RESUMO
Pathogenic bacteria pose a serious threat to public health, and the rapid and cost-effective detection of such bacteria remains a major challenge. Herein, we present a DNAzyme-based fluorescent paper sensor for Klebsiella pneumoniae. The DNAzyme was generated by an in vitro selection technique to cleave a fluorogenic DNA-RNA chimeric substrate in the presence of K.â pneumoniae. The DNAzyme was printed on a paper substrate in a 96-well format to serve as mix-and-read fluorescent assay that exhibits a limit of detection (LOD) 105 â CFUs mL-1 . Evaluated with 20 strains of clinical bacterial isolates, the DNAzyme produced the desired fluorescence signal with the samples of K.â pneumoniae, regardless of their source or drug resistance. The assay is simple to use, rapid, inexpensive, and avoids the complex procedures of sample preparation and equipment. We believe that this DNAzyme-based fluorescent assay has potential for practical applications to identify K.â pneumoniae.
Assuntos
Técnicas de Tipagem Bacteriana/métodos , DNA Catalítico/química , Klebsiella pneumoniae/isolamento & purificação , Técnicas de Tipagem Bacteriana/instrumentação , Corantes Fluorescentes/química , Fluorometria/métodos , Biblioteca Gênica , Limite de Detecção , PapelRESUMO
Mast cells are known as central players in allergy and anaphylaxis, and they play a pivotal role in host defense against certain pathogens. Chlamydia pneumoniae is an important human pathogen, but it is unclear what role mast cells play during C. pneumoniae infection. We infected C57BL/6 (wild-type [WT]) and mast cell-deficient mice (Kit(W-sh/W-sh) [Wsh]) with C. pneumoniae. Wsh mice showed improved survival compared with WT mice, with fewer cells in Wsh bronchoalveolar lavage fluid (BALF), despite similar levels of cytokines and chemokines. We also found a more rapid clearance of bacteria from the lungs of Wsh mice compared with WT mice. Cromolyn, a mast cell stabilizer, reduced BALF cells and bacterial burden similar to the levels seen in Wsh mice; conversely, Compound 48/80, a mast cell degranulator, increased the number of BALF cells and bacterial burden. Histology showed that WT lungs had diffuse inflammation, whereas Wsh mice had patchy accumulations of neutrophils and perivascular accumulations of lymphocytes. Infected Wsh mice had reduced amounts of matrix metalloprotease-9 in BALF and were resistant to epithelial integral membrane protein degradation, suggesting that barrier integrity remains intact in Wsh mice. Mast cell reconstitution in Wsh mice led to enhanced bacterial growth and normal epithelial integral membrane protein degradation, highlighting the specific role of mast cells in this model. These data suggest that mast cells play a detrimental role during C. pneumoniae infection by facilitating immune cell infiltration into the airspace and providing a more favorable replicative environment for C. pneumoniae.
Assuntos
Movimento Celular/imunologia , Infecções por Chlamydophila/imunologia , Chlamydophila pneumoniae/imunologia , Mastócitos/imunologia , Pneumonia Bacteriana/imunologia , Animais , Antiasmáticos/farmacologia , Líquido da Lavagem Broncoalveolar , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Infecções por Chlamydophila/genética , Infecções por Chlamydophila/patologia , Cromolina Sódica/farmacologia , Humanos , Mastócitos/patologia , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/imunologia , Camundongos , Camundongos Transgênicos , Pneumonia Bacteriana/genética , Proteólise/efeitos dos fármacos , p-Metoxi-N-metilfenetilamina/farmacologiaRESUMO
UNLABELLED: The Scc4 protein (CT663) of the pathogenic bacterium Chlamydia has been described as a type III secretion (T3S) chaperone as well as an inhibitor of RNA polymerase. To examine if these roles are connected, we first investigated physical interactions between Chlamydia trachomatis Scc4 and the T3S chaperone Scc1 and a T3S substrate, CopN. In a yeast 3-hybrid assay, Scc4, Scc1, and CopN were all required to detect an interaction, which suggests that these proteins form a trimolecular complex. We also detected interactions between any two of these three T3S proteins in a pulldown assay using only recombinant proteins. We next determined whether these interactions affected the function of Scc4 as an inhibitor of RNA transcription. Using Escherichia coli as a heterologous in vivo system, we demonstrated that expression of C. trachomatis Scc4 led to a drastic decrease in transcript levels for multiple genes. However, coexpression of Scc4 with Scc1, CopN, or both alleviated Scc4-mediated inhibition of transcription. Scc4 expression also severely impaired E. coli growth, but this growth defect was reversed by coexpression of Scc4 with Scc1, CopN, or both, suggesting that the inhibitory effect of Scc4 on transcription and growth can be antagonized by interactions between Scc4, Scc1, and CopN. These findings suggest that the dual functions of Scc4 may serve as a bridge to link T3S and the regulation of gene expression in Chlamydia. IMPORTANCE: This study investigates a novel mechanism for regulating gene expression in the pathogenic bacterium Chlamydia. The Chlamydia type III secretion (T3S) chaperone Scc4 has been shown to inhibit transcription by RNA polymerase. This study describes physical interactions between Scc4 and the T3S proteins Scc1 and CopN. Furthermore, Chlamydia Scc1 and CopN antagonized the inhibitory effects of Scc4 on transcription and growth in a heterologous Escherichia coli system. These results provide evidence that transcription in Chlamydia can be regulated by the T3S system through interactions between T3S proteins.
