RESUMEN
The ribosomal maturation factor P (RimP) is a highly conserved protein in bacteria and has been shown to be important in ribosomal assembly in Escherichia coli Because of its central importance in bacterial metabolism, RimP represents a good potential target for drug design to combat human pathogens such as Mycobacterium tuberculosis However, to date, the only RimP structure available is the NMR structure of the ortholog in another bacterial pathogen, Streptococcus pneumoniae Here, we report a 2.2 Å resolution crystal structure of MSMEG_2624, the RimP ortholog in the close M. tuberculosis relative Mycobacterium smegmatis, and using in vitro binding assays, we show that MSMEG_2624 interacts with the small ribosomal protein S12, also known as RpsL. Further analyses revealed that the conserved residues in the linker region between the N- and C-terminal domains of MSMEG_2624 are essential for binding to RpsL. However, neither of the two domains alone was sufficient to form strong interactions with RpsL. More importantly, the linker region was essential for in vivo ribosomal biogenesis. Our study provides critical mechanistic insights into the role of RimP in ribosome biogenesis. We anticipate that the MSMEG_2624 crystal structure has the potential to be used for drug design to manage M. tuberculosis infections.
Asunto(s)
Proteínas Bacterianas , Mycobacterium smegmatis , Proteínas Ribosómicas , Ribosomas , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , Proteínas de Escherichia coli , Mycobacterium smegmatis/química , Mycobacterium smegmatis/metabolismo , Unión Proteica , Dominios Proteicos , Proteína Ribosómica S9 , Proteínas Ribosómicas/biosíntesis , Proteínas Ribosómicas/química , Ribosomas/química , Ribosomas/metabolismo , Streptococcus pneumoniae/química , Streptococcus pneumoniae/metabolismoRESUMEN
Talaromyces marneffei infection causes talaromycosis (previously known as penicilliosis), a very important opportunistic systematic mycosis in immunocompromised patients. Different virulence mechanisms in T. marneffei have been proposed and investigated. In the sera of patients with talaromycosis, Mp1 protein (Mp1p), a secretory galactomannoprotein antigen with two tandem ligand-binding domains (Mp1p-LBD1 and Mp1p-LBD2), was found to be abundant. Mp1p-LBD2 was reported to possess a hydrophobic cavity to bind copurified palmitic acid (PLM). It was hypothesized that capturing of lipids from human hosts by expressing a large quantity of Mp1p is a virulence mechanism of T. marneffei It was shown that expression of Mp1p enhanced the intracellular survival of T. marneffei by suppressing proinflammatory responses. Mechanistic study of Mp1p-LBD2 suggested that arachidonic acid (AA), a precursor of paracrine signaling molecules for regulation of inflammatory responses, is the major physiological target of Mp1p-LBD2. In this study, we use crystallographic and biochemical techniques to further demonstrate that Mp1p-LBD1, the previously unsolved first lipid binding domain of Mp1p, is also a strong AA-binding domain in Mp1p. These studies on Mp1p-LBD1 support the idea that the highly expressed Mp1p is an effective AA-capturing protein. Each Mp1p can bind up to 4 AA molecules. The crystal structure of Mp1p-LBD1-LBD2 has also been solved, showing that both LBDs are likely to function independently with a flexible linker between them. T. marneffei and potentially other pathogens highly expressing and secreting proteins similar to Mp1p can severely disturb host signaling cascades during proinflammatory responses by reducing the availabilities of important paracrine signaling molecules.
