RESUMEN
Burkholderia pseudomallei is the causative agent of melioidosis and represents a potential bioterrorism threat. In this study, the transcriptomic responses of B. pseudomallei infection of a human macrophage cell model were investigated using whole-genome microarrays. Gene expression profiles were compared between infected THP-1 human monocytic leukemia cells with or without treatment with Daboia russelli russelli daboiatoxin (DRRDbTx) or ceftazidime (antibiotic control). Microarray analyses of infected and treated cells revealed differential upregulation of various inflammatory genes such as interleukin-1 (IL-1), IL-6, tumor necrosis factor-alpha (TNF-α), cyclooxygenase (COX-2), vascular endothelial growth factor (VEGF), chemokine C-X-C motif ligand 4 (CXCL4), transcription factor p65 (NF-kB); and several genes involved in immune and stress responses, cell cycle, and lipid metabolism. Moreover, following DRR-DbTx treatment of infected cells, there was enhanced expression of the tolllike receptor 2 (TLR-2) mediated signaling pathway involved in recognition and initiation of acute inflammatory responses. Importantly, we observed that highly inflammatory cytokine gene responses were similar in infected cells exposed to DRR-DbTx or ceftazidime after 24 h. Additionally, there were increased transcripts associated with cell death by caspase activation that can promote host tissue injury. In summary, the transcriptional responses during B. pseudomallei infection of macrophages highlight a broad range of innate immune mechanisms that are activated within 24 h post-infection. These data provide insights into the transcriptomic kinetics following DRR-DbTx treatment of human macrophages infected with B. pseudomallei.
Asunto(s)
Burkholderia pseudomallei/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Macrófagos/efectos de los fármacos , Proteínas/farmacología , Transcriptoma , Venenos de Víboras/química , Animales , Burkholderia pseudomallei/crecimiento & desarrollo , Burkholderia pseudomallei/ultraestructura , Ceftazidima/farmacología , Línea Celular , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Perfilación de la Expresión Génica , Estudio de Asociación del Genoma Completo , Interacciones Huésped-Patógeno , Humanos , Interleucina-1/genética , Interleucina-1/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiología , Macrófagos/ultraestructura , Análisis por Micromatrices , FN-kappa B/genética , FN-kappa B/metabolismo , Factor Plaquetario 4/genética , Factor Plaquetario 4/metabolismo , Proteínas/aislamiento & purificación , Transducción de Señal , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/metabolismo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , ViperidaeRESUMEN
BACKGROUND: Burkholderia pseudomallei are the causative agent of melioidosis. Increasing resistance of the disease to antibiotics is a severe problem in treatment regime and has led to intensification of the search for new drugs. Antimicrobial peptides are the most ubiquitous in nature as part of the innate immune system and host defense mechanism. METHODS: Here, we investigated a group of venoms (snakes, scorpions and honey bee venoms) for antimicrobial properties against two strains of Gram-negative bacteria Burkholderia pseudomallei by using disc-diffusion assay for in vitro susceptibility testing. The antibacterial activities of the venoms were compared with that of the isolated L-amino acid oxidase (LAAO) and phospholipase A2 (PLA2s) enzymes. MICs were determined using broth dilution method. Bacterial growth was assessed by measurement of optical density at the lowest dilutions (MIC 0.25 mg/ml). The cell viability was measured using tetrazolium salts (XTT) based cytotoxic assay. RESULTS: The studied venoms showed high antimicrobial activity. The venoms of C. adamanteus, Daboia russelli russelli, A. halys, P. australis, B. candidus and P. guttata were equally as effective as Chloramphenicol and Ceftazidime (30 microg/disc). Among those tested, phospholipase A2 enzymes (crotoxin B and daboiatoxin) showed the most potent antibacterial activity against Gram-negative (TES) bacteria. Naturally occurring venom peptides and phospholipase A2 proved to possess highly potent antimicrobial activity against Burkholderia pseudomallei. The XTT-assay results showed that the cell survival decreased with increasing concentrations (0.05-10 mg/mL) of Crotalus adamanteus venom, with no effect on the cell viability evident at 0.5 mg/mL. CONCLUSION: This antibacterial profile of snake venoms reported herein will be useful in the search for potential antibacterial agents against drug resistant microorganisms like B. pseudomallei.
Asunto(s)
Burkholderia pseudomallei/efectos de los fármacos , Melioidosis/microbiología , Venenos de Serpiente/farmacología , Animales , Burkholderia pseudomallei/aislamiento & purificación , Crotoxina/aislamiento & purificación , Crotoxina/farmacología , Farmacorresistencia Microbiana , Humanos , Pruebas de Sensibilidad Microbiana , Fosfolipasas A/aislamiento & purificación , Fosfolipasas A/farmacología , Fosfolipasas A2 , Proteínas/aislamiento & purificación , Proteínas/farmacología , Sepsis/microbiología , Venenos de Serpiente/enzimología , Venenos de VíborasRESUMEN
Candoxin (PDB #1JGK), a three-finger neurotoxin from Bungarus candidus venom, inhibits post-synaptic neuromuscular and neuronal alpha7nACh-receptors, and induces delayed cell-death throughout the glial population. When applied to cultured human glial cell lines, candoxin (CDX) induced cell death in a concentration (EC(50) approximately 1muM) and time dependent manner. Results of TUNEL-histochemistry further confirm CDX-induced brain (hippocampus, frontal cortex, and temporal regions) damage when administered intracerebroventricularly (i.c.v) in adult mice. In this study, we explored differential gene expression profiles following exposure of human glial (Hs 683) cell lines to CDX at various time intervals using Affymetrix-GeneChips. By means of MAS and GeneSpring analyses, 105 genes whose expression was significantly (P<0.01) altered by at least 3-fold were selected. Results of the genome analysis reveal that the potential role of CDX at molecular level involves the regulation of genes in signal transduction, ubiquitin-inflammation, mitochondrial-dysfunction, and damage-response pathways. In addition, using QRT-PCR and rationally designed specific CDX-binding peptide (P-NT.II), we identified the genes-IL7R, IL13RA2, IL-1beta, TNFRSF12A, GADD45A, CD44 and IFI44-that might play an important role in CDX-induced glial inflammation, DNA-damage and degeneration. These findings reveal new insight into the molecular mechanisms of glial-driven neurodegeneration after exposure to neurotoxins.
Asunto(s)
Bungarus , Regulación de la Expresión Génica/efectos de los fármacos , Neuroglía/efectos de los fármacos , Venenos de Serpiente/aislamiento & purificación , Venenos de Serpiente/toxicidad , Análisis de Varianza , Animales , Encéfalo/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Análisis por Conglomerados , Cartilla de ADN , Perfilación de la Expresión Génica , Histocitoquímica , Humanos , Etiquetado Corte-Fin in Situ , Espectrometría de Masas , Mitocondrias/efectos de los fármacos , Neuroglía/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de Proteína , Transducción de Señal/efectos de los fármacos , Venenos de Serpiente/genética , Toxicogenética/métodosRESUMEN
Spirulina fusiformis was tested for its possible in vivo protective effects against cyclophosphamide (CP) and mitomycin-C (MMC) induced genotoxicity and oxidative stress in mice. Pre-treatment with S. fusiformis (250, 500 and 1000 mg kg(-1), p.o., daily for 5 days) significantly reduced the chromosomal damage and lipid peroxidation with concomitant changes in antioxidants and detoxification systems. All the three tested doses were effective in exerting a protective effect against CP and MMC.