduced survival within the biofilm compared to wild-type cells, as indicated by LIVE/DEAD staining. Analyses of an ntrX mutant in a representative alphaproteobacterium, Rhodobacter capsulatus, showed that cytochrome oxidase activity was also reduced compared to that in the wild-type strain SB1003. Taken together, these data provide evidence that the NtrYX two-component system may be a key regulator in the expression of respiratory enzymes and, in particular, cytochrome c oxidase, across a wide range of proteobacteria, including a variety of bacterial pathogens.
Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano/genética , Neisseria gonorrhoeae/enzimologia , Nitrito Redutases/genética , Rhodobacter capsulatus/enzimologia , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Colo do Útero/microbiologia , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Células Epiteliais/microbiologia , Feminino , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Gonorreia/microbiologia , Humanos , Viabilidade Microbiana , Neisseria gonorrhoeae/genética , Nitrito Redutases/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Oxirredutases/metabolismo , Oxigênio/metabolismo , Filogenia , RNA Bacteriano/genética , Rhodobacter capsulatus/genética , Deleção de SequênciaRESUMO
Atypical enteropathogenic Escherichia coli (aEPEC) causes endemic diarrhea, diarrheal outbreaks, and persistent diarrhea in humans, but the mechanism by which aEPEC causes disease is incompletely understood. Virulence regulators and their associated regulons, which often include adhesins, play key roles in the expression of virulence factors in enteric pathogenic bacteria. In this study we identified a transcriptional regulator, RalR, in the rabbit-specific aEPEC strain, E22 (O103:H2) and examined its involvement in the regulation of virulence. Microarray analysis and quantitative real-time reverse transcription-PCR demonstrated that RalR enhances the expression of a number of genes encoding virulence-associated factors, including the Ral fimbria, the Aap dispersin, and its associated transport system, and downregulates several housekeeping genes, including fliC. These observations were confirmed by proteomic analysis of secreted and heat-extracted surface-associated proteins and by adherence and motility assays. To investigate the mechanism of RalR-mediated activation, we focused on its most highly upregulated target operons, ralCDEFGHI and aap. By using primer extension, electrophoretic mobility shift assay, and mutational analysis, we identified the promoter and operator sequences for these two operons. By employing promoter-lacZ reporter systems, we demonstrated that RalR activates the expression of its target genes by binding to one or more 8-bp palindromic sequences (with the consensus of TGTGCACA) located immediately upstream of the promoter core regions. Importantly, we also demonstrated that RalR is essential for virulence since infection of rabbits with E22 carrying a knockout mutation in the ralR gene completely abolished its ability to cause disease.
Assuntos
Escherichia coli Enteropatogênica/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Fatores de Transcrição/metabolismo , Fatores de Virulência/metabolismo , Animais , Modelos Animais de Doenças , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/patologia , Infecções por Escherichia coli/veterinária , Proteínas de Escherichia coli/genética , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Análise em Microsséries , Regiões Promotoras Genéticas , Proteoma/análise , Coelhos , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Transcrição/genética , VirulênciaRESUMO
AraC-like regulators play a key role in the expression of virulence factors in enteric pathogens, such as enteropathogenic Escherichia coli (EPEC), enterotoxigenic E. coli, enteroaggregative E. coli, and Citrobacter rodentium. Bioinformatic analysis of the genome of rabbit-specific EPEC (REPEC) strain E22 (O103:H2) revealed the presence of a gene encoding an AraC-like regulatory protein, RegR, which shares 71% identity to the global virulence regulator, RegA, of C. rodentium. Microarray analysis demonstrated that RegR exerts 25- to 400-fold activation on transcription of several genes encoding putative virulence-associated factors, including a fimbrial operon (SEF14), a serine protease, and an autotransporter adhesin. These observations were confirmed by proteomic analysis of secreted and heat-extracted surface-associated proteins. The mechanism of RegR-mediated activation was investigated by using its most highly upregulated gene target, sefA. Transcriptional analyses and electrophoretic mobility shift assays showed that RegR activates the expression of sefA by binding to a region upstream of the sefA promoter, thereby relieving gene silencing by the global regulatory protein H-NS. Moreover, RegR was found to contribute significantly to virulence in a rabbit infection experiment. Taken together, our findings indicate that RegR controls the expression of a series of accessory adhesins that significantly enhance the virulence of REPEC strain E22.