Asunto(s)
Ácido Araquidónico/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Micosis/microbiología , Talaromyces/metabolismo , Factores de Virulencia/química , Factores de Virulencia/metabolismo , Ácido Araquidónico/química , Proteínas Fúngicas/genética , Interacciones Huésped-Patógeno , Humanos , Espectrometría de Masas , Micosis/genética , Micosis/inmunología , Dominios Proteicos , Talaromyces/química , Talaromyces/genética , Factores de Virulencia/genéticaRESUMEN
Cytotoxin-producing Klebsiella oxytoca is the causative agent of antibiotic-associated hemorrhagic colitis (AAHC). Recently, the cytotoxin associated with AAHC was identified as tilivalline, a known pentacyclic pyrrolobenzodiazepine (PBD) metabolite produced by K. oxytoca Although this assertion of tilivalline's role in AAHC is supported by evidence from animal experiments, some key aspects of this finding appear to be incompatible with toxicity mechanisms of known PBD toxins. We therefore hypothesized that K. oxytoca may produce some other uncharacterized cytotoxins. To address this question, we investigated whether tilivalline alone is indeed necessary and sufficient to induce cytotoxicity or whether K. oxytoca also produces other cytotoxins. LC-MS- and NMR-based metabolomic analyses revealed the presence of an abundant tricyclic PBD, provisionally designated kleboxymycin, in the supernatant of toxigenic K. oxytoca strains. Moreover, by generating multiple mutants with gene deletions affecting tilivalline biosynthesis, we show that a tryptophanase-deficient, tilivalline-negative K. oxytoca mutant induced cytotoxicity in vitro similar to tilivalline-positive K. oxytoca strains. Furthermore, synthetic kleboxymycin exhibited greater than 9-fold higher cytotoxicity than tilivalline in TC50 cell culture assays. We also found that the biosynthetic pathways for kleboxymycin and tilivalline appear to overlap, as tilivalline is an indole derivative of kleboxymycin. In summary, our results indicate that tilivalline is not essential for inducing cytotoxicity observed in K. oxytoca-associated AAHC and that kleboxymycin is a tilivalline-related bacterial metabolite with even higher cytotoxicity.
Asunto(s)
Apoptosis/efectos de los fármacos , Benzodiazepinonas/farmacología , Citotoxinas/farmacología , Enterocolitis Seudomembranosa/patología , Klebsiella oxytoca/metabolismo , Neoplasias Laríngeas/patología , Antibacterianos/efectos adversos , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/microbiología , Carcinoma de Células Escamosas/patología , Enterocolitis Seudomembranosa/inducido químicamente , Enterocolitis Seudomembranosa/microbiología , Humanos , Infecciones por Klebsiella/tratamiento farmacológico , Infecciones por Klebsiella/microbiología , Klebsiella oxytoca/efectos de los fármacos , Neoplasias Laríngeas/tratamiento farmacológico , Neoplasias Laríngeas/microbiología , Péptidos/farmacología , Células Tumorales CultivadasRESUMEN
Seasonal influenza virus remains a common cause of mortality despite the use of neuraminidase inhibitors. This study evaluated the efficacy of a triple combination of zanamivir, clarithromycin and flufenamic acid (FFA) in the treatment of influenza virus A(H1N1) infection. An in vitro cell protection assay and a multiple-cycle growth assay showed that the antiviral activity of zanamivir was enhanced when combined with clarithromycin or FFA. A mouse challenge model was used here for the evaluation of the in vivo efficacy of the triple combination treatment. We found that mice receiving the triple combination of FFA, zanamivir, and clarithromycin had a significantly better survival rate than those receiving the double combination of zanamivir and clarithromycin (88% versus 44%, P = 0.0083) or zanamivir monotherapy (88% versus 26%, P = 0.0002). Mice in the FFA-zanamivir-clarithromycin triple combination group also exhibited significantly less body weight loss than those in the zanamivir-clarithromycin double combination group. There was no significant difference in the lung viral titers among the different groups from day 2 to day 6 postinfection. However, the levels of IL-1ß, TNF-α and RANTES in the FFA-zanamivir-clarithromycin triple combination group were significantly lower than those in the zanamivir-clarithromycin double combination group, zanamivir monotherapy group, or solvent group on day 2 postinfection. Our findings showed that the FFA-zanamivir-clarithromycin triple combination improved the inflammatory markers and survival of severe influenza A(H1N1) infection in mice.
