RESUMO
Phage Culver, with a siphovirus morphology, was isolated using Gordonia terrae CAG3. Culver is assigned to phage cluster CQ1 based on gene content similarity to actinobacteriophages. Notably, Culver is predicted to encode eight tRNAs, lysin A by two adjacent genes, and, unlike other CQ1 phages, two putative integrase genes.
RESUMO
Arthrobacter phage Tokki is a siphovirus isolated from soil in River Falls, Wisconsin. The genome contains 57,652-bp encoding 98 proteins, of which 23 were assigned a function. Tokki's genome structure and content is typical of other AU2 subcluster phages, except for the lack of an identifiable acetyltransferase.
RESUMO
The interaction of macrophages with infectious agents leads to the activation of several signaling cascades, including mitogen-activated protein (MAP) kinases, such as p38. We now demonstrate that p38 MAP kinase-mediated responses are critical components to the immune response to Borrelia burgdorferi. The pharmacological and genetic inhibition of p38 MAP kinase activity during infection with the spirochete results in increased carditis. In transgenic mice that express a dominant negative form of p38 MAP kinase specifically in macrophages, production of the invariant natural killer T (iNKT) cell-attracting chemokine MCP-1 and of the antigen-presenting molecule CD1d are significantly reduced. The expression of the transgene therefore results in the deficient infiltration of iNKT cells, their decreased activation, and a diminished production of interferon γ (IFN-γ), leading to increased bacterial burdens and inflammation. These results show that p38 MAP kinase provides critical checkpoints for the protective immune response to the spirochete during infection of the heart.
Assuntos
Borrelia burgdorferi , Células Matadoras Naturais/fisiologia , Doença de Lyme/imunologia , Macrófagos/enzimologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Antígenos CD1d/genética , Antígenos CD1d/metabolismo , Antígeno CD11b/genética , Antígeno CD11b/metabolismo , Regulação da Expressão Gênica , Cardiopatias/etiologia , Cardiopatias/patologia , Homeostase , Imidazóis/farmacologia , Interferon gama/genética , Interferon gama/metabolismo , Doença de Lyme/complicações , Doença de Lyme/patologia , Camundongos , Camundongos Transgênicos , Piridinas/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
Th17 cells are known to play a critical role in adaptive immune responses to several important extracellular pathogens. Additionally, Th17 cells are implicated in the pathogenesis of several autoimmune and inflammatory disorders as well as in cancer. Therefore, it is essential to understand the mechanisms that regulate Th17 differentiation. Notch signaling is known to be important at several stages of T cell development and differentiation. In this study, we report that Notch1 is activated in both mouse and human in vitro-polarized Th17 cells and that blockade of Notch signaling significantly downregulates the production of Th17-associated cytokines, suggesting an intrinsic requirement for Notch during Th17 differentiation in both species. We also present evidence, using promoter reporter assays, knockdown studies, as well as chromatin immunoprecipitation, that IL-17 and retinoic acid-related orphan receptor γt are direct transcriptional targets of Notch signaling in Th17 cells. Finally, in vivo inhibition of Notch signaling reduced IL-17 production and Th17-mediated disease progression in experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis. Thus, this study highlights the importance of Notch signaling in Th17 differentiation and indicates that selective targeted therapy against Notch may be an important tool to treat autoimmune disorders, including multiple sclerosis.
