RESUMO
External guide sequences (EGSs) signify the short RNAs that induce ribonuclease P (RNase P), an enzyme responsible for processing the 5' termini of tRNA, to specifically cleave a target mRNA by forming a precursor tRNA-like complex. Hence, the EGS technology may serve as a potential strategy for gene-targeting therapy. Our previous studies have revealed that engineered EGS variants induced RNase P to efficiently hydrolyze target mRNAs. In the present research, an EGS variant was designed to be complementary to the mRNA coding for human cytomegalovirus (HCMV) major capsid protein (MCP), which is vital to form the viral capsid. In vitro, the EGS variant was about 80-fold more efficient in inducing human RNase P-mediated cleavage of the target mRNA than a natural tRNA-derived EGS. Moreover, the expressed variant and natural tRNA-originated EGSs led to a decrease of MCP expression by 98% and 73%-74% and a decrease of viral growth by about 10,000- and 200-fold in cells infected with HCMV, respectively. These results reveal direct evidence that the engineered EGS variant has higher efficiency in blocking the expression of HCMV genes and viral growth than the natural tRNA-originated EGS. Therefore, our findings imply that the EGS variant can be a potent candidate agent for the treatment of infections caused by HCMV.
Assuntos
Proteínas do Capsídeo/genética , Citomegalovirus/genética , RNA Guia de Cinetoplastídeos/genética , RNA Mensageiro/genética , RNA de Transferência de Serina/genética , RNA Viral/genética , Ribonuclease P/metabolismo , Pareamento de Bases , Proteínas do Capsídeo/biossíntese , Linhagem Celular Transformada , Linhagem Celular Tumoral , Citomegalovirus/metabolismo , Fibroblastos/metabolismo , Fibroblastos/virologia , Regulação Viral da Expressão Gênica , Marcação de Genes/métodos , Engenharia Genética/métodos , Interações Hospedeiro-Patógeno/genética , Humanos , Terapia de Alvo Molecular , Neuroglia/metabolismo , Neuroglia/virologia , Conformação de Ácido Nucleico , Cultura Primária de Células , Clivagem do RNA , RNA Guia de Cinetoplastídeos/química , RNA Guia de Cinetoplastídeos/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , RNA de Transferência de Serina/química , RNA de Transferência de Serina/metabolismo , RNA Viral/química , RNA Viral/metabolismo , Ribonuclease P/química , Ribonuclease P/genética , Replicação Viral/fisiologiaRESUMO
Interferon-γ (IFN-γ) represents one of the most important innate immunity responses in a host to combat infections of many human viruses including human herpesviruses. Human N-myc interactor (Nmi) protein, which has been shown to interact with signal transducer and activator of transcription (STAT) proteins including STAT1, is important for the activation of IFN-γ induced STAT1-dependent transcription of many genes responsible for IFN-γ immune responses. However, no proteins encoded by herpesviruses have been reported to interact with Nmi and inhibit Nmi-mediated activation of IFN-γ immune responses to achieve immune evasion from IFN-γ responses. In this study, we show strong evidence that the UL23 protein of human cytomegalovirus (HCMV), a human herpesvirus, specifically interacts with Nmi. This interaction was identified through a yeast two-hybrid screen and co-immunoprecipitation in human cells. We observed that Nmi, when bound to UL23, was not associated with STAT1, suggesting that UL23 binding of Nmi disrupts the interaction of Nmi with STAT1. In cells overexpressing UL23, we observed (a) significantly reduced levels of Nmi and STAT1 in the nuclei, the sites where these proteins act to induce transcription of IFN-γ stimulated genes, and (b) decreased levels of the induction of the transcription of IFN-γ stimulated genes. UL23-deficient HCMV mutants induced higher transcription of IFN-γ stimulated genes and exhibited lower titers than parental and control revertant viruses expressing functional UL23 in IFN-γ treated cells. Thus, UL23 appears to interact directly with Nmi and inhibit nuclear translocation of Nmi and its associated protein STAT1, leading to a decrease of IFN-γ induced responses and an increase of viral resistance to IFN-γ. Our results further highlight the roles of UL23-Nmi interactions in facilitating viral immune escape from IFN-γ responses and enhancing viral resistance to IFN antiviral effects.