Assuntos
Chlamydia trachomatis/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Transcrição Gênica/fisiologia , Sistemas de Secreção Tipo III/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Chlamydia trachomatis/genética , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Regulação para Baixo , Regulação Enzimológica da Expressão Gênica , Células HeLa , Humanos , Fator sigma/genética , Fator sigma/metabolismo , Sistemas de Secreção Tipo III/genéticaRESUMO
The salicylidene acylhydrazide INP0341 inhibits growth of Chlamydia in HeLa cells, has negligible cell toxicity, and does not inhibit the growth of lactobacilli. The antichlamydial activity of INP0341 was retained when tested in vaginal and semen simulants. Vaginal tissue from INP0341-treated mice appeared similar to control sham-treated mice. To determine whether INP0341 can protect mice from a vaginal challenge, C3H/HeJ mice were either sham or INP0341 treated intravaginally pre- and postinoculation with 5 × 10(2) inclusion-forming units (IFUs) of Chlamydia trachomatis serovar D. Vaginal cultures taken over a month-long period showed a significant difference in the number of control mice that were culture positive versus the number in the INP0341-treated group, 100% (25/25) and 31% (8/26), respectively (P < .05). The quantity of IFUs shed and antibody titers to Chlamydia were significantly higher for the control group (P < .05). In summary, INP0341 is a promising compound to be considered for formulation as a vaginal microbicide.
Assuntos
Anti-Infecciosos/administração & dosagem , Infecções por Chlamydia/prevenção & controle , Chlamydia trachomatis/efeitos dos fármacos , Hidrazinas/administração & dosagem , Administração Intravaginal , Animais , Carga Bacteriana , Chlamydia trachomatis/isolamento & purificação , Chlamydia trachomatis/patogenicidade , Feminino , Camundongos , Camundongos Endogâmicos C3H , Vagina/microbiologiaRESUMO
Here we investigated the role of the Nod/Rip2 pathway in host responses to Chlamydophila pneumoniae-induced pneumonia in mice. Rip2(-/-) mice infected with C. pneumoniae exhibited impaired iNOS expression and NO production, and delayed neutrophil recruitment to the lungs. Levels of IL-6 and IFN-gamma levels as well as KC and MIP-2 levels in bronchoalveolar lavage fluid (BALF) were significantly decreased in Rip2(-/-) mice compared to wild-type (WT) mice at day 3. Rip2(-/-) mice showed significant delay in bacterial clearance from the lungs and developed more severe and chronic lung inflammation that continued even on day 35 and led to increased mortality, whereas WT mice cleared the bacterial load, recovered from acute pneumonia, and survived. Both Nod1(-/-) and Nod2(-/-) mice also showed delayed bacterial clearance, suggesting that C. pneumoniae is recognized by both of these intracellular receptors. Bone marrow chimera experiments demonstrated that Rip2 in BM-derived cells rather than non-hematopoietic stromal cells played a key role in host responses in the lungs and clearance of C. pneumoniae. Furthermore, adoptive transfer of WT macrophages intratracheally was able to rescue the bacterial clearance defect in Rip2(-/-) mice. These results demonstrate that in addition to the TLR/MyD88 pathway, the Nod/Rip2 signaling pathway also plays a significant role in intracellular recognition, innate immune host responses, and ultimately has a decisive impact on clearance of C. pneumoniae from the lungs and survival of the infectious challenge.
Assuntos
Infecções por Chlamydophila/imunologia , Imunidade Inata , Proteínas Adaptadoras de Sinalização NOD/imunologia , Pneumonia Bacteriana/imunologia , Proteína Serina-Treonina Quinases de Interação com Receptores/imunologia , Animais , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Quimiocinas/imunologia , Quimiocinas/metabolismo , Infecções por Chlamydophila/metabolismo , Infecções por Chlamydophila/patologia , Chlamydophila pneumoniae/imunologia , Chlamydophila pneumoniae/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Macrófagos/imunologia , Camundongos , Camundongos Knockout , Infiltração de Neutrófilos/imunologia , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/biossíntese , Proteínas Adaptadoras de Sinalização NOD/metabolismo , Pneumonia Bacteriana/metabolismo , Pneumonia Bacteriana/patologia , RNA Mensageiro/análise , Proteína Serina-Treonina Quinase 2 de Interação com Receptor , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/fisiologiaRESUMO
Experimental and clinical studies link Chlamydia pneumoniae infection to atherogenesis and atherothrombotic events, but the underlying mechanisms are unclear. We tested the hypothesis that C. pneumoniae-induced acceleration of atherosclerosis in apolipoprotein E (ApoE)(-/-) mice is reciprocally modulated by activation of TLR-mediated innate immune and liver X receptor alpha (LXRalpha) signaling pathways. We infected ApoE(-/-) mice and ApoE(-/-) mice that also lacked TLR2, TLR4, MyD88, or LXRalpha intranasally with C. pneumoniae followed by feeding of a high fat diet for 4 mo. Mock-infected littermates served as controls. Atherosclerosis was assessed in aortic sinuses and in en face preparation of whole aorta. The numbers of activated dendritic cells (DCs) within plaques and the serum levels of cholesterol and proinflammatory cytokines were also measured. C. pneumoniae infection markedly accelerated atherosclerosis in ApoE-deficient mice that was associated with increased numbers of activated DCs in aortic sinus plaques and higher circulating levels of MCP-1, IL-12p40, IL-6, and TNF-alpha. In contrast, C. pneumoniae infection had only a minimal effect on atherosclerosis, accumulation of activated DCs in the sinus plaques, or circulating cytokine increases in ApoE(-/-) mice that were also deficient in TLR2, TLR4, or MyD88. However, C. pneumoniae-induced acceleration of atherosclerosis in ApoE(-/-) mice was further enhanced in ApoE(-/-)LXRalpha(-/-) double knockout mice and was accompanied by higher serum levels of IL-6 and TNF-alpha. We conclude that C. pneumoniae infection accelerates atherosclerosis in hypercholesterolemic mice predominantly through a TLR/MyD88-dependent mechanism and that LXRalpha appears to reciprocally modulate and reduce the proatherogenic effects of C. pneumoniae infection.