Assuntos
Diferenciação Celular/imunologia , Receptor Notch1/fisiologia , Transdução de Sinais/imunologia , Células Th17/imunologia , Animais , Células Cultivadas , Citocinas/antagonistas & inibidores , Citocinas/fisiologia , Regulação para Baixo/imunologia , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Encefalomielite Autoimune Experimental/terapia , Células HEK293 , Humanos , Interleucina-17/antagonistas & inibidores , Interleucina-17/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Esclerose Múltipla/imunologia , Esclerose Múltipla/patologia , Esclerose Múltipla/terapia , Células Th17/metabolismo , Células Th17/patologiaRESUMO
Members of the testis-specific serine/threonine kinases (Tssk) family may have a role in sperm differentiation in the testis and/or fertilization. To gain insight into the functional relevance of these kinases, their expression was examined both at the mRNA and protein levels. Quantitative PCR analysis confirmed that all five Tssk mRNAs are almost exclusively expressed postmeiotically in the testis. Recombinant mouse and human Tssks were cloned and used for validation of an array of commercial and custom-made antibodies against Tssks. Immunolocalization in mouse testis, and in mouse and human sperm, showed that Tssk1, Tssk2, Tssk4 and Tssk6, but not Tssk3, were present in mouse sperm and in germ cells from mouse testis. TSSK1, TSSK2 and TSSK6 were also detected in human sperm, while TSSK3 was absent. In both mouse and human sperm, Tssk1 was partially soluble, while Tssk2, Tssk4 and Tssk6 were insoluble in non-ionic detergents. In vitro recombinant TSSK2 activity assays showed maximum enzymatic activity at 5 mM Mg(2+) and a Km for ATP of â¼10 µM. These, observations together with findings that the Tssk1/Tssk2 double knock-out as well as the Tssk6 null mice are sterile without presenting other detectable defects, suggest that these kinases could be used as targets for male contraception.
Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Espermatozoides/enzimologia , Testículo/enzimologia , Animais , Western Blotting , Eletroforese em Gel de Poliacrilamida , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Masculino , Camundongos , Espermátides/enzimologiaRESUMO
Salp15 is a tick saliva protein that inhibits CD4(+) T cell differentiation through its interaction with CD4. The protein inhibits early signaling events during T cell activation and IL-2 production. Because murine Experimental Autoimmune Encephalomyelitis development is mediated by central nervous system-infiltrating CD4(+) T cells that are specific for myelin-associated proteins, we sought to determine whether the treatment of mice with Salp15 during EAE induction would prevent the generation of proinflammatory T cell responses and the development of the disease. Surprisingly, Salp15-treated mice developed more severe EAE than control animals. The treatment of EAE-induced mice with the tick saliva protein did not result in increased infiltration of T cells to the central nervous system, indicating that Salp15 had not affected the permeability of the blood-brain barrier. Salp15 treatment did not affect the development of antibody responses against the eliciting peptide or the presence of IFNγ in the sera. The treatment with Salp15 resulted, however, in the increased differentiation of Th17 cells in vivo, as evidenced by higher IL-17 production from PLP(139-151)-specific CD4(+) T cells isolated from the central nervous system and the periphery. In vitro, Salp15 was able to induce the differentiation of Th17 cells in the presence of IL-6 and the absence of TGFß These results suggest that a conductive milieu for the differentiation of Th17 cells can be achieved by restriction of the production of IL-2 during T cell differentiation, a role that may be performed by TGFß and other immunosuppressive agents.
Assuntos
Encefalomielite Autoimune Experimental/imunologia , Tolerância Imunológica/efeitos dos fármacos , Imunossupressores/farmacologia , Proteínas e Peptídeos Salivares/imunologia , Proteínas e Peptídeos Salivares/farmacologia , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/imunologia , Barreira Hematoencefálica/metabolismo , Feminino , Interleucina-17/metabolismo , Camundongos , Camundongos Endogâmicos , Permeabilidade , Células Th1/efeitos dos fármacos , Células Th1/imunologiaRESUMO
BACKGROUND & AIMS: MicroRNAs (miRNAs) are members of a class of small noncoding functional RNAs that modulate gene regulation at the post-transcriptional level in a sequence specific manner. miRNA dysfunction has been linked to the pathophysiology of human diseases including those resulting from viral infections. The objective of this study was to investigate changes in miRNA profiles that occur in hepatoma cells expressing hepatitis C virus (HCV) and identify anticorrelated mRNAs, which may be their regulatory targets. METHODS: Microarrays were used to perform global miRNA and mRNA expression analysis. Fold changes and pairwise statistics were computed for the resulting datasets. Hierarchical cluster and pathway analyses were performed to assess the degree of differential expression and identify regulatory networks. Bioinformatics tools were used to integrate mRNA profiling results with miRNA target predictions. RESULTS: Replication of the Con1 strain of HCV virus in hepatoma cells elicited extensive differential expression of both miRNAs and mRNAs. Forty-three differentially expressed miRNAs (P≤0.001) were identified by microarray analysis in HCV expressing cells. Six thousand eight hundred and fifteen differentially expressed mRNAs (P≤0.05) were identified. Computational analyses revealed anticorrelated miRNA:mRNA pairs for each target prediction algorithm used. Pathway analysis generated a filtered pathway with 120 entities, including seven major regulators and nine major targets potentially under the control of at least 11 miRNAs. CONCLUSIONS: The expression of a number of anticorrelated miRNAs:mRNA pairs are affected by the presence of HCV. These miRNAs and their putative targets are attractive candidates for being involved in the pathogenesis and/or progression of HCV-induced chronic hepatitis.