Asunto(s)
Antivirales/administración & dosificación , Claritromicina/administración & dosificación , Ácido Flufenámico/administración & dosificación , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Gripe Humana/tratamiento farmacológico , Gripe Humana/mortalidad , Zanamivir/administración & dosificación , Animales , Aprobación de Drogas/legislación & jurisprudencia , Evaluación Preclínica de Medicamentos , Quimioterapia Combinada , Femenino , Humanos , Subtipo H1N1 del Virus de la Influenza A/fisiología , Gripe Humana/metabolismo , Gripe Humana/virología , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Pulmón/virología , Ratones , Ratones Endogámicos BALB C , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Estados Unidos , United States Food and Drug AdministrationRESUMEN
Immunomodulators have been shown to improve the outcome of severe pneumonia. We have previously shown that mycophenolic acid (MPA), an immunomodulator, has antiviral activity against influenza A/WSN/1933(H1N1) using a high-throughput chemical screening assay. This study further investigated the antiviral activity and mechanism of action of MPA against contemporary clinical isolates of influenza A and B viruses. The 50 % cellular cytotoxicity (CC50) of MPA in Madin Darby canine kidney cell line was over 50 µM. MPA prevented influenza virus-induced cell death in the cell-protection assay, with significantly lower IC50 for influenza B virus B/411 than that of influenza A(H1N1)pdm09 virus H1/415 (0.208 vs 1.510 µM, P=0.0001). For H1/415, MPA interfered with the early stage of viral replication before protein synthesis. For B/411, MPA may also act at a later stage since MPA was active against B/411 even when added 12 h post-infection. Virus-yield reduction assay showed that the replication of B/411 was completely inhibited by MPA at concentrations ≥0.78 µM, while there was a dose-dependent reduction of viral titer for H1/415. The antiviral effect of MPA was completely reverted by guanosine supplementation. Plaque reduction assay showed that MPA had antiviral activity against eight different clinical isolates of A(H1N1), A(H3N2), A(H7N9) and influenza B viruses (IC50 <1 µM). In summary, MPA has broad-spectrum antiviral activity against human and avian-origin influenza viruses, in addition to its immunomodulatory activity. Together with a high chemotherapeutic index, the use of MPA as an antiviral agent should be further investigated in vivo.
Asunto(s)
Antivirales/farmacología , Virus de la Influenza A/efectos de los fármacos , Virus de la Influenza B/efectos de los fármacos , Ácido Micofenólico/farmacología , Animales , Antivirales/toxicidad , Supervivencia Celular/efectos de los fármacos , Perros , Virus de la Influenza A/fisiología , Virus de la Influenza B/fisiología , Concentración 50 Inhibidora , Células de Riñón Canino Madin Darby , Ácido Micofenólico/toxicidad , Carga Viral , Replicación Viral/efectos de los fármacosRESUMEN
OBJECTIVES: The conserved residues 318-483 in the PB2 subunit of influenza A polymerase is an independently folded cap-binding domain (PB2cap) that exhibits a distinct binding mode from other host cap-binding proteins, which suggests that PB2cap might be an ideal drug target. This study aimed to identify a new class of anti-influenza inhibitors that specifically disrupts the interaction between PB2cap and host cap structures. METHODS: An innovative fluorescence polarization assay was established for primary screening, followed by cap-binding inhibitory activity, antiviral efficacy and cytotoxicity evaluations of the selected compounds. The best compound was characterized by multi-cycle virus growth assay, cross-protection test, synergism evaluation, mini-replicon assay, binding affinity analysis, docking simulation and mouse study. RESULTS: Several PB2 cap-binding inhibitors were discovered. The compound 7-(4-hydroxy-2-oxo-2H-chromen-3-yl)-6H,7H,8H-chromeno[3',4':5,6]pyrano[3,2-c]chromene-6,8-dione, designated PB2-39, was identified as a potent inhibitor of replication of multiple subtypes of influenza A virus, including H1N1, H3N2, H5N1, H7N7, H7N9 and H9N2 in vitro and H1N1, H5N1 and H7N9 in vivo. Combinational treatment with the influenza virus release inhibitor zanamivir and PB2-39 exerted a synergistic anti-influenza effect. Mechanistic experiments supported that PB2-39 suppressed viral polymerase activity. Docking and binding affinity analyses demonstrated that PB2-39 interacted with the PB2 cap-binding pocket, suggesting its role as a cap-binding competitor. CONCLUSIONS: Our study provides new insights for the strategic development of novel cap-binding inhibitors of influenza A viruses.
