RESUMO
Virulence of the intracellular pathogen Listeria monocytogenes (Listeria) requires escape from the phagosome into the host cytosol, where the bacteria replicate. Phagosomal escape is a multistep process characterized by perforation, which is dependent on the pore-forming toxin listeriolysin O (LLO), followed by rupture. The contribution of host factors to Listeria phagosomal escape is incompletely defined. Here we show that the cystic fibrosis transmembrane conductance regulator (CFTR) facilitates Listeria cytosolic entry. CFTR inhibition or mutation suppressed Listeria vacuolar escape in culture, and inhibition of CFTR in wild-type mice before oral inoculation of Listeria markedly decreased systemic infection. We provide evidence that high chloride concentrations may facilitate Listeria vacuolar escape by enhancing LLO oligomerization and lytic activity. We propose that CFTR transiently increases phagosomal chloride concentration after infection, potentiating LLO pore formation and vacuole lysis. Our studies suggest that Listeria exploits mechanisms of cellular ion homeostasis to escape the phagosome and emphasize host ion-channel function as a key parameter of bacterial virulence.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Listeria monocytogenes/fisiologia , Listeriose/microbiologia , Fagossomos/microbiologia , Animais , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Benzoatos/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Linhagem Celular , Células Cultivadas , Cloretos/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/antagonistas & inibidores , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Citosol/microbiologia , Feminino , Glicina/análogos & derivados , Glicina/farmacologia , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Proteínas Hemolisinas/química , Proteínas Hemolisinas/metabolismo , Interações Hospedeiro-Patógeno , Hidrazinas/farmacologia , Concentração de Íons de Hidrogênio , Listeria monocytogenes/metabolismo , Listeriose/genética , Listeriose/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Multimerização Proteica , Tiazolidinas/farmacologia , Vacúolos/microbiologia , ortoaminobenzoatos/farmacologiaRESUMO
Methicillin-resistant Staphylococcus aureus is estimated to cause more U.S. deaths annually than HIV/AIDS. The emergence of hypervirulent and multidrug-resistant strains has further amplified public health concern and accentuated the need for new classes of antibiotics. RNA degradation is a required cellular process that could be exploited for novel antimicrobial drug development. However, such discovery efforts have been hindered because components of the Gram-positive RNA turnover machinery are incompletely defined. In the current study we found that the essential S. aureus protein, RnpA, catalyzes rRNA and mRNA digestion in vitro. Exploiting this activity, high through-put and secondary screening assays identified a small molecule inhibitor of RnpA-mediated in vitro RNA degradation. This agent was shown to limit cellular mRNA degradation and exhibited antimicrobial activity against predominant methicillin-resistant S. aureus (MRSA) lineages circulating throughout the U.S., vancomycin intermediate susceptible S. aureus (VISA), vancomycin resistant S. aureus (VRSA) and other Gram-positive bacterial pathogens with high RnpA amino acid conservation. We also found that this RnpA-inhibitor ameliorates disease in a systemic mouse infection model and has antimicrobial activity against biofilm-associated S. aureus. Taken together, these findings indicate that RnpA, either alone, as a component of the RNase P holoenzyme, and/or as a member of a more elaborate complex, may play a role in S. aureus RNA degradation and provide proof of principle for RNA catabolism-based antimicrobial therapy.
Assuntos
Anti-Infecciosos/farmacologia , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ribonuclease P/antagonistas & inibidores , Infecções Estafilocócicas/prevenção & controle , Staphylococcus aureus , Animais , Anti-Infecciosos/uso terapêutico , Feminino , Células Hep G2 , Humanos , Camundongos , Modelos Biológicos , Ribonuclease P/fisiologia , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/uso terapêutico , Infecções Estafilocócicas/genética , Infecções Estafilocócicas/patologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Staphylococcus aureus/patogenicidade , Vancomicina/farmacologia , Vancomicina/uso terapêutico , Virulência/efeitos dos fármacos , Virulência/genéticaRESUMO
The tremendous success of Staphylococcus aureus as a pathogen is due to the controlled expression of a diverse array of virulence factors. The effects of host environments on the expression of virulence factors and the mechanisms by which S. aureus adapts to colonize distinct host tissues are largely unknown. Vertebrates have evolved to sequester nutrient iron from invading bacteria, and iron availability is a signal that alerts pathogenic microorganisms when they enter the hostile host environment. Consistent with this, we report here that S. aureus senses alterations in the iron status via the ferric uptake regulator (Fur) and alters the abundance of a large number of virulence factors. These Fur-mediated changes protect S. aureus against killing by neutrophils, and Fur is required for full staphylococcal virulence in a murine model of infection. A potential mechanistic explanation for the impact of Fur on virulence is provided by the observation that Fur coordinates the reciprocal expression of cytolysins and a subset of immunomodulatory proteins. More specifically, S. aureus lacking fur exhibits decreased expression of immunomodulatory proteins and increased expression of cytolysins. These findings reveal that Fur is involved in initiating a regulatory program that organizes the expression of virulence factors during the pathogenesis of S. aureus pneumonia.