Assuntos
Carcinoma Hepatocelular/genética , Perfilação da Expressão Gênica , Hepacivirus/patogenicidade , Hepatite C Crônica/genética , Neoplasias Hepáticas/genética , MicroRNAs/metabolismo , RNA Mensageiro/metabolismo , Algoritmos , Carcinoma Hepatocelular/virologia , Linhagem Celular Tumoral , Análise por Conglomerados , Biologia Computacional , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Neoplasias Hepáticas/virologia , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
Nanoparticles may be introduced into aquatic environments during production processes and also as a result of release following their use in various commercial formulations and biologic applications. Filter-feeding bivalve mollusks such as oysters are valuable model species for characterizing nanoparticle bioavailability and interactions with basic cellular processes. The adults release their gametes into the environment, so their embryos and larvae are also likely targets of nanoparticles. The purpose of these studies was to characterize the toxicity of metal nanoparticles on embryonic development of oysters, Crassostrea virginica and to compare the relative sensitivity of embryos to adults. Newly-fertilized oyster embryos were exposed to silver nanoparticles (AgNP) and then the percent normal development after 48h was assessed. Studies were conducted with adult oysters in which they were also exposed to AgNP for 48h, and the effects on lysosomal destabilization were determined. The expression of metallothionein (MT) gene expression was also assessed in both embryos and adults. Adverse effects on embryonic development were observed at concentrations similar to those that caused both statistically and biologically significant effects on lysosomal destabilization of adults. Significant increases in MT mRNA levels were observed in both embryos and adult oysters, and MT levels were highly induced in embryos. While we do not know whether the toxicity and gene expression responses observed in this study were due to the nanoparticles themselves or the Ag ions that dissociated from the nanoparticles, these kinds of basic studies are essential for addressing the potential impacts of nanoengineered particles on fundamental cellular processes as well as aquatic organisms.
Assuntos
Crassostrea , Nanopartículas Metálicas/toxicidade , Metalotioneína/metabolismo , Prata/toxicidade , Animais , Embrião não Mamífero/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Lisossomos , RNA Mensageiro/metabolismoRESUMO
The Lyme disease spirochete Borrelia burgdorferi is the only known human pathogen that directly activates invariant NKT (iNKT) cells. The number and activation kinetics of iNKT cells vary greatly among different strains of mice. We now report the role of the iNKT cell response in the pathogenesis of Lyme disease using C57BL/6 mice, a strain with optimal iNKT cell activation that is resistant to the development of spirochetal-induced inflammation. During experimental infection of B6 mice with B. burgdorferi, iNKT cells localize to the inflamed heart where they are activated by CD1d-expressing macrophages. Activation of iNKT cells in vivo results in the production of IFN-gamma, which we demonstrate ameliorates the severity of murine Lyme carditis by at least two mechanisms. First, IFN-gamma enhances the recognition of B. burgdorferi by macrophages, leading to increased phagocytosis of the spirochete. Second, IFN-gamma activation of macrophages increases the surface expression of CD1d, thereby facilitating further iNKT activation. Collectively, our data demonstrate that in the resistant background, B6, iNKT cells modulate the severity of murine Lyme carditis through the action of IFN-gamma, which appears to self-renew through a positive feedback loop during infection.