Assuntos
Citomegalovirus/fisiologia , Evasão da Resposta Imune , Imunidade Inata/efeitos dos fármacos , Interferon gama/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas da Matriz Viral/fisiologia , Células Cultivadas , Citomegalovirus/imunologia , Regulação da Expressão Gênica/imunologia , Células HEK293 , Humanos , Evasão da Resposta Imune/efeitos dos fármacos , Evasão da Resposta Imune/genética , Imunidade Inata/genética , Ligação Proteica , Transdução de Sinais/genética , Transdução de Sinais/imunologiaRESUMO
Antiviral drug development against respiratory syncytial virus (RSV) is urgently needed due to the public health significance of the viral infection. Here, we report the anti-RSV activity of a small molecule, (1S,3R,4R,5R)-3,4- bis{[(E)-3-(3,4-dihydroxyphenyl)prop-2-enoyl]oxy}-1,5-dihydroxycyclohexane-1-carboxylic methyl ester (3,4-DCQAME) or 3,4- O-Dicaffeoylquinic acid methyl ester, which can be isolated from several plants of traditional Chinese medicine. We showed for the first time that compound 3,4-DCQAME potently inhibits RSV entry and infection. In vitro, 3,4-DCQAME can interact with F(ecto), the ectodomain of RSV fusion (F) protein. In cultured cells, the compound can block the interaction of F(ecto) protein with the cellular membrane and inhibit viral fusion during RSV entry, leading to inhibition of viral gene expression and infection. In RSV-infected mice that were treated with 3,4-DCQAME, we observed a reduction of RSV-induced pathologic changes and substantial inhibition of viral infection and growth in the lung tissues. Our results provide the first direct evidence of the anti-RSV activity of 3,4-DCQAME. Furthermore, these results suggest that 3,4-DCQAME represents a promising lead compound for anti-RSV therapeutic development.-Tang, W., Li, M., Liu, Y., Liang, N., Yang, Z., Zhao, Y., Wu, S., Lu, S., Li, Y., Liu, F. Small molecule inhibits respiratory syncytial virus entry and infection by blocking the interaction of the viral fusion protein with the cell membrane.
Assuntos
Antivirais/farmacologia , Membrana Celular/efeitos dos fármacos , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas Virais de Fusão/metabolismo , Animais , Linhagem Celular , Membrana Celular/metabolismo , Membrana Celular/virologia , Expressão Gênica/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/virologia , Masculino , Medicina Tradicional Chinesa/métodos , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Vírus Respiratório Sincicial/metabolismo , Infecções por Vírus Respiratório Sincicial/virologiaRESUMO
Ribonuclease P complexed with external guide sequence (EGS) bound to mRNA represents a unique nucleic acid-based gene interference approach for modulation of gene expression. Compared with other strategies, such as RNA interference, the EGS-based technology is unique because a custom-designed EGS molecule can hybridize with any mRNA and recruit intracellular ribonuclease P for specific degradation of the target mRNA. It has not been reported whether the EGS-based technology can modulate gene expression in mice. In this study, a functional EGS was constructed to target the mRNA encoding the protease (mPR) of murine cytomegalovirus (MCMV), which is essential for viral replication. Furthermore, a unique attenuated strain of Salmonella was generated for gene delivery of EGS in cultured cells and in mice. Efficient expression of EGS was observed in cultured cells treated with the generated Salmonella vector carrying constructs with the EGS expression cassette. Moreover, a significant reduction in mPR expression and viral growth was found in MCMV-infected cells treated with Salmonella carrying the construct with the functional EGS sequence. When MCMV-infected mice were orally treated with Salmonella carrying EGS expression cassettes, viral gene expression and growth in various organs of these animals were reduced and animal survival improved. Our study suggests that EGS RNAs, when expressed following Salmonella-mediated gene transfer, effectively inhibit viral gene expression and infection in mice. Furthermore, these results demonstrate the feasibility of developing Salmonella-mediated delivery of EGS as a unique approach for treatment that reduces viral diseases in vivo.
Assuntos
Infecções por Citomegalovirus/prevenção & controle , Regulação Viral da Expressão Gênica/genética , Muromegalovirus/genética , RNA Mensageiro/metabolismo , Ribonuclease P/metabolismo , Animais , Northern Blotting , Western Blotting , Infecções por Citomegalovirus/genética , Primers do DNA/genética , Técnicas de Transferência de Genes , Terapia Genética/métodos , Camundongos , RNA Mensageiro/genética , Ribonuclease P/genética , Salmonella , Pequeno RNA não TraduzidoRESUMO
Nucleic acid-based gene interfering approaches, such as those mediated by RNA interference and RNase P-associated external guide sequence (EGS), have emerged as promising antiviral strategies. The RNase P-based technology is unique, because a custom-designed EGS can bind to any complementary mRNA sequence and recruit intracellular RNase P for specific degradation of the target mRNA. In this study, a functional EGS was constructed to target hepatitis B virus (HBV) essential transcripts. Furthermore, an attenuated Salmonella strain was constructed and used for delivery of anti-HBV EGS in cells and in mice. Substantial reduction in the levels of HBV gene expression and viral DNA was detected in cells treated with the Salmonella vector carrying the functional EGS construct. Furthermore, oral inoculation of Salmonella carrying the EGS construct led to an inhibition of ~95% in the levels of HBV gene expression and a reduction of ~200,000-fold in viral DNA level in the livers and sera of the treated mice transfected with a HBV plasmid. Our results suggest that EGSs are effective in inhibiting HBV replication in cultured cells and mammalian livers, and demonstrate the use of Salmonella-mediated delivery of EGS as a promising therapeutic approach for human diseases including HBV infection.