Assuntos
Proteínas de Bactérias/biossíntese , Regulação Bacteriana da Expressão Gênica , Pneumonia Estafilocócica/microbiologia , Proteínas Repressoras/fisiologia , Staphylococcus aureus/patogenicidade , Fatores de Virulência/biossíntese , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Cromatografia Líquida , Modelos Animais de Doenças , Eletroforese em Gel Bidimensional , Feminino , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Proteoma/análise , Proteínas Repressoras/genética , Staphylococcus aureus/fisiologiaRESUMO
The regulation of mRNA turnover is a dynamic means by which bacteria regulate gene expression. Although current methodologies allow characterization of the stability of individual transcripts, procedures designed to measure alterations in transcript abundance/turnover on a high throughput scale are lacking. In the current report, we describe the development of a rapid and simplified molecular beacon-based procedure to directly measure the mRNA abundances and mRNA degradation properties of well-characterized Staphylococcus aureus pathogenicity factors. This method does not require any PCR-based amplification, can monitor the abundances of multiple transcripts within a single RNA sample, and was successfully implemented into a high throughput screen of transposon mutant library members to detect isolates with altered mRNA turnover properties. It is expected that the described methodology will provide great utility in characterizing components of bacterial RNA degradation processes and can be used to directly measure the mRNA levels of virtually any bacterial transcript.
Assuntos
Sondas de DNA , Corantes Fluorescentes , Regulação Bacteriana da Expressão Gênica , Técnicas de Sonda Molecular , Estabilidade de RNA/fisiologia , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Staphylococcus aureus/genética , Proteínas de Bactérias/análise , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/análise , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Transcrição Reversa , Sensibilidade e Especificidade , Proteína Estafilocócica A/análise , Proteína Estafilocócica A/genética , Proteína Estafilocócica A/metabolismo , Fatores de Virulência/análise , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
During systemic infection, Staphylococcus aureus acquires nutrient iron from heme, the cofactor of vertebrate myoglobin and hemoglobin. Upon exposure to heme, S. aureus up-regulates the expression of the heme-regulated transporter, HrtAB. Strains lacking hrtAB exhibit increased sensitivity to heme toxicity, and upon heme exposure they elaborate a secreted protein response that interferes with the recruitment of neutrophils to the site of infection. Taken together, these results have led to the suggestion that hrtAB encodes an efflux system responsible for relieving the toxic effects of accumulated heme. Here we extend these observations by demonstrating that HrtA is the ATPase component of the HrtAB transport system. We show that HrtA is an Mn(2+)/Mg(2+)-dependent ATPase that functions at an optimal pH of 7.5 and exhibits in vitro temperature dependence uncommon to ABC transporter ATPases. Furthermore, we identify conserved residues within HrtA that are required for in vitro ATPase activity and are essential for the functionality of HrtA in vivo. Finally, we show that heme induces an alteration in the gene expression pattern of S. aureus Delta hrtA, implying the presence of a novel transcriptional regulatory mechanism responsible for the previously described immunomodulatory characteristics of hrtA mutants exposed to heme.