Assuntos
Proteínas de Bactérias/metabolismo , Escherichia coli Enteropatogênica/genética , Escherichia coli Enteropatogênica/patogenicidade , Regulação Bacteriana da Expressão Gênica , Regulon , Fatores de Transcrição/metabolismo , Fatores de Virulência/biossíntese , Animais , Proteínas de Bactérias/genética , Biologia Computacional , DNA Bacteriano/metabolismo , Modelos Animais de Doenças , Ensaio de Desvio de Mobilidade Eletroforética , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/patologia , Proteínas de Escherichia coli/análise , Proteínas de Fímbrias/biossíntese , Proteínas de Fímbrias/genética , Perfilação da Expressão Gênica , Análise em Microsséries , Regiões Promotoras Genéticas , Ligação Proteica , Proteoma/análise , Coelhos , Fatores de Transcrição/genética , Transcrição Gênica , Fatores de Virulência/genéticaRESUMO
Escherichia coli expresses two L-asparaginase (EC 3.5.1.1) isozymes: L-asparaginse I, which is a low affinity, cytoplasmic enzyme that is expressed constitutively, and L-asparaginase II, a high affinity periplasmic enzyme that is under complex co-transcriptional regulation by both Fnr and Crp. The distinct localisation and regulation of these enzymes suggest different roles. To define these roles, a set of isogenic mutants was constructed that lacked either or both enzymes. Evidence is provided that L-asparaginase II, in contrast to L-asparaginase I, can be used in the provision of an anaerobic electron acceptor when using a non-fermentable carbon source in the presence of excess nitrogen.
Assuntos
Asparaginase/fisiologia , Proteínas de Escherichia coli/fisiologia , Escherichia coli/enzimologia , Anaerobiose , Asparaginase/genética , Asparagina/metabolismo , Meios de Cultura/metabolismo , AMP Cíclico/metabolismo , Proteína Receptora de AMP Cíclico/genética , Proteína Receptora de AMP Cíclico/metabolismo , Citoplasma/enzimologia , Transporte de Elétrons , Escherichia coli/genética , Escherichia coli/fisiologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fumaratos/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/metabolismo , Isoenzimas/genética , Isoenzimas/fisiologia , Nitrogênio/metabolismo , Transcrição GênicaRESUMO
Hospital-acquired diarrhoea (HAD) is common, and often associated with gut microbiota and metabolome dysbiosis following antibiotic administration. Clostridioides difficile is the most significant antibiotic-associated diarrhoeal (AAD) pathogen, but less is known about the microbiota and metabolome associated with AAD and C. difficile infection (CDI) with contrasting antibiotic treatment. We characterised faecal microbiota and metabolome for 169 HAD patients (33 with CDI and 133 non-CDI) to determine dysbiosis biomarkers and gain insights into metabolic strategies C. difficile might use for gut colonisation. The specimen microbial community was analysed using 16 S rRNA gene amplicon sequencing, coupled with untargeted metabolite profiling using gas chromatography-mass spectrometry (GC-MS), and short-chain fatty acid (SCFA) profiling using GC-MS. AAD and CDI patients were associated with a spectrum of dysbiosis reflecting non-antibiotic, short-term, and extended-antibiotic treatment. Notably, extended antibiotic treatment was associated with enterococcal proliferation (mostly vancomycin-resistant Enterococcus faecium) coupled with putative biomarkers of enterococcal tyrosine decarboxylation. We also uncovered unrecognised metabolome dynamics associated with concomitant enterococcal proliferation and CDI, including biomarkers of Stickland fermentation and amino acid competition that could distinguish CDI from non-CDI patients. Here we show, candidate metabolic biomarkers for diagnostic development with possible implications for CDI and vancomycin-resistant enterococci (VRE) treatment.
