RESUMEN
Melioidosis, a human infectious disease with a high mortality rate in many tropical countries, is caused by the pathogen Burkholderia pseudomallei (B. pseudomallei). The function of the B. pseudomallei sigma S (RpoS) transcription factor in survival during the stationary growth phase and conditions of oxidative stress is well documented. Besides the rpoS, bioinformatics analysis of B. pseudomallei genome showed the existence of two rpoN genes, named rpoN1 and rpoN2. In this study, by using the mouse macrophage cell line RAW264.7 as a model of infection, the involvement of B. pseudomallei RpoS and RpoN2 in the invasion, intracellular survival leading to the reduction in multinucleated giant cell (MNGC) formation of RAW264.7 cell line were illustrated. We have demonstrated that the MNGC formation of RAW264.7 cell was dependent on a certain number of intracellular bacteria (at least 5 × 104). In addition, the same MNGC formation (15%) observed in RAW264.7 cells infected with either B. pseudomallei wild type with multiplicity of infection (MOI) 2 or RpoN2 mutant (∆rpoN2) with MOI 10 or RpoS mutant (∆rpoS) with MOI 100. The role of B. pseudomallei RpoS and RpoN2 in the regulation of type III secretion system on bipB-bipC gene expression was also illustrated in this study.
Asunto(s)
Proteínas Bacterianas , Burkholderia pseudomallei , Melioidosis , Factor sigma , Animales , Humanos , Ratones , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Burkholderia pseudomallei/genética , Burkholderia pseudomallei/metabolismo , Línea Celular , Células Gigantes/metabolismo , Células Gigantes/microbiología , Macrófagos/metabolismo , Melioidosis/microbiología , Factor sigma/metabolismoRESUMEN
ß-thalassemia/Hb E is a global health issue, which is characterized by a range of clinical symptoms from a mild and asymptomatic anemia to severe disorders that require transfusions from infancy. Pathological mechanisms of the disease involve the excess of unmatched alpha globin and iron overload, leading to ineffective erythropoiesis and ultimately to the premature death of erythroid precursors in bone marrow (BM) and peripheral organs. However, it is unclear as to how BM microenvironment factors contribute to the defective erythropoiesis in ß-thalassemia/Hb E patients. Here, we employed mass spectrometry-based comparative proteomics to analyze BM plasma that was collected from six ß-thalassemia/Hb E patients and four healthy donors. We identified that the differentially expressed proteins are enriched in secretory or exosome-associated proteins, many of which have putative functions in the oxidative stress response. Using Western blot assay, we confirmed that atypical lipoprotein, Apolipoprotein D (APOD), belonging to the Lipocalin transporter superfamily, was significantly decreased in BM plasma of the tested pediatric ß-thalassemia/Hb E patients. Our results highlight that the disease condition of ineffective erythropoiesis and oxidative stress found in BM microenvironment of ß-thalassemia/Hb E patients is associated with the impaired expression of APOD protein.
RESUMEN
A serious human infectious disease called Melioidosis is a result of Burkholderia pseudomallei infection. Treatment for infected individuals is difficult due to a wide range of ineffective antibiotics including a high level of antibiotic tolerance which has been known to be caused by biofilm production. However, biofilm forming processes of this bacterium are not well documented despite multiple-methodologies being applied. In this study, we utilized a proteomics strategy called whole cell matrix-assisted laser desorption ionization-time of flight mass spectrometry (whole cell MALDI-TOF MS) to discover a potential biomarker relating biofilm forming in B. pseudomallei. The results presented a novel specific type of enzyme amylo-alpha-1, 6-glucosidase, which was demonstrated by a higher level of gene expression during the biofilm development. Our results also suggested a list of candidate markers that might be involved in this scenario. Eventually, this knowledge may expand valuable data to the biofilm study that may increase effective treatments for people infected with B. pseudomallei and possibly other antibiotic tolerant bacteria.