Assuntos
Aterosclerose/microbiologia , Infecções por Chlamydia/complicações , Proteínas de Ligação a DNA/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Transdução de Sinais/fisiologia , Receptores Toll-Like/metabolismo , Animais , Aorta/imunologia , Aorta/patologia , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Aterosclerose/metabolismo , Infecções por Chlamydia/metabolismo , Chlamydophila pneumoniae , Citocinas/sangue , Citocinas/imunologia , Proteínas de Ligação a DNA/genética , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Imunofluorescência , Expressão Gênica , Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Hipercolesterolemia/complicações , Imuno-Histoquímica , Receptores X do Fígado , Camundongos , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Análise de Sequência com Séries de Oligonucleotídeos , Receptores Nucleares Órfãos , Receptores Citoplasmáticos e Nucleares/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptores Toll-Like/genéticaRESUMO
Sepsis due to antimicrobial resistant pathogens is a major health problem worldwide. The inability to rapidly detect and thus treat bacteria with appropriate agents in the early stages of infections leads to excess morbidity, mortality, and healthcare costs. Here we report a rapid diagnostic platform that integrates a novel one-step blood droplet digital PCR assay and a high throughput 3D particle counter system with potential to perform bacterial identification and antibiotic susceptibility profiling directly from whole blood specimens, without requiring culture and sample processing steps. Using CTX-M-9 family ESBLs as a model system, we demonstrated that our technology can simultaneously achieve unprecedented high sensitivity (10 CFU per ml) and rapid sample-to-answer assay time (one hour). In head-to-head studies, by contrast, real time PCR and BioRad ddPCR only exhibited a limit of detection of 1000 CFU per ml and 50-100 CFU per ml, respectively. In a blinded test inoculating clinical isolates into whole blood, we demonstrated 100% sensitivity and specificity in identifying pathogens carrying a particular resistance gene. We further demonstrated that our technology can be broadly applicable for targeted detection of a wide range of antibiotic resistant genes found in both Gram-positive (vanA, nuc, and mecA) and Gram-negative bacteria, including ESBLs (blaCTX-M-1 and blaCTX-M-2 families) and CREs (blaOXA-48 and blaKPC), as well as bacterial speciation (E. coli and Klebsiella spp.) and pan-bacterial detection, without requiring blood culture or sample processing. Our rapid diagnostic technology holds great potential in directing early, appropriate therapy and improved antibiotic stewardship in combating bloodstream infections and antibiotic resistance.
Assuntos
Antibacterianos/farmacologia , Enterobacteriaceae/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Reação em Cadeia da Polimerase , Enterococos Resistentes à Vancomicina/efeitos dos fármacos , Enterobacteriaceae/isolamento & purificação , Humanos , Dispositivos Lab-On-A-Chip , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Técnicas Analíticas Microfluídicas/instrumentação , Tamanho da Partícula , Propriedades de Superfície , Enterococos Resistentes à Vancomicina/isolamento & purificaçãoRESUMO
BACKGROUND: The original hygiene hypothesis predicts that infections should protect against asthma but does not account for increasing evidence that certain infections might also promote asthma development. A mechanistic reconciliation of these findings has not yet emerged. In particular, the role of innate immunity in this context is unclear. OBJECTIVE: We sought to test whether bacterial respiratory tract infection causes airway sensitization toward an antigen encountered in parallel and to elucidate the contribution of innate immune responses. METHODS: Mice were infected with different doses of Chlamydia pneumoniae, followed by exposure to human serum albumin (HSA) and challenge with HSA 2 weeks later. Airway inflammation, immunoglobulins, and lymph node cytokines were assessed. Furthermore, adoptive transfer of dendritic cells (DCs) and depletion of regulatory T (Treg) cells was performed. RESULTS: C pneumoniae-induced lung inflammation triggered sensitization toward HSA, resulting in eosinophilic airway inflammation after HSA challenge. Airway sensitization depended on the severity and timing of infection: low-dose infection and antigen exposure within 5 days of infection induced allergic sensitization, whereas high-dose infection or antigen exposure 10 days after infection did not. Temporal and dose-related effects reflected DC activation and could be reproduced by means of adoptive transfer of HSA-pulsed lung DCs from infected mice. MyD88 deficiency in DCs abolished antigen sensitization, and depletion of Treg cells prolonged the time window in which sensitization could occur. CONCLUSIONS: We conclude that moderate, but not severe, pulmonary bacterial infection can induce allergic sensitization to inert inhaled antigens through a mechanism that requires MyD88-dependent DC activation and is controlled by Treg cells.