Assuntos
Clostridioides difficile , Infecções por Clostridium , Humanos , Clostridioides difficile/genética , Disbiose , Multiômica , Diarreia , Antibacterianos/efeitos adversos , Biomarcadores , Infecções por Clostridium/diagnóstico , Proliferação de Células , HospitaisRESUMO
Many host-adapted bacterial pathogens contain DNA methyltransferases (mod genes) that are subject to phase-variable expression (high-frequency reversible ON/OFF switching of gene expression). In Haemophilus influenzae, the random switching of the modA gene controls expression of a phase-variable regulon of genes (a "phasevarion"), via differential methylation of the genome in the modA ON and OFF states. Phase-variable mod genes are also present in Neisseria meningitidis and Neisseria gonorrhoeae, suggesting that phasevarions may occur in these important human pathogens. Phylogenetic studies on phase-variable mod genes associated with type III restriction modification (R-M) systems revealed that these organisms have two distinct mod genes--modA and modB. There are also distinct alleles of modA (abundant: modA11, 12, 13; minor: modA4, 15, 18) and modB (modB1, 2). These alleles differ only in their DNA recognition domain. ModA11 was only found in N. meningitidis and modA13 only in N. gonorrhoeae. The recognition site for the modA13 methyltransferase in N. gonorrhoeae strain FA1090 was identified as 5'-AGAAA-3'. Mutant strains lacking the modA11, 12 or 13 genes were made in N. meningitidis and N. gonorrhoeae and their phenotype analyzed in comparison to a corresponding mod ON wild-type strain. Microarray analysis revealed that in all three modA alleles multiple genes were either upregulated or downregulated, some of which were virulence-associated. For example, in N. meningitidis MC58 (modA11), differentially expressed genes included those encoding the candidate vaccine antigens lactoferrin binding proteins A and B. Functional studies using N. gonorrhoeae FA1090 and the clinical isolate O1G1370 confirmed that modA13 ON and OFF strains have distinct phenotypes in antimicrobial resistance, in a primary human cervical epithelial cell model of infection, and in biofilm formation. This study, in conjunction with our previous work in H. influenzae, indicates that phasevarions may be a common strategy used by host-adapted bacterial pathogens to randomly switch between "differentiated" cell types.
Assuntos
Metilases de Modificação do DNA/genética , Regulação Bacteriana da Expressão Gênica , Neisseria/genética , Neisseria/patogenicidade , Alelos , Sítios de Ligação , Células Cultivadas , Farmacorresistência Bacteriana/genética , Células Epiteliais/microbiologia , Perfilação da Expressão Gênica , Humanos , Neisseria gonorrhoeae , Neisseria meningitidis , FilogeniaRESUMO
Phase variably expressed (randomly switching) methyltransferases associated with type III restriction-modification (R-M) systems have been identified in a variety of pathogenic bacteria. We have previously shown that a phase variable methyltransferase (Mod) associated with a type III R-M system in Haemophilus influenzae strain Rd coordinates the random switching of expression of multiple genes, and constitutes a phase variable regulon--'phasevarion'. We have now identified the recognition site for the Mod methyltransferase in H. influenzae strain Rd as 5'-CGAAT-3'. This is the same recognition site as the previously described HinfIII system. A survey of 59 H. influenzae strains indicated significant sequence heterogeneity in the central, variable region of the mod gene associated with target site recognition. Intra- and inter-strain transformation experiments using Mod methylated or non-methylated plasmids, and a methylation site assay demonstrated that the sequence heterogeneity seen in the region encoding target site specificity does correlate to distinct target sites. Mutations were identified within the res gene in several strains surveyed indicating that Res is not functional. These data suggest that evolution of this type III R-M system into an epigenetic mechanism for controlling gene expression has, in some strains, resulted in loss of the DNA restriction function.