Assuntos
Regulação Viral da Expressão Gênica , Vírus da Hepatite B/fisiologia , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismo , Ribonuclease P/metabolismo , Replicação Viral , Animais , Linhagem Celular , Expressão Gênica , Técnicas de Transferência de Genes , Genoma Viral , Humanos , Hidrólise , Fígado/metabolismo , Fígado/patologia , Camundongos , Conformação de Ácido Nucleico , Pequeno RNA não Traduzido/química , RNA Viral/metabolismo , Salmonella/genética , Salmonella/metabolismo , TransfecçãoRESUMO
Safe, effective, and tissue-specific delivery is a central issue for the therapeutic application of nucleic-acid-based gene interfering agents, such as ribozymes and siRNAs. In this study, we constructed a functional RNase P-based ribozyme (M1GS RNA) that targets the overlapping mRNA region of M80.5 and protease, two murine cytomegalovirus (MCMV) proteins essential for viral replication. In addition, a novel attenuated strain of Salmonella, which exhibited efficient gene transfer activity and little cytotoxicity and pathogenicity in mice, was constructed and used for delivery of anti-MCMV ribozyme. In MCMV-infected macrophages treated with the constructed attenuated Salmonella strain carrying the functional M1GS RNA construct, we observed an 80-85% reduction in the expression of M80.5/protease and a 2,500-fold reduction in viral growth. Oral inoculation of the attenuated Salmonella strain in mice efficiently delivered antiviral M1GS RNA into spleens and livers, leading to substantial expression of the ribozyme without causing significant adverse effects in the animals. Furthermore, the MCMV-infected mice that were treated orally with Salmonella carrying the functional M1GS sequence displayed reduced viral gene expression, decreased viral titers, and improved survival compared to the untreated mice or mice treated with Salmonella containing control ribozyme sequences. Our results provide direct evidence that oral delivery of M1GS RNA by Salmonella-based vectors effectively inhibits viral gene expression and replication in mice. Moreover, this study demonstrates the utility of Salmonella-mediated oral delivery of RNase P ribozyme for gene-targeting applications in vivo.
Assuntos
Terapia Genética/métodos , RNA Catalítico/administração & dosagem , Ribonuclease P/antagonistas & inibidores , Salmonella/genética , Viroses/terapia , Administração Oral , Animais , Macrófagos/enzimologia , Macrófagos/virologia , Camundongos , RNA Catalítico/farmacologia , Carga Viral/efeitos dos fármacos , Viroses/prevenção & controle , Replicação Viral/efeitos dos fármacosRESUMO
Intracellular cations are essential for the physiology of all living organisms including bacteria. Cations such as potassium ion (K(+)), sodium ion (Na(+)) and proton (H(+)) are involved in nearly all aspects of bacterial growth and survival. K(+) is the most abundant cation and its homeostasis in Escherichia coli and Salmonella is regulated by three major K(+) transporters: high affinity transporter Kdp and low affinity transporters Kup and Trk. Previous studies have demonstrated the roles of cations and cation transport in the physiology of Escherichia coli; their roles in the virulence and physiology of pathogenic bacteria are not well characterized. We have previously reported that the Salmonella K(+) transporter Trk is important for the secretion of effector proteins of the type III secretion system (TTSS) of Salmonella pathogenicity island 1 (SPI-1). Here we further explore the role of Salmonella cation transport in virulence in vitro and pathogenesis in animal models. Impairment of K(+) transport through deletion of K(+) transporters or exposure to the chemical modulators of cation transport, gramicidin and valinomycin, results in a severe defect in the TTSS of SPI-1, and this defect in the TTSS was not due to a failure to regulate intrabacterial pH or ATP. Our results also show that K(+) transporters are critical to the pathogenesis of Salmonella in mice and chicks and are involved in multiple growth and virulence characteristics in vitro, including protein secretion, motility and invasion of epithelial cells. These results suggest that cation transport of the pathogenic bacterium Salmonella, especially K(+) transport, contributes to its virulence in addition to previously characterized roles in maintaining homeostasis of bacteria.