Assuntos
Infecções por Chlamydophila/imunologia , Células Dendríticas/imunologia , Imunidade Inata , Pulmão/imunologia , Hipersensibilidade Respiratória/imunologia , Infecções Respiratórias/imunologia , Linfócitos T Reguladores/imunologia , Animais , Chlamydophila pneumoniae/imunologia , Células Dendríticas/metabolismo , Eosinófilos/imunologia , Eosinófilos/metabolismo , Humanos , Imunoglobulina E/sangue , Inflamação/complicações , Inflamação/imunologia , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/deficiência , Fator 88 de Diferenciação Mieloide/metabolismo , Hipersensibilidade Respiratória/complicações , Infecções Respiratórias/complicações , Infecções Respiratórias/microbiologia , Albumina Sérica/imunologia , Linfócitos T Reguladores/metabolismoRESUMO
Autophagy can either antagonize or promote intracellular bacterial growth, depending on the pathogen. Here, we investigated the role of autophagy during a pulmonary infection with the obligate intracellular pathogen, Chlamydia pneumoniae (CP). In mouse embryonic fibroblasts (MEFs) or macrophages, deficiency of autophagy pathway components led to enhanced CP replication, suggesting that autophagy exerts a bactericidal role. However, in vivo, mice with myeloid-specific deletion of the autophagic protein ATG16L1 suffered increased mortality during CP infection, neutrophilia, and increased inflammasome activation despite no change in bacterial burden. Induction of autophagy led to reduced CP replication in vitro, but impaired survival in CP-infected mice, associated with an initial reduction in IL-1ß production, followed by enhanced neutrophil recruitment, defective CP clearance, and later inflammasome activation and IL-1ß production, which drove the resulting mortality. Taken together, our data suggest that a delicate interplay exists between autophagy and inflammasome activation in determining the outcome of CP infection, perturbation of which can result in inflammatory pathology or unrestricted bacterial growth.
Assuntos
Autofagia , Infecções por Chlamydophila/metabolismo , Infecções por Chlamydophila/microbiologia , Chlamydophila pneumoniae/fisiologia , Inflamassomos/metabolismo , Animais , Biomarcadores , Fibroblastos/metabolismo , Fibroblastos/microbiologia , Citometria de Fluxo , Técnicas de Inativação de Genes , Macrófagos/metabolismo , Macrófagos/microbiologia , CamundongosRESUMO
Infection with Chlamydia trachomatis targets epithelial cells within the genital tract which respond by secreting chemokines and cytokines. Persistent inflammation can lead to fibrosis, tubal infertility and/or ectopic pregnancy; many infections are asymptomatic. Most studies have investigated the inflammatory response in the initial stages of infection, less is known about the later stages of infection, especially with a low, potentially asymptomatic, bacterial load. Our objective was to determine the inflammatory mediators involved in clearance of low-grade infection and the potential involvement in chronic inflammation. Six to eight week old C3H/HeJ mice were pretreated with 2.5 mg medroxyprogesterone acetate on day -10 and -3 before infection. Mice (n=3 for 28 d, n=3 for 35 d) were infected with 5 × 102 inclusion-forming units of C. trachomatis, serovar D; vaginal cultures were obtained weekly to monitor infection. Control mice (n=3 for 28 d, n=3 for 35 d) were sham infected. Mice were killed on day 28 (experiment 1) and day 35 (experiment 2) post-infection and vaginal tissue, uterine horns and oviducts collected for analysis of mRNAs encoding inflammatory cytokines and chemokines. Total RNA was isolated and a superarray analysis performed using mouse Cytokines and Chemokines PCR arrays (Qiagen, Valencia, CA). Statistical differences in gene expression were determined using a paired Students t-test. At 28 days after infection, the expression of mRNA encoding 6, 35 and 3 inflammatory genes differed from controls in vaginal, uterine and oviductal tissues, respectively (P<0.05). At 35 days after infection, the expression of mRNA encoding 16, 38 and 14 inflammatory genes differed from controls in vaginal, uterine and oviductal tissues, respectively (P<0.05). Understanding the mechanisms involved in the inflammatory response at later stages of infection should aid in the development of treatment options that minimize the development of asymptomatic, chronic inflammation-induced infertility.