Assuntos
Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Heme/toxicidade , Staphylococcus aureus/patogenicidade , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/fisiologia , Transporte Biológico/genética , Transporte Biológico/fisiologia , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos/genética , Heme/fisiologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento , Transcrição Gênica/fisiologiaRESUMO
The modulation of mRNA turnover is gaining recognition as a mechanism by which Staphylococcus aureus regulates gene expression, but the factors that orchestrate alterations in transcript degradation are poorly understood. In that regard, we previously found that 138 mRNA species, including transcripts coding for the virulence factors protein A (spa) and collagen-binding protein (cna), are stabilized in a sarA-dependent manner during exponential phase growth, suggesting that SarA directly or indirectly affects the RNA turnover properties of these transcripts. Herein, we expanded our characterization of the effects of sarA on mRNA turnover during late-exponential and stationary phases of growth. Results revealed that the locus affects the RNA degradation properties of cells during both growth phases. Further, using gel mobility shift assays and RIP-Chip, it was found that SarA protein is capable of binding mRNA species that it stabilizes both in vitro and within bacterial cells. Taken together, these results suggest that SarA post-transcriptionally regulates S. aureus gene expression in a manner that involves binding to and consequently altering the mRNA turnover properties of target transcripts.
Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Estabilidade de RNA , Proteínas de Ligação a RNA/metabolismo , Staphylococcus aureus/fisiologia , Imunoprecipitação da Cromatina , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Ligação ProteicaRESUMO
Staphylococcus aureus pathogenesis can be attributed partially to its ability to adapt to otherwise deleterious host-associated stresses. Here, Affymetrix GeneChips® were used to examine the S. aureus responses to inorganic acid and alkaline shock and to assess whether stress-dependent changes in mRNA turnover are likely to facilitate the organism's ability to tolerate a pH challenge. The results indicate that S. aureus adapts to pH shock by eliciting responses expected of cells coping with pH alteration, including neutralizing cellular pH, DNA repair, amino acid biosynthesis, and virulence factor expression. Further, the S. aureus response to alkaline conditions is strikingly similar to that of stringent response-induced cells. Indeed, we show that alkaline shock stimulates the accumulation of the stringent response activator (p)ppGpp. The results also revealed that pH shock significantly alters the mRNA properties of the cell. A comparison of the mRNA degradation properties of transcripts whose titers either increased or decreased in response to a sudden pH change revealed that alterations in mRNA degradation may, in part, account for the changes in the mRNA levels of factors predicted to mediate pH tolerance. A set of small stable RNA molecules were induced in response to acid- or alkaline-shock conditions and may mediate adaptation to pH stress.
Assuntos
Ácidos/toxicidade , Álcalis/toxicidade , Perfilação da Expressão Gênica , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/fisiologia , Estabilidade de RNA , Estresse FisiológicoRESUMO
The regulation of mRNA turnover is a recently appreciated phenomenon by which bacteria modulate gene expression. This review outlines the mechanisms by which three major classes of bacterial trans-acting factors, ribonucleases (RNases), RNA binding proteins, and small noncoding RNAs (sRNA), regulate the transcript stability and protein production of target genes. Because the mechanisms of RNA decay and maturation are best characterized in Escherichia coli, the majority of this review will focus on how these factors modulate mRNA stability in this organism. However, we also address the effects of RNases, RNA binding proteins, sRNAs on mRNA turnover, and gene expression in Bacillus subtilis, which has served as a model for studying RNA processing in gram-positive organisms. We conclude by discussing emerging studies on the role modulating mRNA stability has on gene expression in the important human pathogen Staphylococcus aureus.
RESUMO
Bacterial infection often results in the formation of tissue abscesses, which represent the primary site of interaction between invading bacteria and the innate immune system. We identify the host protein calprotectin as a neutrophil-dependent factor expressed inside Staphylococcus aureus abscesses. Neutrophil-derived calprotectin inhibited S. aureus growth through chelation of nutrient Mn2+ and Zn2+: an activity that results in reprogramming of the bacterial transcriptome. The abscesses of mice lacking calprotectin were enriched in metal, and staphylococcal proliferation was enhanced in these metal-rich abscesses. These results demonstrate that calprotectin is a critical factor in the innate immune response to infection and define metal chelation as a strategy for inhibiting microbial growth inside abscessed tissue.