Assuntos
Interferon gama/biossíntese , Doença de Lyme/imunologia , Doença de Lyme/terapia , Miocardite/imunologia , Miocardite/terapia , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/metabolismo , Doença Aguda , Animais , Antígenos CD1d/biossíntese , Antígenos CD1d/genética , Antígenos CD1d/fisiologia , Borrelia burgdorferi/imunologia , Movimento Celular/genética , Movimento Celular/imunologia , Células Cultivadas , Modelos Animais de Doenças , Retroalimentação Fisiológica/genética , Retroalimentação Fisiológica/imunologia , Interferon gama/fisiologia , Doença de Lyme/metabolismo , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Ativação de Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miocardite/metabolismo , Células T Matadoras Naturais/patologia , Receptores de Antígenos de Linfócitos T alfa-beta/biossíntese , Receptores de Interferon/deficiência , Receptores de Interferon/genética , Receptor de Interferon gamaRESUMO
Ixodes scapularis salivary protein, Salp15, inhibits CD4(+) T cell activation by binding to the most-extracellular domains of the CD4 molecule, potentially overlapping with the gp120-binding region. We now show that Salp15 inhibits the interaction of gp120 and CD4. Furthermore, Salp15 prevents syncytia formation between HL2/3 (a stable HeLa cell line expressing the envelope protein) and CD4-expressing cells. Salp15 prevented gp120-CD4 interaction at least partially through its direct interaction with the envelope glycoprotein. A phage display library screen provided the interacting residues in the C1 domain of gp120. These results provide a potential basis to define exposed gp120 epitopes for the generation of neutralizing vaccines.
Assuntos
Antígenos CD4/metabolismo , Linfócitos T CD4-Positivos/efeitos dos fármacos , Proteína gp120 do Envelope de HIV/metabolismo , HIV/efeitos dos fármacos , Imunossupressores/farmacologia , Proteínas e Peptídeos Salivares/farmacologia , Sequência de Aminoácidos , Antígenos CD4/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linhagem Celular , HIV/imunologia , HIV/metabolismo , Proteína gp120 do Envelope de HIV/imunologia , Células HeLa , Humanos , Dados de Sequência Molecular , Biblioteca de PeptídeosRESUMO
The regulation of innate immune responses to pathogens occurs through the interaction of Toll-like receptors (TLRs) with pathogen-associated molecular patterns and the activation of several signaling pathways whose contribution to the overall innate immune response to pathogens is poorly understood. We demonstrate a mechanism of control of murine macrophage responses mediated by TLR1/2 heterodimers through c-Jun N-terminal kinase 1 (JNK1) activity. JNK controls tumor necrosis factor alpha production and TLR-mediated macrophage responses to Borrelia burgdorferi, the causative agent of Lyme disease, and the TLR1/TLR2-specific agonist PAM(3)CSK(4). JNK1, but not JNK2, activity regulates the expression of the tlr1 gene in the macrophage cell line RAW264.7, as well as in primary CD11b(+) cells. We also show that the proximal promoter region of the human tlr1 gene contains an AP-1 binding site that is subjected to regulation by the kinase and binds two complexes that involve the JNK substrates c-Jun, JunD, and ATF-2. These results demonstrate that JNK1 regulates the response to TLR1/2 ligands and suggest a positive feedback loop that may serve to increase the innate immune response to the spirochete.