Asunto(s)
Biopelículas , Biomarcadores/química , Burkholderia pseudomallei/química , Melioidosis/microbiología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biomarcadores/metabolismo , Burkholderia pseudomallei/genética , Burkholderia pseudomallei/aislamiento & purificación , Burkholderia pseudomallei/fisiología , Sistema de la Enzima Desramificadora del Glucógeno/química , Sistema de la Enzima Desramificadora del Glucógeno/genética , Sistema de la Enzima Desramificadora del Glucógeno/metabolismo , HumanosRESUMEN
Burkholderia pseudomallei is a Gram negative bacterium and the causative agent of melioidosis. Nonetheless, how virulence factors and pathogenic mechanisms are regulated have been elusive. In this study, we determined a role of polyphosphate kinase 1 (Ppk1) in regulation of quorum sensing (QS) and the sigma factor RpoS, and identified genes co-regulated by Ppk1, QS and RpoS. We find that Ppk1 positively controls autoinducer production and expression of rpoS transcript. Proteomic analysis identified 70 protein spots that are differentially expressed between B. pseudomallei wildtype and its ppk1-deficient strain. Within Ppk1regulated proteins, expression of 31 proteins are co-regulated by both RpoS and QS, whose functions of the majority of these proteins are associated with energy production and stress response. Moreover, expression of proteins involved in type III secretion system (T3SS) is also controlled by Ppk1. Quantitative PCR analysis confirmed that the T3SS genes bipB, bsaR and hrpK are down-regulated in ppk1 mutant. In addition, the ppk1-deficient strain exhibits defects in adhesion and invasion into human lung epithelial cells. Our work therefore reveals regulation of virulence factors and a regulatory mechanism of RpoS and QS by Ppk1, which altogether participate in gene expression control, and might be crucial for pathogenicity of B. pseudomallei. SIGNIFICANCE: Polyphosphate kinase1 (Ppk1), which is a key enzyme in polyphosphate biosynthesis, is pivotal for virulence of the melioidosis pathogen B. pseudomallei. This enzyme is not present in human. Therefore, it has been proposed to be a key target for anti-bacterial drugs. An important step toward development of novel antibiotics and therapeutic strategies is an analysis of proteins that are controlled by Ppk1. By using proteomics, we find that Ppk1 co-regulates virulence-associated genes together with quorum sensing (QS) and the sigma factor RpoS. Moreover, we reveal that Ppk1 is critical for bacterial adhesion and host cell invasion, supporting the finding from our proteome analysis.
Asunto(s)
Proteínas Bacterianas , Burkholderia pseudomallei , Regulación Bacteriana de la Expresión Génica , Fosfotransferasas (Aceptor del Grupo Fosfato) , Percepción de Quorum , Factor sigma , Sistemas de Secreción Tipo III , Células A549 , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Burkholderia pseudomallei/genética , Burkholderia pseudomallei/metabolismo , Burkholderia pseudomallei/patogenicidad , Humanos , Fosfotransferasas (Aceptor del Grupo Fosfato)/genética , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Factor sigma/genética , Factor sigma/metabolismo , Sistemas de Secreción Tipo III/genética , Sistemas de Secreción Tipo III/metabolismoRESUMEN
BACKGROUND: Burkholderia pseudomallei is an intracellular bacteria causing Melioidosis, the disease widely disseminates in Southeast Asia and Northern Australia. B. pseudomallei has ability to invade various types of host cell and to interfere with host defense mechanisms, such as nitric oxide (NO). Due to the cross-talk among alternative killing mechanisms in host immune response against invading microbes, autophagy is the molecular mechanism belonging to intracellular elimination of eukaryotic cells that has been widely discussed. However, bacterial evasion strategy of B. pseudomallei and host-bacterial protein-protein interaction within autophagic machinery remain unknown. METHODS: Here, we demonstrated the protein-protein interaction study between different strains of B. pseudomallei, including wild type PP844 and rpoS mutant, with autophagy-related protein LC3 that has been constructed, using the modified immunoaffinity hydrophobic chromatography based-technique. Liquid chromatography tandem-mass spectrometry (LC-MS/MS) analysis was utilized for identifying the eluted proteins obtained from the established column. In addition, the expression level of gene encoding candidate protein was predicted prior to verification using real-time quantitative reverse transcription PCR assay (RT-qPCR). RESULTS: LC3 recombinant proteins could be entrapped inside the column before encountering their bacterial interacting partners. Based on affinity interaction, the binding capacity of LC3 with antibody displayed over 50% readily for hydrophobically binding with bacterial proteins. Following protein identification, bacterial ATP-binding cassette (ABC) transporter periplasmic substrate-binding protein (BPSL2203) was identified as a candidate LC3-interacting protein, which was found only in B. pseudomallei wild type. Gene expression analysis and bioinformatics of BPSL2203 were validated the proteomic result which are suggesting the role of RpoS-dependent gene regulation. CONCLUSIONS: Remarkably, utilization of the modified immunoaffinity hydrophobic chromatography with LC-MS/MS is a convenient and reliable approach to a study in B. pseudomallei-LC3 protein-protein interaction.