Assuntos
Proteínas de Bactérias/metabolismo , Metilases de Modificação do DNA/metabolismo , Epigênese Genética , Regulação Bacteriana da Expressão Gênica , Haemophilus influenzae/enzimologia , Haemophilus influenzae/genética , Proteínas de Bactérias/genética , Sequência de Bases , Metilação de DNA , Metilases de Modificação do DNA/genética , DNA Bacteriano/química , Desoxirribonucleases de Sítio Específico do Tipo III/genética , Evolução Molecular , Variação Genética , Mutação , Plasmídeos/metabolismo , RegulonRESUMO
Spore-forming bacteria encompass a diverse range of genera and species, including important human and animal pathogens, and food contaminants. Clostridioides difficile is one such bacterium and is a global health threat because it is the leading cause of antibiotic-associated diarrhoea in hospitals. A crucial mediator of C. difficile disease initiation, dissemination and re-infection is the formation of spores that are resistant to current therapeutics, which do not target sporulation. Here, we show that cephamycin antibiotics inhibit C. difficile sporulation by targeting spore-specific penicillin-binding proteins. Using a mouse disease model, we show that combined treatment with the current standard-of-care antibiotic, vancomycin, and a cephamycin prevents disease recurrence. Cephamycins were found to have broad applicability as an anti-sporulation strategy, as they inhibited sporulation in other spore-forming pathogens, including the food contaminant Bacillus cereus. This study could directly and immediately affect treatment of C. difficile infection and advance drug development to control other important spore-forming bacteria that are problematic in the food industry (B. cereus), are potential bioterrorism agents (Bacillus anthracis) and cause other animal and human infections.
Assuntos
Antibacterianos/farmacologia , Cefamicinas/farmacologia , Clostridioides difficile/efeitos dos fármacos , Infecções por Clostridium/prevenção & controle , Animais , Toxinas Bacterianas/genética , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Clostridioides difficile/genética , Clostridioides difficile/crescimento & desenvolvimento , Infecções por Clostridium/microbiologia , Modelos Animais de Doenças , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Ligação às Penicilinas/efeitos dos fármacos , Proteínas de Ligação às Penicilinas/genética , Esporos Bacterianos/efeitos dos fármacos , Vancomicina/farmacologia , Células Vero/efeitos dos fármacosRESUMO
Bacterial infection a leading cause of death among patients with stroke, with elderly patients often presenting with more debilitating outcomes. The findings from our retrospective study, supported by previous clinical reports, showed that increasing age is an early predictor for developing fatal infectious complications after stroke. However, exactly how and why older individuals are more susceptible to infection after stroke remains unclear. Using a mouse model of transient ischaemic stroke, we demonstrate that older mice (>12 months) present with greater spontaneous bacterial lung infections compared to their younger counterparts (7-10 weeks) after stroke. Importantly, we provide evidence that older poststroke mice exhibited elevated intestinal inflammation and disruption in gut barriers critical in maintaining colonic integrity following stroke, including reduced expression of mucin and tight junction proteins. In addition, our data support the notion that the localized pro-inflammatory microenvironment driven by increased tumour necrosis factor-α production in the colon of older mice facilitates the translocation and dissemination of orally inoculated bacteria to the lung following stroke onset. Therefore, findings of this study demonstrate that exacerbated dysfunction of the intestinal barrier in advanced age promotes translocation of gut-derived bacteria and contributes to the increased risk to poststroke bacterial infection.