Assuntos
Abscesso/microbiologia , Quelantes/metabolismo , Complexo Antígeno L1 Leucocitário/metabolismo , Manganês/metabolismo , Neutrófilos/metabolismo , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/crescimento & desenvolvimento , Abscesso/imunologia , Abscesso/metabolismo , Animais , Cálcio/metabolismo , Quelantes/farmacologia , Dimerização , Perfilação da Expressão Gênica , Nefropatias/imunologia , Nefropatias/metabolismo , Nefropatias/microbiologia , Complexo Antígeno L1 Leucocitário/genética , Complexo Antígeno L1 Leucocitário/farmacologia , Abscesso Hepático/metabolismo , Abscesso Hepático/microbiologia , Abscesso Hepático/patologia , Espectrometria de Massas , Camundongos , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Zinco/metabolismoRESUMO
Staphylococcus aureus, a bacterium responsible for tremendous morbidity and mortality, exists as a harmless commensal in approximately 25% of humans. Identifying the molecular machinery activated upon infection is central to understanding staphylococcal pathogenesis. We describe the heme sensor system (HssRS) that responds to heme exposure and activates expression of the heme-regulated transporter (HrtAB). Inactivation of the Hss or Hrt systems leads to increased virulence in a vertebrate infection model, a phenotype that is associated with an inhibited innate immune response. We suggest that the coordinated activity of Hss and Hrt allows S. aureus to sense internal host tissues, resulting in tempered virulence to avoid excessive host tissue damage. Further, genomic analyses have identified orthologous Hss and Hrt systems in Bacillus anthracis, Listeria monocytogenes, and Enterococcus faecalis, suggesting a conserved regulatory system by which Gram-positive pathogens sense heme as a molecular marker of internal host tissue and modulate virulence.
Assuntos
Meio Ambiente , Heme/fisiologia , Heme/toxicidade , Staphylococcus aureus/patogenicidade , Aclimatação , Ferro/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento , VirulênciaRESUMO
Bacterial pathogens regulate virulence factor expression at both the level of transcription initiation and mRNA processing/turnover. Within Staphylococcus aureus, virulence factor transcript synthesis is regulated by a number of two-component regulatory systems, the DNA binding protein SarA, and the SarA family of homologues. However, little is known about the factors that modulate mRNA stability or influence transcript degradation within the organism. As our entree to characterizing these processes, S. aureus GeneChips were used to simultaneously determine the mRNA half-lives of all transcripts produced during log-phase growth. It was found that the majority of log-phase transcripts (90%) have a short half-life (<5 min), whereas others are more stable, suggesting that cis- and/or trans-acting factors influence S. aureus mRNA stability. In support of this, it was found that two virulence factor transcripts, cna and spa, were stabilized in a sarA-dependent manner. These results were validated by complementation and real-time PCR and suggest that SarA may regulate target gene expression in a previously unrecognized manner by posttranscriptionally modulating mRNA turnover. Additionally, it was found that S. aureus produces a set of stable RNA molecules with no predicted open reading frame. Based on the importance of the S. aureus agr RNA molecule, RNAIII, and small stable RNA molecules within other pathogens, it is possible that these RNA molecules influence biological processes within the organism.
Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Estabilidade de RNA , RNA Mensageiro/metabolismo , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Fatores de Virulência/metabolismo , Proteínas de Bactérias/genética , Meia-Vida , RNA Bacteriano/metabolismo , Fatores de Virulência/genéticaRESUMO
Despite its being a leading cause of nosocomal and community-acquired infections, surprisingly little is known about Staphylococcus aureus stress responses. In the current study, Affymetrix S. aureus GeneChips were used to define transcriptome changes in response to cold shock, heat shock, stringent, and SOS response-inducing conditions. Additionally, the RNA turnover properties of each response were measured. Each stress response induced distinct biological processes, subsets of virulence factors, and antibiotic determinants. The results were validated by real-time PCR and stress-mediated changes in antimicrobial agent susceptibility. Collectively, many S. aureus stress-responsive functions are conserved across bacteria, whereas others are unique to the organism. Sets of small stable RNA molecules with no open reading frames were also components of each response. Induction of the stringent, cold shock, and heat shock responses dramatically stabilized most mRNA species. Correlations between mRNA turnover properties and transcript titers suggest that S. aureus stress response-dependent alterations in transcript abundances can, in part, be attributed to alterations in RNA stability. This phenomenon was not observed within SOS-responsive cells.