RESUMEN
Burkholderia pseudomallei is an intracellular Gram-negative bacterial pathogen and the causative agent of melioidosis, a widespread disease in Southeast Asia. Reactive nitrogen, in an intermediate form of nitric oxide (NO), is one of the first lines of defense used by host cells to eliminate intracellular pathogens, through the stimulation of inducible nitric oxide synthase (iNOS). Studies in phagocytotic cells have shown that the iNOS response is muted in B. pseudomallei infection, and implicated the rpoS sigma factor as a key regulatory factor mediating suppression. The liver is a main visceral organ affected by B. pseudomallei, and there is little knowledge about the interaction of liver cells and B. pseudomallei This study investigated the induction of iNOS, as well as autophagic flux and light-chain 3 (LC3) localization in human liver (HC04) cells in response to infection with B. pseudomallei and its rpoS deficient mutant. Results showed that the rpoS mutant was unable to suppress iNOS induction and that the mutant showed less induction of autophagy and lower co-localization with LC3, and this was coupled with a lower intracellular growth rate. Combining these results suggest that B. pseudomallei rpoS is an important factor in establishing infection in liver cells.
Asunto(s)
Proteínas Bacterianas/metabolismo , Burkholderia pseudomallei/genética , Hepatocitos/enzimología , Hepatocitos/microbiología , Interacciones Huésped-Patógeno , Óxido Nítrico Sintasa de Tipo II/biosíntesis , Factor sigma/metabolismo , Autofagia , Proteínas Bacterianas/genética , Burkholderia pseudomallei/crecimiento & desarrollo , Burkholderia pseudomallei/metabolismo , Línea Celular , Inducción Enzimática , Humanos , Melioidosis/microbiología , Viabilidad Microbiana , Proteínas Asociadas a Microtúbulos/metabolismo , Mutación , Óxido Nítrico Sintasa de Tipo II/genética , Factor sigma/genéticaRESUMEN
Whole-cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (whole-cell MALDI-TOF MS) has been widely adopted as a useful technology in the identification and typing of microorganisms. This study employed the whole-cell MALDI-TOF MS to identify and differentiate wild-type and mutants containing constructed single gene mutations of Burkholderia pseudomallei, a pathogenic bacterium causing melioidosis disease in both humans and animals. Candidate biomarkers for the B. pseudomallei mutants, including rpoS, ppk, and bpsI isolates, were determined. Taxon-specific and clinical isolate-specific biomarkers of B. pseudomallei were consistently found and conserved across all average mass spectra. Cluster analysis of MALDI spectra of all isolates exhibited separate distribution. A total of twelve potential mass peaks discriminating between wild-type and mutant isolates were identified using ClinProTools analysis. Two peaks (m/z 2721 and 2748 Da) were specific for the rpoS isolate, three (m/z 3150, 3378, and 7994 Da) for ppk, and seven (m/z 3420, 3520, 3587, 3688, 4623, 4708, and 5450 Da) for bpsI. Our findings demonstrated that the rapid, accurate, and reproducible mass profiling technology could have new implications in laboratory-based rapid differentiation of extensive libraries of genetically altered bacteria.