Assuntos
Envelhecimento/metabolismo , Colo/metabolismo , Pneumonia/metabolismo , Acidente Vascular Cerebral/metabolismo , Infecções Urinárias/metabolismo , Doença Aguda , Idoso , Animais , Estudos de Coortes , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estudos Retrospectivos , Fatores de RiscoRESUMO
Neisseria meningitidis express numerous virulence factors that enable it to interact with diverse microenvironments within the host, during both asymptomatic nasopharyngeal colonization and invasive disease. Many of these interactions involve bacterial or host glycans. In order to characterise the meningococcal glycointeractome, glycan arrays representative of structures found on human cells, were used as a screening tool to investigate host glycans bound by N. meningitidis. Arrays probed with fluorescently labelled wild-type MC58 revealed binding to 223 glycans, including blood group antigens, mucins, gangliosides and glycosaminoglycans. Mutant strains lacking surface components, including capsule, lipooligosaccharide (LOS), Opc and pili, were investigated to identify the factors responsible for glycan binding. Surface plasmon resonance and isothermal calorimetry were used to confirm binding and determine affinities between surface components and host glycans. We observed that the L3 LOS immunotype (whole cells and purified LOS) bound 26 structures, while L8 only bound 5 structures. We further demonstrated a direct glycan-glycan interaction between purified L3 LOS and Thomsen-Friedenreich (TF) antigen, with a KD of 13 nM. This is the highest affinity glycan-glycan interaction reported to date. These findings highlight the diverse glycointeractions that may occur during different stages of meningococcal disease, which could be exploited for development of novel preventative and therapeutic strategies.
Assuntos
Neisseria meningitidis Sorogrupo B/metabolismo , Polissacarídeos/química , Polissacarídeos/metabolismo , Calorimetria/métodos , Técnicas de Inativação de Genes , Glicômica , Interações Hospedeiro-Patógeno , Humanos , Infecções Meningocócicas/metabolismo , Mutação , Neisseria meningitidis Sorogrupo B/química , Neisseria meningitidis Sorogrupo B/genética , Neisseria meningitidis Sorogrupo B/patogenicidade , Ressonância de Plasmônio de Superfície , Fatores de VirulênciaRESUMO
The Helicobacter pylori phase variable gene modH, typified by gene HP1522 in strain 26695, encodes a N6-adenosine type III DNA methyltransferase. Our previous studies identified multiple strain-specific modH variants (modH1 - modH19) and showed that phase variation of modH5 in H. pylori P12 influenced expression of motility-associated genes and outer membrane protein gene hopG. However, the ModH5 DNA recognition motif and the mechanism by which ModH5 controls gene expression were unknown. Here, using comparative single molecule real-time sequencing, we identify the DNA site methylated by ModH5 as 5'-Gm6ACC-3'. This motif is vastly underrepresented in H. pylori genomes, but overrepresented in a number of virulence genes, including motility-associated genes, and outer membrane protein genes. Motility and the number of flagella of H. pylori P12 wild-type were significantly higher than that of isogenic modH5 OFF or ΔmodH5 mutants, indicating that phase variable switching of modH5 expression plays a role in regulating H. pylori motility phenotypes. Using the flagellin A (flaA) gene as a model, we show that ModH5 modulates flaA promoter activity in a GACC methylation-dependent manner. These findings provide novel insights into the role of ModH5 in gene regulation and how it mediates epigenetic regulation of H. pylori motility.
Assuntos
Proteínas de Bactérias/metabolismo , Helicobacter pylori/metabolismo , Proteínas de Bactérias/genética , Epigênese Genética/genética , Regulação Bacteriana da Expressão Gênica/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Helicobacter pylori/genéticaRESUMO
Neisseria meningitidis is a human-specific bacterium that varies in invasive potential. All meningococci are carried in the nasopharynx, and most genotypes are very infrequently associated with invasive meningococcal disease; however, those belonging to the 'hyperinvasive lineages' are more frequently associated with sepsis or meningitis. Genome content is highly conserved between carriage and disease isolates, and differential gene expression has been proposed as a major determinant of the hyperinvasive phenotype. Three phase variable DNA methyltransferases (ModA, ModB and ModD), which mediate epigenetic regulation of distinct phase variable regulons (phasevarions), have been identified in N. meningitidis. Each mod gene has distinct alleles, defined by their Mod DNA recognition domain, and these target and methylate different DNA sequences, thereby regulating distinct gene sets. Here 211 meningococcal carriage and >1,400 disease isolates were surveyed for the distribution of meningococcal mod alleles. While modA11-12 and modB1-2 were found in most isolates, rarer alleles (e.g., modA15, modB4, modD1-6) were specific to particular genotypes as defined by clonal complex. This suggests that phase variable Mod proteins may be associated with distinct phenotypes and hence invasive potential of N. meningitidis strains.