Asunto(s)
Antivirales/aislamiento & purificación , Antivirales/farmacología , Virus de la Influenza A/efectos de los fármacos , Virus de la Influenza A/fisiología , Proteínas de Unión a Caperuzas de ARN/antagonistas & inhibidores , Proteínas Virales/antagonistas & inhibidores , Replicación Viral/efectos de los fármacos , Animales , Antivirales/toxicidad , Línea Celular , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Sinergismo Farmacológico , Femenino , Polarización de Fluorescencia , Humanos , Ratones Endogámicos BALB C , Infecciones por Orthomyxoviridae/tratamiento farmacológico , Resultado del TratamientoRESUMEN
Although the major causes of isoniazid (INH) resistance in Mycobacterium tuberculosis are confined to structural mutations in katG and promoter mutations in the mabA-inhA operon, a significant proportion of INH-resistant strains have unknown resistance mechanisms. Recently, we identified a high-level INH-resistant M. tuberculosis clinical isolate, GB005, with no known resistance-associated mutations. A comprehensive study was performed to investigate the molecular basis of drug resistance in this strain. Although no mutations were found throughout the katG and furA-katG intergenic region, the katG expression and the catalase activity were greatly diminished compared to those in H37Rv (P < 0.01). Northern blotting revealed that the katG transcript from the isolate was smaller than that of H37Rv. Sequencing analysis of furA and upstream genes discovered a 7.2-kb truncation extended from the 96th base preceding the initiation codon of katG. Complementation of the M. tuberculosis Δ(furA-katG) strain with katG and different portions of the truncated region identified a 134-bp upstream fragment of furA that was essential for full catalase activity and INH susceptibility in M. tuberculosis. The promoter activity of this fragment was also shown to be stronger than that of the furA-katG intergenic region (P < 0.01). Collectively, these findings demonstrate that deletion of the 134-bp furA upstream fragment is responsible for the reduction in katG expression, resulting in INH resistance in GB005. To our knowledge, this is the first report showing that deletion of the upstream region preceding the furA-katG operon causes high-level INH resistance in a clinical isolate of M. tuberculosis.
Asunto(s)
Antituberculosos/farmacología , Proteínas Bacterianas/metabolismo , Isoniazida/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/metabolismo , Operón/genética , Proteínas Bacterianas/genética , Catalasa/metabolismo , Mutación , Mycobacterium tuberculosis/genética , Tuberculosis Resistente a Múltiples Medicamentos/genéticaRESUMEN
Penicillium marneffei is an opportunistic fungal pathogen endemic in Southeast Asia, causing lethal systemic infections in immunocompromised patients. P. marneffei grows in a mycelial form at the ambient temperature of 25°C and transitions to a yeast form at 37°C. The ability to alternate between the mycelial and yeast forms at different temperatures, namely, thermal dimorphism, has long been considered critical for the pathogenicity of P. marneffei, yet the underlying genetic mechanisms remain elusive. Here we employed high-throughput sequencing to unravel global transcriptional profiles of P. marneffei PM1 grown at 25 and 37°C. Among â¼11,000 protein-coding genes, 1,447 were overexpressed and 1,414 were underexpressed at 37°C. Counterintuitively, heat-responsive genes, predicted in P. marneffei through sequence comparison, did not tend to be overexpressed at 37°C. These results suggest that P. marneffei may take a distinct strategy of genetic regulation at the elevated temperature; the current knowledge concerning fungal heat response, based on studies of model fungal organisms, may not be applicable to P. marneffei. Our results further showed that the tandem repeat sequences (TRSs) are overrepresented in coding regions of P. marneffei genes, and TRS-containing genes tend to be overexpressed at 37°C. Furthermore, genomic sequences and expression data were integrated to characterize gene clusters, multigene families, and species-specific genes of P. marneffei. In sum, we present an integrated analysis and a comprehensive resource toward a better understanding of temperature-dependent genetic regulation in P. marneffei.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Respuesta al Choque Térmico/genética , Penicillium/metabolismo , Transcripción Genética , Genes Fúngicos , Proteínas de Choque Térmico/genética , Calor , Penicillium/genética , Secuencias Repetidas en Tándem , TranscriptomaRESUMEN
The opportunistic pathogen Pseudomonas aeruginosa (P. aeruginosa) causes infections that are difficult to treat, which is due to the bacterial natural resistance to antibiotics. The bacterium is also able to form a biofilm that protects the bacterium from clearance by the human immune system and leads to chronic infection. Herein, we synthesized and characterized a novel gallium compound that interferes with both the iron metabolism and quorum sensing system of P. aeruginosa to achieve a significant bactericidal activity. The compound could substantially reduce the secretion of bacterial virulence factors as well as eliminate biofilm formation. Integrative omics analysis indicates that this compound can significantly disturb the gene transcription and metabolism of P. aeruginosa. The effectiveness of the gallium compound was further validated in mammalian cell and murine skin infection models. Our study offers a new strategy to design new gallium-based antimicrobials to combat P. aeruginosa infection.