Asunto(s)
Burkholderia pseudomallei , Marcadores Genéticos/genética , Mutación/genética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Burkholderia pseudomallei/clasificación , Burkholderia pseudomallei/genética , Burkholderia pseudomallei/aislamiento & purificación , Genotipo , Humanos , Melioidosis/microbiología , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Proteínas Recombinantes/genética , Factor sigma/genéticaRESUMEN
Burkholderia pseudomallei is the cause of melioidosis, a fatal tropical infectious disease, which has been reported to have a high rate of recurrence, even when an intensive dose of antibiotics is used. Biofilm formation is believed to be one of the possible causes of relapse because of its ability to increase drug resistance. EPS in biofilms have been reported to be related to the limitation of antibiotic penetration in B. pseudomallei. However, the mechanisms by which biofilms restrict the diffusion of antibiotics remain unclear. The present study presents a correlation between exopolysaccharide production in biofilm matrix and antibiotic resistance in B. pseudomallei using bpsI, ppk, and rpoS mutant strains. CLSM revealed a reduction in exopolysaccharide production and disabled micro-colony formation in B. pseudomallei mutants, which paralleled the antibiotic resistance. Different ratios of carbohydrate contents in the exopolysaccharides of the mutants were detected, although they have the same components, including glucose, galactose, mannose, and rhamnose, with the exception being that no detectable rhamnose peak was observed in the bpsI mutant. These results indicate that the correlation between these phenomena in the B. pseudomallei biofilm at least results from the exopolysaccharide, which may be under the regulation of bpsI, ppk, or rpoS genes.
Asunto(s)
Antibacterianos/farmacología , Biopelículas/crecimiento & desarrollo , Burkholderia pseudomallei/fisiología , Resistencia a Medicamentos/fisiología , Polisacáridos Bacterianos/química , Humanos , Melioidosis , MutaciónRESUMEN
Burkholderia pseudomallei is the causative agent of melioidosis. The complete genome sequences of this pathogen have been revealed, which explain some pathogenic mechanisms. In various hostile conditions, for example, during nitrogen and amino acid starvation, bacteria can utilize alternative sigma factors such as RpoS and RpoN to modulate genes expression for their adaptation and survival. In this study, we demonstrate that mutagenesis of rpoN2, which lies on chromosome 2 of B. pseudomallei and encodes a homologue of the sigma factor RpoN, did not alter nitrogen and amino acid utilization of the bacterium. However, introduction of B. pseudomallei rpoN2 into E. coli strain deficient for rpoN restored the ability to utilize amino acids. Moreover, comparative partial proteomic analysis of the B. pseudomallei wild type and its rpoN2 isogenic mutant was performed to elucidate its amino acids utilization property which was comparable to its function found in the complementation assay. By contrast, the rpoN2 mutant exhibited decreased katE expression at the transcriptional and translational levels. Our finding indicates that B. pseudomallei RpoN2 is involved in a specific function in the regulation of catalase E expression.
RESUMEN
Burkholderia pseudomallei is the causative agent of melioidosis, which is an endemic disease in Northeast Thailand and Northern Australia. Environmental reservoirs, including wet soils and muddy water, serve as the major sources for contributing bacterial infection to both humans and animals. The whole-cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (whole-cell MALDI-TOF MS) has recently been applied as a rapid, accurate, and high-throughput tool for clinical diagnosis and microbiological research. In this present study, we employed a whole-cell MALDI-TOF MS approach for assessing its potency in clustering a total of 11 different B. pseudomallei isolates (consisting of 5 environmental and 6 clinical isolates) with respect to their origins and to further investigate the source-identifying biomarker ions belonging to each bacterial group. The cluster analysis demonstrated that six out of eleven isolates were grouped correctly to their sources. Our results revealed a total of ten source-identifying biomarker ions, which exhibited statistically significant differences in peak intensity between average environmental and clinical mass spectra using ClinProTools software. Six out of ten mass ions were assigned as environmental-identifying biomarker ions (EIBIs), including, m/z 4,056, 4,214, 5,814, 7,545, 7,895, and 8,112, whereas the remaining four mass ions were defined as clinical-identifying biomarker ions (CIBIs) consisting of m/z 3,658, 6,322, 7,035, and 7,984. Hence, our findings represented, for the first time, the source-specific biomarkers of environmental and clinical B. pseudomallei.