Assuntos
Proteínas de Bactérias/biossíntese , Metilases de Modificação do DNA/biossíntese , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Neisseria meningitidis/enzimologia , Neisseria meningitidis/patogenicidade , Fatores de Virulência/biossíntese , Proteínas de Bactérias/genética , Metilases de Modificação do DNA/genética , Humanos , Neisseria meningitidis/genética , Fatores de Virulência/genéticaRESUMO
Bacterial infection is highly prevalent in patients who have had a stroke. Despite the potential contribution of micro-aspiration in post-stroke pneumonia, we found that the majority of the microorganisms detected in the patients who developed infections after having a stroke were common commensal bacteria that normally reside in the intestinal tracts. In a mouse model of ischemic stroke, post-stroke infection was only observed in mice that were born and raised in specific-pathogen-free facilities; this was not seen in mice that were born and raised in germ-free facilities. Using high-throughput 16S rRNA gene amplicon sequencing and bioinformatics analyses, we provide evidence demonstrating that the source of the bacteria forming the microbial community in the lungs of post-stroke mice was indeed the host small intestine. Additionally, stroke-induced gut barrier permeability and dysfunction preceded the dissemination of orally inoculated bacteria to peripheral tissues. This study identifies a novel pathway in which stroke promotes the translocation and dissemination of selective strains of bacteria that originated from the host gut microbiota.
Assuntos
Infecções Bacterianas/imunologia , Translocação Bacteriana/imunologia , Microbioma Gastrointestinal/genética , Infecções por Bactérias Gram-Positivas/imunologia , Intestino Delgado/metabolismo , RNA Ribossômico 16S/genética , Acidente Vascular Cerebral/imunologia , Antagonistas Adrenérgicos beta/farmacologia , Idoso , Idoso de 80 Anos ou mais , Animais , Bacteriemia/imunologia , Bacteriemia/metabolismo , Bacteriemia/microbiologia , Infecções Bacterianas/metabolismo , Infecções Bacterianas/microbiologia , Hemocultura , Biologia Computacional , Modelos Animais de Doenças , Enterococcus faecalis , Feminino , Células Caliciformes/citologia , Células Caliciformes/metabolismo , Infecções por Bactérias Gram-Positivas/metabolismo , Infecções por Bactérias Gram-Positivas/microbiologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Infarto da Artéria Cerebral Média/imunologia , Intestino Delgado/citologia , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/microbiologia , Masculino , Camundongos , Microbiota/genética , Pessoa de Meia-Idade , Permeabilidade/efeitos dos fármacos , Pneumonia Bacteriana/imunologia , Pneumonia Bacteriana/metabolismo , Pneumonia Bacteriana/microbiologia , Receptores Adrenérgicos beta/metabolismo , Análise de Sequência de RNA , Organismos Livres de Patógenos Específicos , Infecções Urinárias/imunologia , Infecções Urinárias/metabolismo , Infecções Urinárias/microbiologia , Proteína da Zônula de Oclusão-1/metabolismoRESUMO
Non-typeable Haemophilus influenzae contains an N(6)-adenine DNA-methyltransferase (ModA) that is subject to phase-variable expression (random ON/OFF switching). Five modA alleles, modA2, modA4, modA5, modA9 and modA10, account for over two-thirds of clinical otitis media isolates surveyed. Here, we use single molecule, real-time (SMRT) methylome analysis to identify the DNA-recognition motifs for all five of these modA alleles. Phase variation of these alleles regulates multiple proteins including vaccine candidates, and key virulence phenotypes such as antibiotic resistance (modA2, modA5, modA10), biofilm formation (modA2) and immunoevasion (modA4). Analyses of a modA2 strain in the chinchilla model of otitis media show a clear selection for ON switching of modA2 in the middle ear. Our results indicate that a biphasic epigenetic switch can control bacterial virulence, immunoevasion and niche adaptation in an animal model system.