Assuntos
Adaptação Fisiológica , Temperatura Baixa , Temperatura Alta , RNA Mensageiro/metabolismo , Resposta SOS em Genética , Staphylococcus aureus/fisiologia , Contagem de Colônia Microbiana , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Testes de Sensibilidade Microbiana , Análise de Sequência com Séries de Oligonucleotídeos , Estabilidade de RNA , RNA Bacteriano/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Staphylococcus aureus/genéticaRESUMO
A growing body of evidence obtained using in vitro model systems indicates that the deposition of fibrillar beta-amyloid (Abeta) results in neurite degeneration and cell death in central neurons. Little is known, however, about the molecular mechanisms underlying these neurotoxic effects. We have shown previously that fibrillar Abeta induced sustained activation of the mitogen-activated protein kinase (MAPK) followed by hyperphosphorylation of tau proteins in aging hippocampal neurons. Furthermore, the blockage of MAPK activation using specific inhibitors prevented neurite degeneration in these cells. These results suggested that the MAPK signal transduction pathway could play a key role in Abeta-induced neurite degeneration. We sought to identify upstream elements of the MAPK signaling cascade activated by Abeta deposition. We evaluated the participation of the integrins in this pathway by monitoring the activation of MAPK in the presence of specific integrin inhibitors. Our results indicate that pretreatment of mature hippocampal neurons with either echistatin or alpha(1) integrin-blocking antibodies prevented Abeta-induced MAPK activation. In addition, the blockage of alpha(1) activation prevented cell death induced by Abeta. Similar results were obtained when alpha(1) and beta(1) integrin blocking antibodies were used combined. Taken collectively, these results identify alpha(1) integrin and the alpha(1) plus beta(1) integrin complexes as potential targets for therapeutic intervention in the Abeta signaling pathway in aging neurons.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Hipocampo/metabolismo , Integrina alfa1/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neurônios/metabolismo , Envelhecimento/metabolismo , Envelhecimento/patologia , Animais , Morte Celular/fisiologia , Inibidores Enzimáticos/farmacologia , Hipocampo/patologia , Integrina alfa1/efeitos dos fármacos , Integrina alfa1beta1/efeitos dos fármacos , Integrina alfa1beta1/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Proteínas Quinases Ativadas por Mitógeno/efeitos dos fármacos , Neurofibrilas/metabolismo , Neurônios/patologia , Peptídeos/farmacologia , Ratos , Transdução de Sinais/efeitos dos fármacosRESUMO
Ror1 and Ror2 belong to a family of tyrosine kinase receptors that are highly conserved among species. They are expressed throughout the organism, including the central nervous system. In the present study, we analyzed the expression and subcellular localization of Ror1 and Ror2 in astrocytes by means of reverse transcription-polymerase chain reaction, Western blot analysis, and immunocytochemistry. Our results indicated that both Ror1 and Ror2 are readily detectable in cultured astrocytes. They also showed that Ror1 and Ror2 are associated with different components of the cytoskeleton. While Ror1 co-localized with F-actin along stress fibers, Ror2 partially co-localized with microtubules. In addition, our results suggest that Ror1 and Ror2 undergo different posttranslational modifications in cultured astrocytes. Ror1 is highly glycosylated in these cells. In contrast, no glycosylation was detected in Ror2. Taken together, these results suggest distinct roles for these tyrosine kinase receptors in astrocytes.
Assuntos
Astrócitos/enzimologia , Citoesqueleto/metabolismo , Glicosilação , Processamento de Proteína Pós-Traducional/fisiologia , Receptores Proteína Tirosina Quinases/metabolismo , Actinas/metabolismo , Animais , Astrócitos/ultraestrutura , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/embriologia , Citoesqueleto/ultraestrutura , Imuno-Histoquímica , Microtúbulos/metabolismo , RNA Mensageiro/metabolismo , Ratos , Receptores Proteína Tirosina Quinases/genética , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase , Fibras de Estresse/metabolismoRESUMO
A growing body of evidence suggests that estrogen has beneficial effects on Alzheimer's disease. However, the mechanisms underlying estrogen's neuroprotective effects are not completely understood. In the present study, we analyzed first whether estrogen protects mature hippocampal neurons against fibrillar Abeta-induced neurotoxicity. 17alpha-Estradiol and 17beta-estradiol partially prevented neuronal death induced by fibrillar Abeta. Estrogen-induced neuroprotection correlated with the formation of a more dynamic microtubular system, including an increase in the pool of unstable microtubules and the expression of juvenile microtubule-associated proteins MAP2c and MAP1b. These results provide further evidence that experimental conditions capable of increasing the pool of unstable microtubules might render mature hippocampal neurons resistant to the degeneration caused by fibrillar Abeta deposits.