Assuntos
Borrelia burgdorferi/imunologia , Regulação da Expressão Gênica , Macrófagos/imunologia , Proteína Quinase 8 Ativada por Mitógeno/fisiologia , Receptor 1 Toll-Like/genética , Animais , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular , Células Cultivadas , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Regiões Promotoras GenéticasRESUMO
The interaction of Borrelia burgdorferi, the causative agent of Lyme borreliosis, with phagocytic cells induces the activation of NF-kappaB and the expression of proinflammatory cytokines including tumor necrosis factor alpha (TNF-alpha). B. burgdorferi-induced TNF-alpha production is also dependent on the activation of p38 mitogen-activated protein (MAP) kinase. The specific contribution of these signaling pathways to the response of phagocytic cells to the spirochete and the molecular mechanisms underlying this response remain unresolved. We now show that p38 MAP kinase activity regulates the transcriptional activation of NF-kappaB in response to spirochetal lysate stimulation of phagocytic cells. The regulation occurs at the nuclear level and is independent of the translocation of the transcription factor to the nucleus or its capacity to bind to specific DNA target sequences. In RAW264.7 cells, p38alpha MAP kinase regulates the phosphorylation of NF-kappaB RelA. p38 MAP kinase phosphorylates the nuclear kinase mitogen- and stress-activated protein kinase 1 (MSK1). MSK1 in turn phosphorylates the transcriptionally active subunit of NF-kappaB, RelA. The repression of MSK1 expression with small interfering RNA results in reduced RelA phosphorylation and a significant decrease in the production of TNF-alpha in response to B. burgdorferi lysates. Overall, these results clarify the contribution of the signaling pathways that are activated in response to the interaction of spirochetes with phagocytic cells to TNF-alpha production. Our results situate p38 MAP kinase activity as a central regulator of the phagocytic proinflammatory response through MSK1-mediated transcriptional activation of the transcription factor NF-kappaB.
Assuntos
Antígenos de Bactérias/imunologia , NF-kappa B/genética , Proteínas Quinases/metabolismo , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/biossíntese , Proteínas Quinases p38 Ativadas por Mitógeno/imunologia , Animais , Western Blotting , Borrelia burgdorferi/imunologia , Ensaio de Desvio de Mobilidade Eletroforética , Interações Hospedeiro-Parasita/imunologia , Camundongos , Camundongos Transgênicos , Proteína Quinase 8 Ativada por Mitógeno/imunologia , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/imunologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fagócitos/imunologia , Fagócitos/microbiologia , Fosforilação , Proteínas Quinases/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/imunologia , Fator de Transcrição RelA/imunologia , Transcrição Gênica , Transfecção , Fator de Necrose Tumoral alfa/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Infection with Borrelia burgdorferi, the causative agent of Lyme disease, results in a Th1 response and proinflammatory cytokine production. Mice deficient for MKK3, an upstream activator of p38 mitogen-activated protein (MAP) kinase, develop a lower Th1 response and exhibit an impaired ability to produce proinflammatory cytokines upon infection with the spirochete. We investigated the contribution of p38 MAP kinase activity in gamma interferon (IFN-gamma) production in CD4+ T cells in response to specific antigen through T-cell receptor (TCR)- and interleukin-12 (IL-12)-mediated signals. The specific inhibition of p38 MAP kinase in T cells and the administration of a pharmacological inhibitor of the kinase during the course of infection with the spirochete resulted in reduced levels of IFN-gamma in the sera of infected mice. Our results also demonstrate that although p38 MAP kinase activity is not required for the differentiation of B. burgdorferi-specific CD4+ T cells, the production of IFN-gamma by Th1 effector cells is regulated by the kinase. Both TCR engagement and IL-12 induced the production of the Th1 cytokine through the activation of the p38 MAP kinase pathway. Thus, the inhibition of this pathway in vitro resulted in decreased levels of IFN-gamma during restimulation of B. burgdorferi-specific T cells in response to anti-CD3 and IL-12 stimulation. These results clarify the specific contribution of the p38 MAP kinase in the overall immune response to the spirochete and its role in the effector function of B. burgdorferi-specific T cells.