Asunto(s)
Biomarcadores/análisis , Burkholderia pseudomallei/aislamiento & purificación , Microbiología Ambiental , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Análisis por Conglomerados , Iones , Análisis de Componente PrincipalRESUMEN
Burkholderia pseudomallei, a pathogenic gram-negative bacterium, causes the severe human disease melioidosis. This organism can survive in eukaryotic host cells by escaping reactive oxygen species via the regulation of stress responsive sigma factors, including RpoS. In B. pseudomallei, RpoS has been reported to play a role in the oxidative stress response through enhanced activity of OxyR and catalase. In this study, the RpoS dependent oxidative stress responsive system was further characterized using comparative proteomic analysis. The proteomic profiles of wild-type B. pseudomallei following exposure to H2 O2 and between wild-type and the rpoS mutant strains were analyzed. Using stringent criteria, 13 oxidative responsive proteins, eight of which are regulated by RpoS, were identified with high confidence. It was observed that ScoA, a subunit of the SCOT enzyme not previously shown to be involved directly in the oxidative stress response, is significantly down-regulated after hydrogen peroxide treatment. ScoA and ScoB have been predicted to be organized in a single operon using computational methods: in this study it was confirmed by RT-PCR that these genes are indeed co-transcribed as a single mRNA. The present study is the first to report a role for RpoS in the down-regulation of SCOT expression in response to oxidative stress in B. pseudomallei.
Asunto(s)
Proteínas Bacterianas/metabolismo , Burkholderia pseudomallei/enzimología , Coenzima A Transferasas/metabolismo , Regulación Enzimológica de la Expresión Génica , Estrés Oxidativo , Factor sigma/metabolismo , Proteínas Bacterianas/genética , Burkholderia pseudomallei/química , Burkholderia pseudomallei/efectos de los fármacos , Burkholderia pseudomallei/genética , Coenzima A Transferasas/genética , Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Peróxido de Hidrógeno/farmacología , Melioidosis/microbiología , Estrés Oxidativo/efectos de los fármacos , Factor sigma/genéticaRESUMEN
Melioidosis is a severe infectious disease that is endemic in Southeast Asia and Northern Australia. Burkholderia pseudomallei, the causative agent of this disease, has developed resistance to an increasing list of antibiotics, demanding a search for novel agents. Lactoferricin and lactoferrampin are two antimicrobial domains of lactoferrin with a broad spectrum of antimicrobial activity. A hybrid peptide (LFchimera) containing lactoferrampin (LFampin265-284) and a part of lactoferricin (LFcin17-30) has strikingly higher antimicrobial activities compared to the individual peptides. In this study, the antimicrobial activities of this chimeric construct (LFchimera1), as well as of another one containing LFcin17-30 and LFampin268-284, a shorter fragment of LFampin265-284 (LFchimera2), and the constituent peptides were tested against 7 isolates of B. pseudomallei and compared to the preferential antibiotic ceftazidime (CAZ). All isolates including B. pseudomallei 979b shown to be resistant to CAZ, at a density of 10(5) CFU/ml, could be killed by 5-10 µM of LFchimera1 within 2 h, while the other peptides as well as the antibiotic CAZ only inhibited the B. pseudomallei strains resulting in an overgrowth in 24 h. These data indicate that LFchimera1 could be considered for development of therapeutic agents against B. pseudomallei.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/metabolismo , Burkholderia pseudomallei/efectos de los fármacos , Lactoferrina/metabolismo , Animales , Péptidos Catiónicos Antimicrobianos/genética , Bovinos , Lactoferrina/genética , Pruebas de Sensibilidad Microbiana , Viabilidad Microbiana/efectos de los fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
AGC2, a member of the mitochondrial carrier protein family, is as an aspartate-glutamate carrier and is important for urea synthesis and the maintenance of the malate-aspartate shuttle. Mutations in SLC25A13, the gene encoding AGC2, result in two age dependent disorders: neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) and type II citrullinemia (CTLN2). The clinical features of CTLN2 are very similar to those of other urea cycle disorders making a clear diagnosis difficult. Analysis of the SLC25A13 gene sequence can provide a definitive diagnosis, however the predictive value of DNA sequencing requires that the disease association of variants be characterized. We utilized the yeast Saccharomyces cerevisiae lacking AGC1 as a model system to study the effect on the function of AGC2 variants and confirmed that this system is capable of distinguishing between AGC2 variants with normal (p.Pro632Leu) or impaired function (p.Gly437Glu, p.Gly531Asp, p.Thr546Met, p.Leu598Arg and p.Glu601Lys). Three novel AGC2 genetic variants, p.Met1? (c.2T>C), p.Pro502Leu (c.1505C>T), and p.Arg605Gln (c.1814G>A) were investigated and our analysis revealed that p.Pro502Leu and p.Arg605Gln substitutions in the AGC2 protein were without effect and these variants were fully functional. The p.Met1? mutant is capable of expressing a truncated p.Met1_Phe34del AGC2 variant, however this protein is not functional due to disruptions in a calcium binding EF hand as well as incorrect intracellular localization. Our study demonstrates that the characterization of AGC2 expressed in yeast cells is a powerful technique to investigate AGC2 variants, and this analysis should aid in establishing the disease association of novel variants.