RESUMEN
Penicillium marneffei is the most important thermal dimorphic, pathogenic fungus endemic in China and Southeast Asia and is particularly important in HIV-positive patients. We report the 28,887,485-bp draft genome sequence of P. marneffei, which contains its complete mitochondrial genome, sexual cycle genes, a high diversity of Mp1p homologues, and polyketide synthase genes.
Asunto(s)
Genoma Fúngico , Penicillium/genética , Secuencia de Bases , Datos de Secuencia Molecular , Análisis de Secuencia de ADNRESUMEN
A PCR-sequencing assay was evaluated for direct detection of mutations in the quinolone resistance-determining region (QRDR) of gyrase A (gyrA) gene in fluoroquinolone-resistant Mycobacterium tuberculosis in respiratory specimens. As determined by gyrA QRDR analysis, complete concordance of genotypic and phenotypic fluoroquinolone resistance was demonstrated. Our results indicate that the assay is a rapid and reliable method for the diagnosis of fluoroquinolone-resistant tuberculosis, facilitating timely clinical management and public health control. Using the assay, we detected a novel gyrA Ala74Ser mutation in M. tuberculosis directly from sputum specimens. The functional effect of the Ala74Ser mutant was verified through the study of the DNA supercoiling inhibitory activity of fluoroquinolones against the recombinant gyrase. The drug-mediated gyrase-DNA cleavage complex model suggests perturbation of the gyrA-gyrA dimer interface caused by the Ala74Ser mutation probably disturbs the putative quinolone binding pocket and leads to the reduction of the drug binding affinity. A number of gyrA mutations (Glu21Gln, Ser95Thr, and Gly668Asp) were also characterized to be natural polymorphisms not associated with fluoroquinolone resistance.
Asunto(s)
Antiinfecciosos/farmacología , Girasa de ADN/genética , Farmacorresistencia Bacteriana/genética , Fluoroquinolonas/farmacología , Mutación , Mycobacterium tuberculosis/efectos de los fármacos , Girasa de ADN/química , Humanos , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/genética , Reacción en Cadena de la Polimerasa/métodos , Análisis de Secuencia de ADN , Tuberculosis/diagnóstico , Tuberculosis/microbiologíaRESUMEN
Candida albicans is an opportunistic fungal pathogen that is highly resistant to contemporary antifungals, due to their biofilm lifestyle. The ability of C. albicans to invade human tissues is due to its filamentation. Therefore, inhibition of biofilms and filamentation of the yeast are high value targets to develop the next-generation antifungals. Curcumin (CU) is a natural polyphenol with excellent pharmacological attributes, but limitations such as poor solubility, acid, and enzyme tolerance have impeded its practical utility. Sophorolipids (SL) are biologically-derived surfactants that serve as efficient carriers of hydrophobic molecules such as curcumin into biofilms. Here, we synthesised a curcumin-sophorolipid nanocomplex (CUSL), and comprehensively evaluated its effects on C. albicans biofilms and filamentation. Our results demonstrated that sub-inhibitory concentration of CUSL (9.37 µg/mL) significantly inhibited fungal adhesion to substrates, and subsequent biofilm development, maturation, and filamentation. This effect was associated with significant downregulation of a select group of biofilm, adhesins, and hyphal regulatory genes. In conclusion, the curcumin-sophorolipid nanocomplex is a potent inhibitor of the two major virulence attributes of C. albicans, biofilm formation and filamentation, thus highlighting its promise as a putative anti-fungal agent with biofilm penetrative potential.