Assuntos
Borrelia burgdorferi , Interferon gama/biossíntese , Interleucina-12/fisiologia , Doença de Lyme/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Células Th1/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Antígenos CD4/análise , Feminino , Interferon gama/sangue , Doença de Lyme/enzimologia , MAP Quinase Quinase 3/genética , Camundongos , Camundongos Endogâmicos , Mutação , Inibidores de Proteínas Quinases/farmacologia , Receptores de Antígenos de Linfócitos T/genética , Células Th1/efeitos dos fármacos , Células Th1/enzimologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
Subcutaneous inoculation of mice with Borrelia burgdorferi, the causative agent of Lyme disease, results in established infection and the development of acute arthritis and carditis, hallmarks of human disease. Because conflicting results may originate from the site of subcutaneous inoculation, we addressed the dissemination capacity of spirochetes injected in the shoulder region versus the footpad. Spirochetes inoculated in the footpad disseminated to a lesser extent to distant organs, such as the ear and the heart. This resulted in distinct degrees of joint and cardiac inflammation at the peak of the disease. The differences eventually leveled out. These results suggest that caution must be exercised in the interpretation of results obtained with routes of inoculation that do not closely represent the natural site of infection.
Assuntos
Borrelia burgdorferi/patogenicidade , Doença de Lyme/etiologia , Doença de Lyme/microbiologia , Animais , Anticorpos Antibacterianos/sangue , Sequência de Bases , Borrelia burgdorferi/genética , Borrelia burgdorferi/imunologia , Borrelia burgdorferi/isolamento & purificação , Linfócitos T CD4-Positivos/imunologia , DNA Bacteriano/análise , DNA Bacteriano/genética , Modelos Animais de Doenças , Feminino , Humanos , Inflamação/etiologia , Inflamação/microbiologia , Injeções Subcutâneas , Doença de Lyme/imunologia , Doença de Lyme/patologia , Camundongos , Camundongos Endogâmicos C3H , Miocardite/etiologia , Miocardite/microbiologia , Especificidade de Órgãos , Reação em Cadeia da PolimeraseRESUMO
Pseudomonas fluorescens is a normal inhabitant of the soil rhizosphere. The use of genetically altered strains of P. fluorescens in bioremediation has led to the need for effective monitoring of such cells released into the environment. In this study, we present data on the persistence in soil of P. fluorescens harboring gfp (green fluorescent protein) or lux (bioluminescence) genes. Comparisons were made between strains marked chromosomally and strains carrying these markers on a plasmid. Overall effects of plasmid carriage on culturability were also examined. Sterile soil microcosms were inoculated with washed cells to a final concentration of ca. 10(6) CFU.g(-1) and placed at 5, 23, and 35-37 degrees C. Samples were taken periodically and examined for culturability and viability, using the substrate responsiveness assay. Our results indicated no significant loss of culturability at 5 and 23 degrees C for a period of over one year. In contrast, cells of P. fluorescens incubated at 35-37 degrees C entered the viable but nonculturable state within 7 days. All cells labeled with gfp retained fluorescence regardless of culturability, suggesting that the green fluorescent protein can be of value in monitoring the presence of cells following their release to the environment. Because fluorescence was maintained regardless of the cells' physiological state, this protein may also be an indicator of cell viability.