Assuntos
Sistemas de Secreção Bacterianos , Potássio/metabolismo , Salmonella/fisiologia , Animais , Transporte Biológico , Cátions/metabolismo , Linhagem Celular , Galinhas , Camundongos , Salmonella/metabolismo , Salmonella/patogenicidade , Salmonelose Animal/microbiologia , Salmonelose Animal/patologia , VirulênciaRESUMO
Small non-coding RNAs (sRNAs) that act as regulators of gene expression have been identified in all kingdoms of life, including microRNA (miRNA) and small interfering RNA (siRNA) in eukaryotic cells. Numerous sRNAs identified in Salmonella are encoded by genes located at Salmonella pathogenicity islands (SPIs) that are commonly found in pathogenic strains. Whether these sRNAs are important for Salmonella pathogenesis and virulence in animals has not been reported. In this study, we provide the first direct evidence that a pathogenicity island-encoded sRNA, IsrM, is important for Salmonella invasion of epithelial cells, intracellular replication inside macrophages, and virulence and colonization in mice. IsrM RNA is expressed in vitro under conditions resembling those during infection in the gastrointestinal tract. Furthermore, IsrM is found to be differentially expressed in vivo, with higher expression in the ileum than in the spleen. IsrM targets the mRNAs coding for SopA, a SPI-1 effector, and HilE, a global regulator of the expression of SPI-1 proteins, which are major virulence factors essential for bacterial invasion. Mutations in IsrM result in disregulation of expression of HilE and SopA, as well as other SPI-1 genes whose expression is regulated by HilE. Salmonella with deletion of isrM is defective in bacteria invasion of epithelial cells and intracellular replication/survival in macrophages. Moreover, Salmonella with mutations in isrM is attenuated in killing animals and defective in growth in the ileum and spleen in mice. Our study has shown that IsrM sRNA functions as a pathogenicity island-encoded sRNA directly involved in Salmonella pathogenesis in animals. Our results also suggest that sRNAs may represent a distinct class of virulence factors that are important for bacterial infection in vivo.
Assuntos
Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismo , Salmonelose Animal/microbiologia , Salmonella typhimurium/patogenicidade , Fatores de Virulência/biossíntese , Regiões 5' não Traduzidas , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Feminino , Trato Gastrointestinal/citologia , Trato Gastrointestinal/microbiologia , Regulação Bacteriana da Expressão Gênica , Ilhas Genômicas , Íleo/citologia , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Regiões Promotoras Genéticas , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/genética , Salmonelose Animal/patologia , Salmonella typhimurium/genética , Salmonella typhimurium/crescimento & desenvolvimento , Salmonella typhimurium/metabolismo , Alinhamento de Sequência , Análise de Sequência , Baço/citologia , Fatores de Virulência/genéticaRESUMO
BACKGROUND: Adenosine triphosphate (ATP) is used as an intracellular energy source by all living organisms. It plays a central role in the respiration and metabolism, and is the most important energy supplier in many enzymatic reactions. Its critical role as the energy storage molecule makes it extremely valuable to all cells. RESULTS: We report here the detection of extracellular ATP in the cultures of a variety of bacterial species. The levels of the extracellular ATP in bacterial cultures peaked around the end of the log phase and decreased in the stationary phase of growth. Extracellular ATP levels were dependent on the cellular respiration as bacterial mutants lacking cytochrome bo oxidase displayed lower extracellular ATP levels. We have also shown that Escherichia coli (E. coli) and Salmonella actively depleted extracellular ATP and an ATP supplement in culture media enhanced the stationary survival of E. coli and Salmonella. In addition to E. coli and Salmonella the presence of the extracellular ATP was observed in a variety of bacterial species that contain human pathogens such as Acinetobacter, Pseudomonas, Klebsiella and Staphylococcus. CONCLUSION: Our results indicate that extracellular ATP is produced by many bacterial species during growth and extracellular ATP may serve a role in the bacterial physiology.