Asunto(s)
Sistemas de Transporte de Aminoácidos Acídicos/deficiencia , Sistemas de Transporte de Aminoácidos Acídicos/genética , Antiportadores/deficiencia , Antiportadores/genética , Proteínas de Transporte de Membrana Mitocondrial/genética , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Colestasis Intrahepática/genética , Citrulinemia/genética , Variación Genética , Humanos , Mitocondrias/genética , Proteínas de Transporte de Membrana Mitocondrial/deficiencia , Datos de Secuencia Molecular , Alineación de Secuencia , Análisis de Secuencia de ADN/métodosRESUMEN
Burkholderia pseudomallei is the causative agent of a fatal disease, melioidosis. However, the mechanisms of pathogenesis and genes involved in its virulence are not understood. In the current study, whether stationary phase and stress response sigma factor RpoS and BpsI-mediated quorum sensing (QS) system co-regulate its target genes was investigated. Positive regulation of RpoS on bpsI expression and autoinducer production in stationary phase, but not on fliC and ppk expression, was observed. In contrast, expression of rpoS was not affected by deletion of bpsI. The present results also indicate that production of extracellular protease and siderophore, two QS-controlled exo products, is regulated via RpoS supporting the previously known inhibitory role of QS on those two factors. Proteomic analysis revealed that expression of 74 protein spots representing 60 genes is controlled by QS. Of those, 45 genes are co-regulated by both RpoS and QS, and regulation of genes involved in transcription and translation is favored by QS. Taken together, our findings indicate major target genes expression in stationary phase that is influenced by hierarchical control of RpoS over QS. We propose that this regulation may play an important role in the pathogenicity of B. pseudomallei.
Asunto(s)
Proteínas Bacterianas/genética , Burkholderia pseudomallei/fisiología , Regulación Bacteriana de la Expresión Génica , Melioidosis/microbiología , Percepción de Quorum , Factor sigma/metabolismo , Proteínas Bacterianas/metabolismo , Burkholderia pseudomallei/genética , Burkholderia pseudomallei/crecimiento & desarrollo , Humanos , Datos de Secuencia Molecular , Factor sigma/genéticaRESUMEN
BACKGROUND: Dengue is the world's most common mosquito-borne viral disease. Poor proofreading by RNA polymerase during its replication results in the accumulation of mutations in its genome. This leads to a diversity of genotypes in the viral population termed quasispecies. Quasispecies play an important role in disease severity. The study of quasispecies in dengue has been hindered because of the requirement for large amounts of cloning and sequencing, which could be overcome by 454 pyrosequencing. In this study, we attempted to demonstrate the feasibility of using 454 pyrosequencing to study genome diversity of dengue virus quasispecies by sequencing a pool of known dengue viral strains. RESULTS: Two sets of dengue DNA templates were sequenced by 454/Roche GS FLX. The total number of reads for data 1 and data 2 were 54,440 and 134,441, with average lengths of 221 and 232 bp, respectively. Reads containing ambiguous base Ns were excluded (6.00% in data 1, 7.05% in data 2). More than 99% of reads could be aligned back to the correct serotypes by BLAST. The reads covered the whole genome without any gaps, and the minimum coverage depth was 50×. Frequencies of known strains detected from each data set were highly correlated with the input ratios. We also explored criteria for filtering error reads and artifacts from true variations. CONCLUSIONS: This study showed that 454 pyrosequencing, coupled with our analysis procedure, could sequence the whole genome of dengue virus with good coverage. The ratio of detected variants in the sequencing data reflected the starting ratio, proving that the proposed technique could be used to study the frequencies of variants in quasispecies.