Asunto(s)
Candida albicans , Curcumina , Antifúngicos/farmacología , Biopelículas , Curcumina/farmacología , Humanos , Hifa , Ácidos OléicosRESUMEN
Implant-associated infections is one of the most challenging post-operative complications in bone-related implantations. To tackle this clinical issue, we developed a low-cost and durable surface coating for medical grade titanium implants that uses positively charged silane molecules. The in vitro antimicrobial tests revealed that the titanium surface coated with (3-aminopropyl) triethoxysilane, which has the appropriate length of hydrophobic alkyl chain and positive charged amino group, suppressed more than 90% of the initial bacterial adhesion of S. aureus, P. aeruginosa, and E. coli after 30 min of incubation. In terms of growth inhibitory rate, the treated surface was able to reduce 75.7% ± 11.9% of bacterial growth after a 24-hour culturing, thereby exhibiting superior anti-biofilm formation in the late stage. When implanted into the rat model infected by S. aureus, the treated surface eliminated the implant-associated infection through the mechanism of inhibition of bacterial adhesion on the implant surface. Additionally, the treated surface was highly compatible with mammalian cells. In general, our design demonstrated its potential for human clinical trials as a low-cost and effective antibacterial strategy to minimize post-operative implant-related bacterial infection.
Asunto(s)
Materiales Biocompatibles Revestidos , Staphylococcus aureus , Animales , Antibacterianos/uso terapéutico , Materiales Biocompatibles Revestidos/farmacología , Escherichia coli , Prótesis e Implantes , Ratas , Propiedades de Superficie , Titanio/farmacologíaRESUMEN
Staphylococcus aureus is one of the most common pathogenic bacteria that causes human infectious diseases. The emergence of antibiotic-resistant strains of S. aureus promotes the development of new anti-bacterial strategies. Silver ions (Ag+) have attracted profound attention due to their broad-spectrum antimicrobial activities. Although the antibacterial properties of silver have been well known for many centuries, its mechanism of action remains unclear and its protein targets are rarely reported. Herein, we identify the catabolite control protein A (CcpA) of S. aureus as a putative target for Ag+. CcpA binds 2 molar equivalents of Ag+via its two cysteine residues (Cys216 and Cys242). Importantly, Ag+ binding induces CcpA oligomerization and abolishes its DNA binding capability, which further attenuates S. aureus growth and suppresses α-hemolysin toxicity. This study extends our understanding of the bactericidal effects of silver.
RESUMEN
The PB1 C-terminal domain and PB2 N-terminal domain interaction of the influenza A polymerase, which modulates the assembly of PB1 and PB2 subunits, may serve as a valuable target for the development of novel anti-influenza therapeutics. In this study, we performed a systematic screening of a chemical library, followed by the antiviral evaluation of primary hits and their analogues. Eventually, a novel small-molecule compound PP7 that abrogated the PB1-PB2 association and impaired viral polymerase activity was identified. PP7 exhibited antiviral activities against influenza virus subtypes A (H1N1)pdm09, A(H7N9) and A(H9N2) in cell cultures and partially protected mice against lethal challenge of mouse-adapted influenza A (H1N1)pdm09 virus. Surprisingly, a panel of other subtypes of influenza virus, including A(H5N1) and A(H7N7), showed various degrees of resistance to the compound. Biochemical studies revealed a similar pattern of resistance on the impairment of polymerase activity. Molecular docking analyses suggested a PP7-binding site that appeared to be completely conserved among the subtypes of the virus mentioned above. Thus, we propose that alternative/additional binding site (s) may exist for the regulation of PB1-PB2 subunits assembly of influenza A virus.