Assuntos
Trifosfato de Adenosina/metabolismo , Enterobacteriaceae/crescimento & desenvolvimento , Pseudomonas/crescimento & desenvolvimento , Staphylococcus/crescimento & desenvolvimento , Enterobacteriaceae/metabolismo , Viabilidade Microbiana/efeitos dos fármacos , Pseudomonas/metabolismo , Staphylococcus/metabolismoRESUMO
A fundamental challenge in gene therapy is to develop approaches for delivering nucleic acid-based gene interfering agents, such as small interfering RNAs and ribozymes, to the appropriate cells in a way that is tissue/cell specific, efficient, and safe. Using human cytomegalovirus (HCMV) infection of differentiated macrophages as the model, we showed that Salmonella can efficiently deliver RNase P-based ribozyme sequence in specific human cells, leading to substantial ribozyme expression and effective inhibition of viral infection. We constructed a functional RNase P ribozyme (M1GS RNA) that targets the overlapping mRNA region of two HCMV capsid proteins, the capsid scaffolding protein (CSP) and assemblin, which are essential for viral capsid formation. Substantial expression of ribozymes was observed in human differentiated macrophages that were treated with attenuated Salmonella strains carrying the ribozyme sequence constructs. A reduction of 87-90% in viral CSP expression and a reduction of about 5,000-fold in viral growth were observed in cells that were treated with Salmonella carrying the sequence of the functional ribozyme but not with those carrying the sequence of a control ribozyme that contained mutations abolishing the catalytic activity. To our knowledge, this study showed for the first time that ribozymes expressed following targeted gene transfer with Salmonella-based vectors are highly active and specific in blocking viral infection. Moreover, these results demonstrate the feasibility to develop Salmonella-mediated gene transfer of RNase P ribozymes as an effective approach for gene-targeting applications.
Assuntos
Regulação Viral da Expressão Gênica , RNA Catalítico/química , Ribonuclease P/química , Salmonella/metabolismo , Diferenciação Celular , Infecções por Citomegalovirus/terapia , Fibroblastos/metabolismo , Técnicas de Transferência de Genes , Terapia Genética/métodos , Vetores Genéticos , Humanos , Cinética , Conformação de Ácido Nucleico , Mutação Puntual , Replicação ViralRESUMO
Ribonuclease P (RNase P) complexed with external guide sequence (EGS) represents a novel nucleic acid-based gene interference approach to modulate gene expression. We have previously used an in vitro selection procedure to generate EGS variants that efficiently direct human RNase P to cleave a target mRNA in vitro. In this study, a variant was used to target the mRNA encoding the protease of human cytomegalovirus (HCMV), which is essential for viral capsid formation and replication. The EGS variant was about 35-fold more active in inducing human RNase P to cleave the mRNA in vitro than the EGS derived from a natural tRNA. Moreover, a reduction of 95% in the expression of the protease and a reduction of 4,000-fold in viral growth were observed in HCMV-infected cells that expressed the EGS variant, whereas a reduction of 80% in the protease expression and an inhibition of 150-fold in viral growth were detected in cells that expressed the EGS derived from a natural tRNA sequence. No significant reduction in viral protease expression or viral growth was observed in cells that either did not express an EGS or produced a "disabled" EGS, which carried nucleotide mutations that precluded RNase P recognition. Our results provide direct evidence that engineered EGS variant is highly effective in blocking HCMV expression and growth by targeting the viral protease. Furthermore, these results demonstrate the utility of engineered EGS RNAs in gene targeting applications, including the inhibition of HCMV infection by blocking the expression of virus-encoded essential proteins.
Assuntos
Citomegalovirus/genética , Citomegalovirus/fisiologia , Regulação Viral da Expressão Gênica/genética , Engenharia Genética/métodos , Interferência de RNA , Replicação Viral/genética , Sequência de Bases , Capsídeo/enzimologia , Capsídeo/metabolismo , Citomegalovirus/enzimologia , Citomegalovirus/crescimento & desenvolvimento , Replicação do DNA/genética , Estudos de Viabilidade , Genes Virais/genética , Células HeLa , Humanos , Peptídeo Hidrolases/deficiência , Peptídeo Hidrolases/genética , RNA Mensageiro/genética , Ribonuclease P/metabolismoRESUMO
External guide sequences (EGSs) are RNA molecules that can bind to a target mRNA and direct ribonuclease P (RNase P), a tRNA processing enzyme, for specific cleavage of the target mRNA. Using an in vitro selection procedure, we have previously generated EGS variants that efficiently direct human RNase P to cleave a target mRNA in vitro. In this study, we constructed EGSs from a variant to target the overlapping region of the mRNAs coding for human cytomegalovirus (HCMV) capsid scaffolding protein (CSP) and assemblin, which are essential for viral capsid formation. The EGS variant was about 40-fold more active in directing human RNase P to cleave the mRNA in vitro than the EGS derived from a natural tRNA. Moreover, a reduction of about 98% and 75% in CSP/assemblin gene expression and a reduction of 7000- and 250-fold in viral growth were observed in HCMV-infected cells that expressed the variant and the tRNA-derived EGS, respectively. Our study shows that the EGS variant is more effective in blocking HCMV gene expression and growth than the tRNA-derived EGS. Moreover, these results demonstrate the utility of highly active EGS RNA variants in gene targeting applications including anti-HCMV therapy.