RESUMO
The salicylidene acylhydrazide (SA) compounds have exhibited promising microbicidal properties. Previous reports have shown the SA compounds, using cell cultures, to exhibit activity against Chlamydia trachomatis, herpes simplex virus and HIV-1. In addition, using an animal model of a vaginal infection the SA compound INP0341, when dissolved in a liquid, was able to significantly protect mice from a vaginal infection with C. trachomatis. To expand upon this finding, in this report INP0341 was formulated as a vaginal gel, suitable for use in humans. Gelling agents (polymers) with inherent antimicrobial properties were chosen to maximize the total antimicrobial effect of the gel. In vitro formulation work generated a gel with suitable rheology and sustained drug release. A formulation containing 1 mM INP0341, 1.6 wt% Cremophor ELP (solubility enhancer) and 1.5 wt% poly(acrylic acid) (gelling and antimicrobial agent), was chosen for studies of efficacy and toxicity using a mouse model of a vaginal infection. The gel formulation was able to attenuate a vaginal challenge with C. trachomatis, serovar D. Formulations with and without INP0341 afforded protection, but the inclusion of INP0341 increased the protection. Mouse vaginal tissue treated with the formulation showed no indication of gel toxicity. The lack of toxicity was confirmed by in vitro assays using EpiVaginal tissues, which showed that a 24 h exposure to the gel formulation did not decrease the cell viability or the barrier function of the tissue. Therefore, the gel formulation described here appears to be a promising vaginal microbicide to prevent a C. trachomatis infection with the potential to be expanded to other sexually transmitted diseases.
Assuntos
Anti-Infecciosos/farmacologia , Chlamydia trachomatis , Hidrazinas/farmacologia , Linfogranuloma Venéreo/tratamento farmacológico , Cremes, Espumas e Géis Vaginais/farmacologia , Animais , Anti-Infecciosos/química , Preparações de Ação Retardada/química , Preparações de Ação Retardada/farmacologia , Modelos Animais de Doenças , Feminino , Células HeLa , Humanos , Hidrazinas/química , Camundongos , Vagina/microbiologia , Cremes, Espumas e Géis Vaginais/químicaRESUMO
Chlamydia pneumoniae (CP) lung infection can induce chronic lung inflammation and is associated with not only acute asthma but also COPD exacerbations. However, in mouse models of CP infection, most studies have investigated specifically the acute phase of the infection and not the longer-term chronic changes in the lungs. We infected C57BL/6 mice with 5 × 10(5) CP intratracheally and monitored inflammation, cellular infiltrates and cytokine levels over time to investigate the chronic inflammatory lung changes. While bacteria numbers declined by day 28, macrophage numbers remained high through day 35. Immune cell clusters were detected as early as day 14 and persisted through day 35, and stained positive for B, T, and follicular dendritic cells, indicating these clusters were inducible bronchus associated lymphoid tissues (iBALTs). Classically activated inflammatory M1 macrophages were the predominant subtype early on while alternatively activated M2 macrophages increased later during infection. Adoptive transfer of M1 but not M2 macrophages intratracheally 1 week after infection resulted in greater lung inflammation, severe fibrosis, and increased numbers of iBALTS 35 days after infection. In summary, we show that CP lung infection in mice induces chronic inflammatory changes including iBALT formations as well as fibrosis. These observations suggest that the M1 macrophages, which are part of the normal response to clear acute C. pneumoniae lung infection, result in an enhanced acute response when present in excess numbers, with greater inflammation, tissue injury, and severe fibrosis.
Assuntos
Infecções por Chlamydia/patologia , Pneumonia por Clamídia/patologia , Chlamydophila pneumoniae/patogenicidade , Pulmão/patologia , Macrófagos/patologia , Transferência Adotiva , Animais , Linfócitos B/imunologia , Linfócitos B/patologia , Carga Bacteriana , Contagem de Células , Infecções por Chlamydia/imunologia , Infecções por Chlamydia/microbiologia , Pneumonia por Clamídia/imunologia , Pneumonia por Clamídia/microbiologia , Chlamydophila pneumoniae/imunologia , Doença Crônica , Citocinas/biossíntese , Citocinas/imunologia , Células Dendríticas/imunologia , Células Dendríticas/patologia , Fibrose , Pulmão/imunologia , Pulmão/microbiologia , Macrófagos/classificação , Macrófagos/imunologia , Macrófagos/transplante , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T/imunologia , Linfócitos T/patologiaRESUMO
Plasmacytoid dendritic cells (pDCs) are known for their robust antiviral response and their pro-tolerance effects towards allergic diseases and tissue engraftments. However, little is known about the role pDCs may play during a bacterial infection, including pulmonary Chlamydia pneumoniae (CP). In this study, we investigated the role of pDCs during pulmonary CP infection. Our results revealed that depletion of pDCs during acute CP infection in mice results in delayed and reduced lung inflammation, with an early delay in cellular recruitment and significant reduction in early cytokine production in the lungs. This was followed by impaired and delayed bacterial clearance from the lungs which then resulted in a severe and prolonged chronic inflammation and iBALT like structures containing large numbers of B and T cells in these animals. We also observed that increasing the pDC numbers in the lung by FLT3L treatment experimentally results in greater lung inflammation during acute CP infection. In contrast to these results, restimulation of T-cells in the draining lymph nodes of pDC-depleted mice induced greater amounts of proinflammatory cytokines than we observed in control mice. These results suggest that pDCs in the lung may provide critical proinflammatory innate immune responses in response to CP infection, but are suppressive towards adaptive immune responses in the lymph node. Thus pDCs in the lung and the draining lymph node appear to have different roles and phenotypes during acute CP infection and may play a role in host immune responses.