Asunto(s)
Antivirales/farmacología , Virus de la Influenza A/química , Virus de la Influenza A/efectos de los fármacos , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , Proteínas Virales/antagonistas & inhibidores , Animales , Antivirales/administración & dosificación , Antivirales/aislamiento & purificación , Antivirales/uso terapéutico , Sitios de Unión , Línea Celular , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Subtipo H5N1 del Virus de la Influenza A/efectos de los fármacos , Subtipo H7N7 del Virus de la Influenza A/efectos de los fármacos , Subtipo H7N9 del Virus de la Influenza A/efectos de los fármacos , Subtipo H9N2 del Virus de la Influenza A/efectos de los fármacos , Virus de la Influenza A/enzimología , Ratones , Simulación del Acoplamiento Molecular , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Mapeo de Interacción de Proteínas , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/metabolismo , Bibliotecas de Moléculas Pequeñas , Proteínas Virales/química , Proteínas Virales/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
Talaromyces (Penicillium) marneffei is one of the leading causes of systemic mycosis in immunosuppressed or AIDS patients in Southeast Asia. How this intracellular pathogen evades the host immune defense remains unclear. We provide evidence that T. marneffei depletes levels of a key proinflammatory lipid mediator arachidonic acid (AA) to evade the host innate immune defense. Mechanistically, an abundant secretory mannoprotein Mp1p, shown previously to be a virulence factor, does so by binding AA with high affinity via a long hydrophobic central cavity found in the LBD2 domain. This sequesters a critical proinflammatory signaling lipid, and we see evidence that AA, AA's downstream metabolites, and the cytokines interleukin-6 and tumor necrosis factor α are downregulated in T. marneffei-infected J774 macrophages. Given that Mp1p-LBD2 homologs are identified in other fungal pathogens, we expect that this novel class of fatty-acid-binding proteins sequestering key proinflammatory lipid mediators represents a general virulence mechanism of pathogenic fungi.
Asunto(s)
Antígenos Fúngicos/inmunología , Inmunidad Innata/inmunología , Inflamación/inmunología , Lípidos/inmunología , Talaromyces/inmunología , Factores de Virulencia/inmunología , Animales , Ácido Araquidónico/química , Ácido Araquidónico/inmunología , Ácido Araquidónico/metabolismo , Células Cultivadas , Inflamación/metabolismo , Mediadores de Inflamación/química , Mediadores de Inflamación/inmunología , Mediadores de Inflamación/metabolismo , Macrófagos/inmunología , Macrófagos/microbiología , Ratones , Factores de Virulencia/química , Factores de Virulencia/aislamiento & purificaciónRESUMEN
The RNA-dependent RNA polymerase of influenza A virus comprises conserved and independently-folded subdomains with defined functionalities. The N-terminal domain of the PA subunit (PA(N)) harbors the endonuclease function so that it can serve as a desired target for drug discovery. To identify a class of anti-influenza inhibitors that impedes PA(N) endonuclease activity, a screening approach that integrated the fluorescence resonance energy transfer based endonuclease inhibitory assay with the DNA gel-based endonuclease inhibitory assay was conducted, followed by the evaluation of antiviral efficacies and potential cytotoxicity of the primary hits in vitro and in vivo. A small-molecule compound ANA-0 was identified as a potent inhibitor against the replication of multiple subtypes of influenza A virus, including H1N1, H3N2, H5N1, H7N7, H7N9 and H9N2, in cell cultures. Combinational treatment of zanamivir and ANA-0 exerted synergistic anti-influenza effect in vitro. Intranasal administration of ANA-0 protected mice from lethal challenge and reduced lung viral loads in H1N1 virus infected BALB/c mice. In summary, ANA-0 shows potential to be developed to novel anti-influenza agents.
Asunto(s)
Antivirales/farmacología , Virus de la Influenza A/efectos de los fármacos , Virus de la Influenza A/enzimología , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , Proteínas Virales/antagonistas & inhibidores , Replicación Viral/efectos de los fármacos , Administración Intranasal , Animales , Antivirales/administración & dosificación , Antivirales/aislamiento & purificación , Evaluación Preclínica de Medicamentos , Sinergismo Farmacológico , Femenino , Virus de la Influenza A/fisiología , Pulmón/virología , Ratones Endogámicos BALB C , Infecciones por Orthomyxoviridae/patología , Infecciones por Orthomyxoviridae/prevención & control , Infecciones por Orthomyxoviridae/virología , Análisis de Supervivencia , Resultado del Tratamiento , Carga ViralRESUMEN
Assembly of the heterotrimeric influenza virus polymerase complex from the individual subunits PB1, PA, and PB2 is a prerequisite for viral replication, in which the interaction between the C terminal of PA (PAC) and the N-terminal of PB1 (PB1N) may be a desired target for antiviral development. In this study, we compared the feasibility of high throughput screening by enzyme-linked immunosorbent assay (ELISA) and fluorescence polarization assay. Among the two, ELISA was demonstrated to own broader dynamic range so that it was used for screening inhibitors that blocked PAC and PB1N interaction. Several binding inhibitors of PAC-PB1N were identified and subsequently tested for the antiviral efficacy. Apparently, 3-(2-chlorophenyl)-6-ethyl-7-methyl[1,2,4]triazolo[4,3-a]pyrimidin-5-ol, designated ANA-1, was found to be a strong inhibitor of viral polymerase activity and act as a potent antiviral agent against the infections of multiple subtypes of influenza A virus, including H1N1, H3N2, H5N1, H7N7, H7N9 and H9N2 subtypes, in cell cultures. Intranasal administration of ANA-1 protected mice from lethal challenge and reduced lung viral loads in H1N1 virus infected BALB/c mice. Docking analyses predicted that ANA-1 bound to an allosteric site of PAC, which might cause conformational changes thereby disrupting the PAC-PB1N interaction. Overall, our study has identified a novel compound with potential to be developed as an anti-influenza drug.