Assuntos
Citomegalovirus/metabolismo , Regulação Viral da Expressão Gênica , RNA Mensageiro/biossíntese , RNA Viral/biossíntese , Ribonuclease P/metabolismo , Linhagem Celular Tumoral , Citomegalovirus/genética , Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/metabolismo , Infecções por Citomegalovirus/terapia , Terapia Genética/métodos , Humanos , RNA Mensageiro/genética , RNA Viral/genética , Ribonuclease P/genética , Pequeno RNA não TraduzidoRESUMO
The type III secretion system (T3SS) encoded by Salmonella pathogenicity island 1 (SPI-1) is important for the invasion of epithelial cells during development of Salmonella-associated enterocolitis. It has been suggested that the level and timing of the expression of the SPI-1 T3SS proteins and effectors dictate the consequences of bacterial infection and pathogenesis. However, the expression of these proteins has not been extensively studied in vivo, especially during the later stages of salmonellosis when the infection is established. We have constructed recombinant Salmonella strains that contain a FLAG epitope inserted in-frame to genes invJ, prgJ, sipC, sipD, sopA and sopB, and investigated the expression of the tagged proteins both in vitro and in vivo during murine salmonellosis. Mice were inoculated intraperitoneally or intragastrically with the tagged Salmonella strains. At different time points post-infection, bacteria were recovered from various organs, and the expression of the tagged proteins was determined. Our results provide direct evidence that PrgJ and SipD are expressed in Salmonella colonizing the liver and ileum of infected animals at both the early and late stages of infection. Furthermore, our study has shown that the InvJ protein is expressed preferentially in Salmonella colonizing the ileum but not the liver, while SipC is expressed preferentially in Salmonella colonizing the liver but not the ileum. Thus, Salmonella appears to express different SPI-1 proteins and effectors when colonizing specific tissues. Our results suggest that differential expression of these proteins may be important for tissue-specific aspects of bacterial pathogenesis such as gastroenterititis in the ileum and systemic infection in the liver.
Assuntos
Proteínas de Bactérias/genética , Salmonelose Animal/microbiologia , Salmonella/genética , Animais , Contagem de Colônia Microbiana , Epitopos/genética , Feminino , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Íleo/microbiologia , Fígado/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Salmonella/crescimento & desenvolvimento , Salmonella/patogenicidade , VirulênciaRESUMO
BACKGROUND: Salmonella enterica, a common food-borne bacterial pathogen, is believed to change its protein expression profile in the presence of different environmental stress such as that caused by the exposure to hydrogen peroxide (H2O2), which can be generated by phagocytes during infection and represents an important antibacterial mechanism of host cells. Among Salmonella proteins, the effectors of Salmonella pathogenicity island 1 and 2 (SPI-1 and SPI-2) are of particular interest since they are expressed during host infection in vivo and are important for invasion of epithelial cells and for replication in organs during systemic infection, respectively. However, the expression profiles of these proteins upon exposure to H2O2 or to host cells in vivo during the established phase of systemic infection have not been extensively studied. RESULTS: Using stable isotope labeling coupled with mass spectrometry, we performed quantitative proteomic analysis of Salmonella enterica serovar Enteritidis and identified 76 proteins whose expression is modulated upon exposure to H2O2. SPI-1 effector SipC was expressed about 3-fold higher and SopB was expressed approximately 2-fold lower in the presence of H2O2, while no significant change in the expression of another SPI-1 protein SipA was observed. The relative abundance of SipA, SipC, and SopB was confirmed by Western analyses, validating the accuracy and reproducibility of our approach for quantitative analysis of protein expression. Furthermore, immuno-detection showed substantial expression of SipA and SipC but not SopB in the late phase of infection in macrophages and in the spleen of infected mice. CONCLUSIONS: We have identified Salmonella proteins whose expression is modulated in the presence of H2O2. Our results also provide the first direct evidence that SipC is highly expressed in the spleen at late stage of salmonellosis in vivo. These results suggest a possible role of SipC and other regulated proteins in supporting survival and replication of Salmonella under oxidative stress and during its systemic infection in vivo.
Assuntos
Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Proteômica/métodos , Salmonella enteritidis/metabolismo , Animais , Linhagem Celular , Macrófagos/microbiologia , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos BALB C , Estresse Oxidativo , Salmonelose Animal/microbiologia , Salmonella enteritidis/genética , Baço/microbiologiaRESUMO
BACKGROUND: Child mortality is a major global health challenge, especially in regions of limited resources. Accessibility to lifesaving medicine and adequate nutrition is essential to reduce child mortality and improve the health and well-being of the world's most vulnerable children. METHODS: We have developed NutMox, a novel pediatric formulation of the ß-lactam antibiotic amoxicillin in a matrix of peanut-based ready-to-use therapeutic food (RUTF) consisting of peanut butter, sugar, vegetable oil, dry milk and vitamins. NutMox is ready to use and thermostable, requires no chewing or pill swallowing and provides both an antibiotic and nutrition. RESULTS: Amoxicillin in NutMox formulations was stable for at least 12 months at storage temperatures of 4°C, 25°C and 37°C. Amoxicillin formulated in NutMox displayed similar pharmacokinetics in mice to that in suspension. CONCLUSIONS: Our results demonstrated the feasibility of a peanut butter-based matrix for pediatric formulations of amoxicillin, suggesting that such a matrix can serve as a base for delivering medications in addition to its current use as an RUTF.