Assuntos
Pneumonia por Clamídia/imunologia , Chlamydophila pneumoniae/imunologia , Células Dendríticas/imunologia , Imunidade Inata/imunologia , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Líquido da Lavagem Broncoalveolar/imunologia , Líquido da Lavagem Broncoalveolar/microbiologia , Linhagem Celular Tumoral , Pneumonia por Clamídia/microbiologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Toxina Diftérica/imunologia , Toxina Diftérica/farmacologia , Feminino , Citometria de Fluxo , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-6/imunologia , Interleucina-6/metabolismo , Ligantes , Pulmão/imunologia , Pulmão/microbiologia , Pulmão/patologia , Linfonodos/imunologia , Linfonodos/metabolismo , Linfonodos/microbiologia , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Salicylidene acylhydrazides belong to a class of compounds shown to inhibit bacterial type III secretion (T3S) in pathogenic Gram-negative bacteria. This class of compounds also inhibits growth and replication of Chlamydiae, strict intracellular bacteria that possess a T3S system. In this study a library of 58 salicylidene acylhydrazides was screened to identify inhibitors of Chlamydia growth. Compounds inhibiting growth of both Chlamydia trachomatis and Chlamydophila pneumoniae were tested for cell toxicity and seven compounds were selected for preliminary pharmacokinetic analysis in mice using cassette dosing. Two compounds, ME0177 and ME0192, were further investigated by individual pharmacokinetic analysis. Compound ME0177 had a relatively high peak plasma concentration (C(max)) and area under curve and therefore may be considered for systemic treatment of Chlamydia infections. The other compound, ME0192, had poor pharmacokinetic properties but the highest anti-chlamydial activity in vitro and therefore was tested for topical treatment in a mouse vaginal infection model. ME0192 administered vaginally significantly reduced the infectious burden of C. trachomatis and the number of infected mice.
Assuntos
Antibacterianos/farmacologia , Infecções por Chlamydia/tratamento farmacológico , Chlamydia trachomatis/efeitos dos fármacos , Chlamydophila pneumoniae/efeitos dos fármacos , Hidrazinas/farmacologia , Animais , Antibacterianos/farmacocinética , Antibacterianos/uso terapêutico , Área Sob a Curva , Sobrevivência Celular/efeitos dos fármacos , Infecções por Chlamydia/metabolismo , Infecções por Chlamydia/microbiologia , Chlamydia trachomatis/crescimento & desenvolvimento , Chlamydophila pneumoniae/crescimento & desenvolvimento , Feminino , Células HeLa , Humanos , Hidrazinas/farmacocinética , Hidrazinas/uso terapêutico , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade MicrobianaRESUMO
Salicylidene acylhydrazide compounds have been shown to inhibit bacterial pathogens, including Chlamydia and Neisseria gonorrhoeae. If such compounds could also target HIV-1, their potential use as topical microbicides to prevent sexually transmitted infections would be considerable. In this study, the in vitro anti-HIV-1 activity, cytotoxicity and mechanism of action of several salicylidene acylhydrazides were determined. Inhibitory activity was assessed using TZM-bl cells and primary peripheral blood mononuclear cells (PBMCs) as targets for HIV-1 infection. Antiviral activity was measured against cell-free and cell-associated virus and in vaginal fluid and semen simulants. Since the antibacterial activity of salicylidene acylhydrazides is reversible by Fe(2+), the ability of Fe(2+) and other cations to reverse the anti-HIV-1 activity of the compounds was determined. Real-time PCR was also employed to determine the stage affected in the HIV-1 replication cycle. Four compounds with 50% inhibitory concentrations against HIV-1 of 1-7 µM were identified. In vitro toxicity varied but was generally limited. Activity was similar against three R5 clade B primary isolates and whether the target for virus replication was TZM-bl cells or PBMCs. Compounds inhibited cell-free and cell-associated virus and were active in vaginal fluid and semen simulants. Fe(2+), but not other cations, reversed the anti-HIV-1 effect. Finally, the inhibitory effect of the compounds occurred at a post-integration step. In conclusion, salicylidene acylhydrazides were identified with in vitro anti-HIV-1 activity in the micromolar range. The activity of these compounds against other sexually transmitted pathogens makes them potential candidates to formulate for use as a broad-spectrum topical genital microbicide.