Assuntos
Proteínas de Fluorescência Verde/genética , Proteínas Luminescentes/genética , Organismos Geneticamente Modificados , Pseudomonas fluorescens/genética , Técnicas Bacteriológicas , Biodegradação Ambiental , Cromossomos Bacterianos , Monitoramento Ambiental/métodos , Marcadores Genéticos , Proteínas de Fluorescência Verde/análise , Proteínas Luminescentes/análise , Raízes de Plantas , Plasmídeos/genética , Microbiologia do Solo , TemperaturaRESUMO
Ingestion of shellfish-associated Vibrio parahaemolyticus is the primary cause of potentially severe gastroenteritis in many countries. However, only Kanagawa phenomenon (hemolysin) positive (KP+) strains of V. parahaemolyticus are isolated from patients, whereas >99% of strains isolated from the environment do not produce this hemolysin (i.e. are KP-). The reasons for these differences are not known. Following a temperature downshift, Vibrio parahaemolyticus enters the viable but nonculturable (VBNC) state wherein cells maintain viability but cannot be cultured on routine microbiological media We speculated that KP+ and KP- strains may respond differently to the temperature and salinity conditions of seawater by entering into this state which might account for the low numbers of culturable KP+ strains isolated from estuarine waters. The response of eleven KP+ and KP- strains of V. parahaemolyticus following exposure to a nutrient and temperature downshift in different salinities, similar to conditions encountered in their environment, was examined. The strains included those from which the KP+ genes had been selectively removed or added. Our results indicated that the ability to produce hemolysin did not affect entrance into the VBNC state. Further, VBNC cells of both biotypes could be restored to the culturable state following an overnight temperature upshift.
Assuntos
Proteínas Hemolisinas/biossíntese , Vibrio parahaemolyticus/fisiologia , Adaptação Fisiológica , Proteínas de Bactérias , Toxinas Bacterianas , Temperatura Baixa , Contagem de Colônia Microbiana , Meios de Cultura/química , Proteínas Hemolisinas/genética , Água do Mar/química , Água do Mar/microbiologia , Cloreto de Sódio , Temperatura , Fatores de Tempo , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/crescimento & desenvolvimentoRESUMO
Vibrio vulnificus has proven difficult to culture from water or shellfish during winter months, which is attributed to the viable but nonculturable (VBNC) state. Because reactive oxygen species were found to be involved in the low temperature-induced entrance of V. vulnificus into this state, we generated an oxyR mutant which lacks catalase activity. This strain is nonculturable on solid media even at ambient temperature, due to the presence of H(2)O(2) in such media. Low temperature incubation of the parent resulted in loss of catalase activity, making the cells H(2)O(2) sensitive, and paralleling the loss of culturability (entry into the VBNC state). Thus, cells of V. vulnificus in the VBNC state are likely exhibiting this response to low in situ temperature and only when the artificial condition of laboratory culture is attempted are the cells nonculturable due to cold-induced loss of catalase activity. To our knowledge, this is the first study providing direct evidence for the metabolic basis of nonculturability and the viable but nonculturable state.
Assuntos
Proteínas de Bactérias/metabolismo , Catalase/metabolismo , Temperatura Baixa , Viabilidade Microbiana , Transativadores/metabolismo , Vibrio vulnificus/fisiologia , Proteínas de Bactérias/genética , Contagem de Colônia Microbiana , DNA Bacteriano/química , DNA Bacteriano/genética , Deleção de Genes , Peróxido de Hidrogênio/toxicidade , Dados de Sequência Molecular , Análise de Sequência de DNA , Transativadores/genética , Vibrio vulnificus/efeitos dos fármacos , Vibrio vulnificus/genética , Vibrio vulnificus/crescimento & desenvolvimentoRESUMO
Representative encapsulated strains of Vibrio vulnificus from market oysters and oyster-associated primary septicemia cases (25 isolates each) were tested in a blinded fashion for potential virulence markers that may distinguish strains from these two sources. These isolates were analyzed for plasmid content, for the presence of a 460-bp amplicon by randomly amplified polymorphic DNA PCR, and for virulence in subcutaneously (s.c.) inoculated, iron-dextran-treated mice. Similar percentages of market oyster and clinical isolates possessed detectable plasmids (24 and 36%, respectively), produced the 460-bp amplicon (45 and 50%, respectively), and were judged to be virulent in the mouse s.c. inoculation-iron-dextran model (88% for each). Therefore, it appears that nearly all V. vulnificus strains in oysters are virulent and that genetic tests for plasmids and specific PCR size amplicons cannot distinguish between fully virulent and less virulent strains or between clinical and environmental isolates. The inability of these methods to distinguish food and clinical V. vulnificus isolates demonstrates the need for alternative subtyping approaches and virulence assays.