Assuntos
Amoxicilina , Arachis , Composição de Medicamentos/métodos , Alimentos Fortificados , Criança , Estudos de Viabilidade , HumanosRESUMO
Potassium (K(+)) is the most abundant intracellular cation and is essential for many physiological functions of all living organisms; however, its role in the pathogenesis of human pathogens is not well understood. In this study, we characterized the functions of the bacterial Trk K(+) transport system and external K(+) in the pathogenesis of Salmonella enterica, a major food-borne bacterial pathogen. Here we report that Trk is important for Salmonella to invade and grow inside epithelial cells. It is also necessary for the full virulence of Salmonella in an animal infection model. Analysis of proteins of Salmonella indicated that Trk is involved in the expression and secretion of effector proteins of the type III secretion system (TTSS) encoded by Salmonella pathogenicity island 1 (SPI1) that were previously shown to be necessary for Salmonella invasion. In addition to the role of the Trk transporter in the pathogenesis of Salmonella, we discovered that external K(+) modulates the pathogenic properties of Salmonella by increasing the expression and secretion of effector proteins of the SPI1-encoded TTSS and by enhancing epithelial cell invasion. Our studies demonstrated that K(+) is actively involved in the pathogenesis of Salmonella and indicated that Salmonella may take advantage of the high K(+) content inside host cells and in the intestinal fluid during diarrhea to become more virulent.
Assuntos
Proteínas de Bactérias/metabolismo , Canais de Potássio/genética , Canais de Potássio/metabolismo , Potássio/metabolismo , Salmonella enterica/metabolismo , Salmonella enterica/patogenicidade , Animais , Células Epiteliais/microbiologia , Regulação Bacteriana da Expressão Gênica/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Células HeLa , Humanos , Camundongos , Mutação , Peritonite/microbiologia , Potássio/farmacologia , Infecções por Salmonella/microbiologia , Salmonella enterica/efeitos dos fármacos , Cloreto de Sódio/farmacologia , Gastropatias/microbiologia , Fatores de Tempo , VirulênciaRESUMO
BACKGROUND: The global regulatory system ArcAB controls the anaerobic growth of E. coli, however, its role in aerobic conditions is not well characterized. We have previously reported that ArcA was necessary for Salmonella to resist reactive oxygen species (ROS) in aerobic conditions. RESULTS: To investigate the mechanism of ROS resistance mediated by ArcAB, we generated deletion mutants of ArcA and ArcB in E. coli. Our results demonstrated that both ArcA and ArcB were necessary for resistance to hydrogen peroxide (H2O2), a type of ROS, and their function in this resistance was independent from H2O2 scavenge. Mutagenesis analysis of ArcA indicated that ROS resistance was mediated through a distinct signaling pathway from that used in anaerobic conditions. An abundant protein flagellin was elevated at both the protein and mRNA levels in the DeltaarcA mutant as compared to the wild type E. coli, and deletion of flagellin restored the resistance of the DeltaarcA mutant to H2O2. The resistance of the DeltaarcA mutant E. coli to H2O2 can also be restored by amino acid supplementation, suggesting that a deficiency in amino acid and/or protein synthesis in the mutant contributed to its susceptibility to H2O2, which is consistent with the notion that protein synthesis is necessary for ROS resistance. CONCLUSION: Our results suggest that in addition to its role as a global regulator for anaerobic growth of bacteria, ArcAB system is also important for bacterial resistance to ROS in aerobic conditions, possibly through its influence on bacterial metabolism, especially amino acid and/or protein assimilation and synthesis.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Peróxido de Hidrogênio/metabolismo , Proteínas de Membrana/metabolismo , Estresse Oxidativo , Proteínas Quinases/metabolismo , Proteínas Repressoras/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Flagelina/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana/genética , Mutagênese , Fosforilação , Proteínas Quinases/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , Proteínas Repressoras/genéticaRESUMO
BACKGROUND: The type III secretion systems (T3SSs) encoded by Salmonella pathogenicity island 1 and 2 (SPI-1 and SPI-2) are important for invasion of epithelial cells during development of Salmonella-associated enterocolitis and for replication in macrophages during systemic infection, respectively. In vitro studies have previously revealed hierarchical transport of different SPI-1 factors and ordered synergistic/antagonistic relationships between these proteins during Salmonella entry. These results suggest that the level and timing of the expression of these proteins dictate the consequences of bacterial infection and pathogenesis. However, the expression of these proteins has not been extensively studied in vivo, especially during the later stages of salmonellosis when the infection is established. RESULTS: In this study, we have constructed bacterial strains that contain a FLAG epitope inserted in frame to SPI-1 genes prgI, sipA, sipB, sopE2, spaO, and sptP, and investigated the expression of the tagged proteins both in vitro and in vivo during murine salmonellosis. The tagged Salmonella strains were inoculated intraperitoneally or intragastrically into mice and recovered from various organs. Our results provide direct evidence that PrgI and SipB are expressed in Salmonella colonizing the spleen and cecum of the infected animals at early and late stages of infection. Furthermore, this study demonstrates that the SpaO protein is expressed preferably in Salmonella colonizing the cecum but not the spleen and that SptP is expressed preferably in Salmonella colonizing the spleen but not the cecum. CONCLUSION: These results suggest that Salmonella may express different SPI-1 proteins when they colonize specific tissues and that differential expression of these proteins may be important for tissue-specific aspects of bacterial pathogenesis such as gastroenterititis in the cecum and systemic infection in the spleen.