Asunto(s)
Antivirales/farmacología , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Orthomyxoviridae/efectos de los fármacos , Orthomyxoviridae/enzimología , ARN Polimerasa Dependiente del ARN/metabolismo , Proteínas Virales/metabolismo , Replicación Viral/efectos de los fármacos , Sitio Alostérico/efectos de los fármacos , Animales , Línea Celular , Perros , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H1N1 del Virus de la Influenza A/metabolismo , Subtipo H1N1 del Virus de la Influenza A/fisiología , Células de Riñón Canino Madin Darby , Ratones , Ratones Endogámicos BALB C , Simulación del Acoplamiento Molecular , Orthomyxoviridae/clasificación , Orthomyxoviridae/fisiología , Unión Proteica/efectos de los fármacos , Conformación Proteica , Estructura Terciaria de Proteína , Bibliotecas de Moléculas Pequeñas/farmacología , Replicación Viral/genéticaRESUMEN
To prevent the attachment of bacteria to implant surfaces, the 1D zinc oxide nanowire-coating has been successfully developed on material surfaces by using a custom-made hydrothermal approach. The chemical nature, surface topography and wettability of spike-like 1D ZnO nanowire-coating are comprehensively investigated. The anti-adhesive and antimicrobial properties of 1D nanowire-coating are tested against Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli by using in vitro live/dead staining and scanning electron microscopy. We find that the adhesion of bacteria can be reduced via the special spike-like topography and that the release of Zn(2+) ions can help suppress the growth of attached bacteria. Furthermore, the antimicrobial effect is also evaluated under in vivo conditions by using a rat model infected with bioluminescent S. aureus. The amount of live bacteria in the rat implanted with a nanowire-coated sample is less than that of the control at various time points. Hence, it is believed that the nanowire-coated material is promising for application in orthopaedic implantation after the long-term animal studies have been completed.
Asunto(s)
Antibacterianos/farmacología , Materiales Biocompatibles Revestidos/farmacología , Nanocables/química , Prótesis e Implantes , Óxido de Zinc/química , Animales , Antibacterianos/química , Adhesión Bacteriana/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Materiales Biocompatibles Revestidos/química , Escherichia coli/efectos de los fármacos , Escherichia coli/ultraestructura , Femenino , Interacciones Huésped-Patógeno/efectos de los fármacos , Ratones , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Nanocables/ultraestructura , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/ultraestructura , Ratas Sprague-Dawley , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/prevención & control , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/fisiología , Staphylococcus aureus/ultraestructuraRESUMEN
An Escherichia coli-Laribacter hongkongensis shuttle vector (pPW380) was constructed by ligating the 4701-bp EcoRI digested fragment of pHLHK8 to EcoRI digested pBK-CMV. An E. coli-L. hongkongensis inducible expression shuttle vector was further constructed by ligating a 2105-bp fragment that contains the tetracycline repressor and tetracycline-inducible promoter region of pALC2084 to the 8897-bp fragment of pPW380, deletion of the green fluorescent protein gene, and insertion of a multiple cloning site. This inducible expression system was able to express two commonly used reporter genes, the green fluorescent protein gene and the glutathione S-transferase gene, efficiently in E. coli and L. hongkongensis.