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Virus del Dengue/genética , Genoma Viral , Análisis de Secuencia de ARN , Mapeo Cromosómico , Secuencia de Consenso , Genotipo , SerotipificaciónRESUMEN
Burkholderia pseudomallei is the etiologic agent of melioidosis. Using 2DE and MALDI-TOF MS, we report here a proteome reference map constructed from early stationary phase, a bacterial adaptation process. We identified 282 protein spots representing 220 ORFs; many of them have been implicated in bacterial pathogenesis. Up to 20% of identified ORFs belong to post-translational modification and stress responses. The proteome reference map will support future analysis of the bacterial gene and environmental regulation and facilitate comparative proteomics with its sibling species.
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Proteínas Bacterianas/metabolismo , Burkholderia pseudomallei/metabolismo , Melioidosis/microbiología , Proteoma/metabolismo , Proteínas Bacterianas/genética , Burkholderia pseudomallei/genética , Electroforesis en Gel Bidimensional/métodos , Humanos , Procesamiento Proteico-Postraduccional/genética , Proteoma/genética , Estándares de Referencia , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Estrés Fisiológico/genéticaRESUMEN
BACKGROUND: Hemoglobin E/ß-thalassemia is particularly common in Southeast Asia and has variable symptoms ranging from mild to severe anemia. Previous investigations demonstrated the remarkable symptoms of ß-thalassemia in terms of the acceleration of apoptotic cell death. Ineffective erythropoiesis has been studied in human hematopoietic stem cells, however the distinct apoptotic mechanism was unclear. METHODS: The phosphoproteome of bone marrow HSCs/CD34⺠cells from HbE/ß-thalassemic patients was analyzed using IMAC phosphoprotein isolation followed by LC-MS/MS detection. Decyder MS software was used to quantitate differentially expressed proteins in 3 patients and 2 normal donors. The differentially expressed proteins from HSCs/CD34⺠cells were compared with HbE/ß-thalassemia and normal HSCs. RESULTS: A significant change in abundance of 229 phosphoproteins was demonstrated. Importantly, the analysis of the candidate proteins revealed a high abundance of proteins that are commonly found in apoptotic cells including cytochrome C, caspase 6 and apoptosis inducing factors. Moreover, in the HSCs patients a significant increase was observed in a specific type of phosphoserine/threonine binding protein, which is known to act as an important signal mediator for the regulation of cell survival and apoptosis in HbE/ß-thalassemia. CONCLUSIONS: Our study used a novel method to investigate proteins that influence a particular pathway in a given disease or physiological condition. Ultimately, phosphoproteome profiling in HbE/ß-thalassemic stem cells is an effective method to further investigate the cell death mechanism of ineffective erythropoiesis in ß-thalassemia. Our report provides a comprehensive phosphoproteome, an important resource for the study of ineffective erythropoiesis and developing therapies for HbE/ß-thalassemia.
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Apoptosis , Células Madre Hematopoyéticas/metabolismo , Hemoglobina E/metabolismo , Fosfoproteínas/metabolismo , Proteómica/métodos , Talasemia beta/metabolismo , Talasemia beta/patología , Antígenos CD34/metabolismo , Supervivencia Celular , Células Cultivadas , Niño , Preescolar , Células Madre Hematopoyéticas/patología , Humanos , Espectrometría de Masas , Modelos Biológicos , Fosfoproteínas/química , Transducción de Señal , Donantes de TejidosRESUMEN
Burkholderia pseudomallei, the causative agent of the potentially fatal tropical disease melioidosis, is known to be highly resistant to oxidative stress although the mechanism of this resistance remains to be fully elucidated. Previous studies have shown that an OxyR is involved in the regulation of oxidative stress via the katG and dpsA genes encoding KatG and DpsA and that the alternative sigma factor, RpoS, plays a critical role in resistance to oxidative stress by regulating katG and katE genes. Here it is shown that RpoS is essential for expression of the oxidative stress regulator OxyR, since a mutant strain lacking RpoS failed to induce oxyR expression both during normal growth and under conditions of oxidative stress. It is further demonstrated that the RpoS acts as a positive transcriptional regulator of oxyR and dpsA expression, while OxyR acts as a negative transcriptional regulator of the katG-dpsA operon via OxyR repressor under normal growth conditions, and as a positive transcriptional regulator via OxyR under conditions of oxidative stress. Therefore both RpoS and OxyR are required to promote expression of both the katG-dpsA operon and dpsA gene.