Assuntos
Fármacos Anti-HIV/farmacologia , HIV-1/efeitos dos fármacos , Hidrazinas/farmacologia , Bases de Schiff/farmacologia , Secreções Corporais/virologia , Células Cultivadas , Feminino , HIV-1/fisiologia , Humanos , Concentração Inibidora 50 , Masculino , Testes de Sensibilidade Microbiana , Replicação Viral/efeitos dos fármacosRESUMO
Chlamydia pneumoniae (CP) is associated with induction and exacerbation of asthma. CP infection can induce allergic airway sensitization in mice in a dose- and time-dependent manner. Allergen exposure 5 days after a low dose (mild-moderate), but not a high dose (severe) CP infection induces antigen sensitization in mice. Innate immune signals play a critical role in controlling CP infection induced allergic airway sensitization, however these mechanisms have not been fully elucidated. Wild-type, TLR2-/-, and TLR4-/- mice were infected intranasally (i.n.) with a low dose of CP, followed by i.n. exposure to human serum albumin (HSA) and challenged with HSA 2 weeks later. Airway inflammation, immunoglobulins, eosinophils, and goblet cells were measured. Low dose CP infection induced allergic sensitization in TLR2-/- mice, but not in TLR4-/- mice, due to differential Treg responses in these genotypes. TLR2-/- mice had reduced numbers of Tregs in the lung during CP infection while TLR4-/- mice had increased numbers. High dose CP infection resulted in an increase in Tregs and pDCs in lungs, which prevented antigen sensitization in WT mice. Depletion of Tregs or pDCs resulted in allergic airway sensitization. We conclude that Tregs and pDCs are critical determinants regulating CP infection-induced allergic sensitization. Furthermore, TLR2 and TLR4 signaling during CP infection may play a regulatory role through the modulation of Tregs.
Assuntos
Chlamydophila pneumoniae/imunologia , Células Dendríticas/imunologia , Imunização , Pneumonia Bacteriana/imunologia , Pneumonia Bacteriana/microbiologia , Hipersensibilidade Respiratória/imunologia , Linfócitos T Reguladores/imunologia , Animais , Antígenos de Bactérias/imunologia , Bordetella/imunologia , Bordetella/efeitos da radiação , Chlamydophila pneumoniae/efeitos da radiação , Eosinófilos/patologia , Humanos , Inflamação/complicações , Inflamação/microbiologia , Inflamação/patologia , Pulmão/imunologia , Pulmão/microbiologia , Pulmão/patologia , Depleção Linfocítica , Camundongos , Viabilidade Microbiana/efeitos da radiação , Pneumonia Bacteriana/complicações , Pneumonia Bacteriana/patologia , Hipersensibilidade Respiratória/complicações , Hipersensibilidade Respiratória/microbiologia , Hipersensibilidade Respiratória/patologia , Albumina Sérica/imunologia , Transdução de Sinais/imunologia , Transdução de Sinais/efeitos da radiação , Fatores de Tempo , Receptor 2 Toll-Like/deficiência , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/deficiência , Receptor 4 Toll-Like/metabolismo , Raios UltravioletaRESUMO
Vaginal microbicides with activity towards organisms that cause sexually transmitted infections have been proposed as a strategy to reduce transmission. Small-molecule inhibitors of Chlamydia trachomatis serovar D belonging to the class of salicylidene acylhydrazides (INPs) have been shown to work through a mechanism that involves iron restriction. Expanding on this work, ten INPs were tested against a lymphogranuloma venereum strain of C. trachomatis (serovar L2), Neisseria gonorrhoeae, and hydrogen peroxide-producing Lactobacillus crispatus and Lactobacillus jensenii. Seven INPs had minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations of <50 microM towards C. trachomatis L2. Three INPs had a MIC <12.5 microM against N. gonorrhoeae. Inhibition was reversed by iron, holo-transferrin and holo-lactoferrin but not by the iron-poor forms of these compounds. The compounds exhibited no bactericidal activity toward Lactobacillus. The INPs were not cytotoxic to HeLa 229 cells. When INP 0341 was tested in a mouse model of a Chlamydia vaginal infection there was a significant reduction in the number of mice shedding C. trachomatis up to 4 days after infection (P<0.01). In summary, select INPs are promising vaginal microbicide candidates as they inhibit the growth of two common sexually transmitted organisms in vitro, are active in a mouse model against C. trachomatis, are not cytotoxic and do not inhibit organisms that compose the normal vaginal flora.