Assuntos
Proteínas de Bactérias/metabolismo , Salmonelose Animal/microbiologia , Salmonella typhimurium/metabolismo , Animais , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Ceco/microbiologia , Contagem de Colônia Microbiana , Feminino , Regulação Bacteriana da Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Mutagênese Insercional , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/metabolismo , Salmonella typhimurium/genética , Salmonella typhimurium/crescimento & desenvolvimento , Baço/microbiologiaRESUMO
Fresh produce, including salad, is increasingly implicated in foodborne outbreaks. Although studies have been carried out to detect specific human pathogens from fresh produce, the total bacterial community associated with fresh produce is poorly understood. In this study, we characterized the bacterial community associated with alfalfa sprouts, using a culture-independent method. Four retail-purchased alfalfa sprout samples were obtained from different producers, and the bacterial community associated with each sample was determined by 16S rDNA profiling. Our results indicate that alfalfa sprouts sampled in our study shared significant similarities in their bacterial communities. Proteobacteria was the dominant phylum detected from all alfalfa sprout samples, with Enterobacteriaceae, Oxalobacteraceae, Moraxellaceae, and Sphingomonadaceae as the most frequently detected families. These results indicate that growth conditions of alfalfa sprouts should be taken into consideration to prevent the proliferation of pathogenic proteobacteria such as Escherichia coli O157 and Salmonella.
Assuntos
Qualidade de Produtos para o Consumidor , Enterobacteriaceae/isolamento & purificação , Contaminação de Alimentos/análise , Medicago sativa/microbiologia , Proteobactérias/isolamento & purificação , Contagem de Colônia Microbiana , DNA Bacteriano/análise , Enterobacteriaceae/crescimento & desenvolvimento , Escherichia coli O157/crescimento & desenvolvimento , Escherichia coli O157/isolamento & purificação , Microbiologia de Alimentos , Humanos , Proteobactérias/crescimento & desenvolvimento , Medição de Risco , Salmonella/crescimento & desenvolvimento , Salmonella/isolamento & purificaçãoRESUMO
Rationales: Gene-targeting ribozymes represent promising nucleic acid-based gene interference agents for therapeutic application. We previously used an in vitro selection procedure to engineer novel RNase P-based ribozyme variants with enhanced targeting activity. However, it has not been reported whether these ribozyme variants also exhibit improved activity in blocking gene expression in animals. Methods and Results: In this report, R388-AS, a new engineered ribozyme variant, was designed to target the mRNA of assemblin (AS) of murine cytomegalovirus (MCMV), which is essential for viral progeny production. Variant R338-AS cleaved AS mRNA sequence in vitro at least 200 times more efficiently than ribozyme M1-AS, which originated from the wild type RNase P catalytic RNA sequence. In cultured MCMV-infected cells, R338-AS exhibited better antiviral activity than M1-AS and decreased viral AS expression by 98-99% and virus production by 15,000 fold. In MCMV-infected mice, R388-AS was more active in inhibiting AS expression, blocking viral replication, and improving animal survival than M1-AS. Conclusions: Our results provide the first direct evidence that novel engineered RNase P ribozyme variants with more active catalytic activity in vitro are also more effective in inhibiting viral gene expression in animals. Moreover, our studies imply the potential of engineering novel RNase P ribozyme variants with unique mutations to improve ribozyme activity for therapeutic application.