Asunto(s)
Proteínas Bacterianas/metabolismo , Burkholderia pseudomallei/metabolismo , Catalasa/genética , Regulación Bacteriana de la Expresión Génica , Estrés Oxidativo , Proteínas Represoras/metabolismo , Factor sigma/metabolismo , Proteínas Bacterianas/genética , Burkholderia pseudomallei/genética , Catalasa/metabolismo , Operón , Proteínas Represoras/genética , Factor sigma/genéticaRESUMEN
Burkholderia pseudomallei, a motile and rod Gram-negative bacterium, is the causative agent of melioidosis. The bacterium is an intracellular pathogen and that motility is generally crucial for their survival in a natural environment and for systemic infection inside a host. We report here a role of B. pseudomallei polyphosphate kinase in virulence, such as an oxidative stress response, motilities and biofilm formation. The polyphosphate kinase (ppk) mutant is susceptible to hydrogen peroxide in an oxidative stress condition, unable to perform swimming, swarming motilities, and has lower density biofilm forming capacity than the wild-type strain. We also demonstrated that both polyphosphate kinase and motile flagella are essential and independently involved in biofilm formation. The B. pseudomallei flagellin (fliC) mutant and B. mallei, a nonmotile species, are shown to produce higher density biofilm formation than the ppk mutant, but less than wild type B. pseudomallei.
Asunto(s)
Biopelículas/crecimiento & desarrollo , Burkholderia pseudomallei/enzimología , Estrés Oxidativo/fisiología , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Burkholderia pseudomallei/efectos de los fármacos , Burkholderia pseudomallei/genética , Burkholderia pseudomallei/fisiología , Peróxido de Hidrógeno/farmacología , Microscopía Confocal , Mutación , Fosfotransferasas (Aceptor del Grupo Fosfato)/genéticaRESUMEN
BACKGROUND: Burkholderia pseudomallei, a gram-negative bacterium that causes melioidosis, was reported to produce biofilm. As the disease causes high relapse rate when compared to other bacterial infections, it therefore might be due to the reactivation of the biofilm forming bacteria which also provided resistance to antimicrobial agents. However, the mechanism on how biofilm can provide tolerance to antimicrobials is still unclear. METHODOLOGY/PRINCIPAL FINDINGS: The change in resistance of B. pseudomallei to doxycycline, ceftazidime, imipenem, and trimethoprim/sulfamethoxazole during biofilm formation were measured as minimum biofilm elimination concentration (MBEC) in 50 soil and clinical isolates and also in capsule, flagellin, LPS and biofilm mutants. Almost all planktonic isolates were susceptible to all agents studied. In contrast, when they were grown in the condition that induced biofilm formation, they were markedly resistant to all antimicrobial agents even though the amount of biofilm production was not the same. The capsule and O-side chains of LPS mutants had no effect on biofilm formation whereas the flagellin-defective mutant markedly reduced in biofilm production. No alteration of LPS profiles was observed when susceptible form was changed to resistance. The higher amount of N-acyl homoserine lactones (AHLs) was detected in the high biofilm-producing isolates. Interestingly, the biofilm mutant which produced a very low amount of biofilm and was sensitive to antimicrobial agents significantly resisted those agents when grown in biofilm inducing condition. CONCLUSIONS/SIGNIFICANCE: The possible drug resistance mechanism of biofilm mutants and other isolates is not by having biofilm but rather from some factors that up-regulated when biofilm formation genes were stimulated. The understanding of genes related to this situation may lead us to prevent B. pseudomallei biofilms leading to the relapse of melioidosis.