Assuntos
Anti-Infecciosos/farmacologia , Infecções por Chlamydia/prevenção & controle , Chlamydia trachomatis/efeitos dos fármacos , Gonorreia/prevenção & controle , Hidrazinas/farmacologia , Neisseria gonorrhoeae/efeitos dos fármacos , Vagina/microbiologia , Administração Intravaginal , Animais , Anti-Infecciosos/uso terapêutico , Infecções por Chlamydia/transmissão , Modelos Animais de Doenças , Feminino , Gonorreia/transmissão , Células HeLa , Humanos , Hidrazinas/uso terapêutico , Lactobacillus/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C3H , Testes de Sensibilidade MicrobianaRESUMO
INPs, which are chemically synthesized compounds belonging to a class of acylated hydrazones of salicylaldehydes, can inhibit the growth of Chlamydiaceae. Evidence has been presented that in Yersinia and Chlamydia INPs may affect the type III secretion (T3S) system. In the present study 25 INPs were screened for antichlamydial activity at a concentration of 50 muM, and 14 were able to completely inhibit the growth of Chlamydia trachomatis serovar D in McCoy and HeLa 229 cells. The antichlamydial activities of two of these INPs, INPs 0341 and 0400, were further characterized due to their low cytotoxicity. These compounds were found to inhibit C. trachomatis in a dose-dependent manner; were not toxic to elementary bodies; were cidal at a concentration of > or =20 microM; inhibited all Chlamydiaceae tested; and could inhibit the development of C. trachomatis as determined by the yield of progeny when they were added up to 24 h postinfection. INP 0341 was able to affect the expression of several T3S genes. Compared to the expression in control cultures, lcrH-1, copB, and incA, all middle- to late-expressed T3S genes, were not expressed in the INP 0341-treated cultures 24 to 36 h postinfection. Iron, supplied as ferrous sulfate, as ferric chloride, or as holo-transferrin, was able to negate the antichlamydial properties of the INPs. In contrast, apo-transferrin and other divalent metal ions tested were not able to reverse the inhibitory effect of the INPs. In conclusion, the potent antichlamydial activity of INPs is directly or indirectly linked with iron, and this inhibition of Chlamydia has an effect on the T3S system of this intracellular pathogen.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Chlamydiaceae/efeitos dos fármacos , Hidrazonas/farmacologia , Ferro/farmacologia , Animais , Antibacterianos/toxicidade , Proteínas de Bactérias/genética , Linhagem Celular , Chlamydia trachomatis/efeitos dos fármacos , Chlamydia trachomatis/crescimento & desenvolvimento , Chlamydia trachomatis/patogenicidade , Chlamydiaceae/classificação , Chlamydiaceae/crescimento & desenvolvimento , Chlamydiaceae/patogenicidade , Relação Dose-Resposta a Droga , Regulação Bacteriana da Expressão Gênica , Células HeLa , Humanos , Hidrazonas/toxicidade , Ferro/metabolismo , Testes de Sensibilidade Microbiana/métodosRESUMO
Members of the family Chlamydiaceae possess at least 13 genes, distributed throughout the chromosome, that are homologous with genes of known type III secretion systems (TTS). The aim of this study was to use putative TTS proteins of Chlamydophila pneumoniae, whose equivalents in other bacterial TTS function as chaperones, to identify interactions between chlamydial proteins. Using the BacterioMatch Two-Hybrid Vector system (Stratagene, La Jolla, Calif.), lcrH-2 and sycE, positions 1021 and 0325, respectively, from C. pneumoniae CM-1 were used as "bait" to identify target genes (positions 0324, 0705, 0708, 0808 to 0810, 1016 to 1020, and 1022) in close proximity on the chromosome. Interaction between the products of the lcrH-2 (1021) and lcrE (copN) (0324) genes was detected and confirmed by pull-down experiments and enzyme immunoassays using recombinant LcrH-2 and LcrE. As further confirmation of this interaction, the homologous genes from Chlamydia trachomatis, serovar E, and Chlamydophila psittaci, Texas turkey, were also cloned in the two-hybrid system to determine if LcrH-2 and LcrE would interact with their orthologs in other species. Consistent with their genetic relatedness, LcrH-2 from C. pneumoniae interacted with LcrE produced from the three species of Chlamydiaceae; LcrH-2 from C. psittaci reacted with LcrE from C. pneumoniae but not from C. trachomatis; and C. trachomatis LcrH-2 did not react with LcrE from the other two species. Deletions from the N and C termini of LcrE from C. pneumoniae identified the 50 C-terminal amino acids as essential for the interaction with LcrH-2. Thus, it appears that in the Chlamydiaceae TTS, LcrH-2 interacts with LcrE, and therefore it may serve as a chaperone for this protein.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Chlamydiaceae/metabolismo , Chaperonas Moleculares/metabolismo , Fatores de Virulência/genética , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Chaperoninas/genética , Chaperoninas/metabolismo , Chlamydia trachomatis/genética , Chlamydia trachomatis/metabolismo , Chlamydiaceae/genética , Chlamydophila pneumoniae/genética , Chlamydophila pneumoniae/metabolismo , Chlamydophila psittaci/genética , Chlamydophila psittaci/metabolismo , Chaperonas Moleculares/genética , Ligação Proteica , Mapeamento de Interação de Proteínas , Transporte Proteico , Deleção de Sequência , Técnicas do Sistema de Duplo-Híbrido , Fatores de Virulência/fisiologiaRESUMO
Chlamydia pneumoniae is the causative agent of respiratory tract infections and a number of chronic diseases. Here we investigated the involvement of the common TLR adaptor molecule MyD88 in host responses to C. pneumoniae-induced pneumonia in mice. MyD88-deficient mice were severely impaired in their ability to mount an acute early inflammatory response toward C. pneumoniae. Although the bacterial burden in the lungs was comparable 5 days after infection, MyD88-deficient mice exhibited only minor signs of pneumonia and reduced expression of inflammatory mediators. MyD88-deficient mice were unable to up-regulate proinflammatory cytokines and chemokines, demonstrated delayed recruitment of CD8+ and CD4+ T cells to the lungs, and were unable to clear the pathogen from their lungs at day 14. At day 14 the MyD88-deficent mice developed a severe, chronic lung inflammation with elevated IL-1beta and IFN-gamma leading to increased mortality, whereas wild-type mice as well as TLR2- or TLR4-deficient mice recovered from acute pneumonia and did not show delayed bacterial clearance. Thus, MyD88 is essential to recognize C. pneumoniae infection and initiate a prompt and effective immune host response against this organism leading to clearance of bacteria from infected lungs.