RESUMEN
Certain environmental microorganisms can cause severe human infections, even in the absence of an obvious requirement for transition through an animal host for replication ("accidental virulence"). To understand this process, we compared eleven isolate genomes of Burkholderia pseudomallei (Bp), a tropical soil microbe and causative agent of the human and animal disease melioidosis. We found evidence for the existence of several new genes in the Bp reference genome, identifying 282 novel genes supported by at least two independent lines of supporting evidence (mRNA transcripts, database homologs, and presence of ribosomal binding sites) and 81 novel genes supported by all three lines. Within the Bp core genome, 211 genes exhibited significant levels of positive selection (4.5%), distributed across many cellular pathways including carbohydrate and secondary metabolism. Functional experiments revealed that certain positively selected genes might enhance mammalian virulence by interacting with host cellular pathways or utilizing host nutrients. Evolutionary modifications improving Bp environmental fitness may thus have indirectly facilitated the ability of Bp to colonize and survive in mammalian hosts. These findings improve our understanding of the pathogenesis of melioidosis, and establish Bp as a model system for studying the genetics of accidental virulence.
Asunto(s)
Evolución Biológica , Burkholderia pseudomallei/genética , Burkholderia pseudomallei/patogenicidad , Genes Bacterianos , Animales , Secuencia de Bases , Femenino , Técnica del Anticuerpo Fluorescente , Perfilación de la Expresión Génica , Genoma Bacteriano , Melioidosis/genética , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Virulencia/genéticaRESUMEN
Previous studies have shown that the O polysaccharides (OPS) expressed by Burkholderia mallei are similar to those produced by Burkholderia thailandensis except that they lack the 4-O-acetyl modifications on their 6-deoxy-α-l-talopyranosyl residues. In the present study, we describe the identification and characterization of an open reading frame, designated oacA, expressed by B. thailandensis that accounts for this phenomenon. Utilizing the B. thailandensis and B. mallei lipopolysaccharide (LPS)-specific monoclonal antibodies Pp-PS-W and 3D11, Western immunoblot analyses demonstrated that the LPS antigens expressed by the oacA mutant, B. thailandensis ZT0715, were antigenically similar to those produced by B. mallei ATCC 23344. In addition, immunoblot analyses demonstrated that when B. mallei ATCC 23344 was complemented in trans with oacA, it synthesized B. thailandensis-like LPS antigens. To elucidate the structure of the OPS moieties expressed by ZT0715, purified samples were analyzed via nuclear magnetic resonance spectroscopy. As predicted, these studies demonstrated that the loss of OacA activity influenced the O acetylation phenotype of the OPS moieties. Unexpectedly, however, the results indicated that the O methylation status of the OPS antigens was also affected by the loss of OacA activity. Nonetheless, it was revealed that the LPS moieties expressed by the oacA mutant reacted strongly with the B. mallei LPS-specific protective monoclonal antibody 9C1-2. Based on these findings, it appears that OacA is required for the 4-O acetylation and 2-O methylation of B. thailandensis OPS antigens and that ZT0715 may provide a safe and cost-effective source of B. mallei-like OPS to facilitate the synthesis of glanders subunit vaccine candidates.
Asunto(s)
Proteínas Bacterianas/metabolismo , Burkholderia/clasificación , Burkholderia/genética , Regulación Bacteriana de la Expresión Génica/fisiología , Antígenos O/metabolismo , Proteínas Bacterianas/genética , Conformación de Carbohidratos , Mutación , Antígenos O/genéticaRESUMEN
The stringent response is a regulatory system that allows bacteria to sense and adapt to nutrient-poor environments. The central mediator of the stringent response is the molecule guanosine 3',5'-bispyrophosphate (ppGpp), which is synthesized by the enzymes RelA and SpoT and which is also degraded by SpoT. Our laboratory previously demonstrated that a relA mutant of Pseudomonas aeruginosa, the principal cause of lung infections in cystic fibrosis patients, was attenuated in virulence in a Drosophila melanogaster feeding model of infection. In this study, we examined the role of spoT in P. aeruginosa virulence. We generated an insertion mutation in spoT within the previously constructed relA mutant, thereby producing a ppGpp-devoid strain. The relA spoT double mutant was unable to establish a chronic infection in D. melanogaster and was also avirulent in the rat lung agar bead model of infection, a model in which the relA mutant is fully virulent. Synthesis of the virulence determinants pyocyanin, elastase, protease, and siderophores was impaired in the relA spoT double mutant. This mutant was also defective in swarming and twitching, but not in swimming motility. The relA spoT mutant and, to a lesser extent, the relA mutant were less able to withstand stresses such as heat shock and oxidative stress than the wild-type strain PAO1, which may partially account for the inability of the relA spoT mutant to successfully colonize the rat lung. Our results indicate that the stringent response, and SpoT in particular, is a crucial regulator of virulence processes in P. aeruginosa.
Asunto(s)
Proteínas Bacterianas/metabolismo , Drosophila melanogaster/microbiología , Regulación Bacteriana de la Expresión Génica , Respuesta al Choque Térmico/fisiología , Pulmón/microbiología , Pseudomonas aeruginosa/patogenicidad , Agar , Animales , Carga Bacteriana , Proteínas Bacterianas/genética , Modelos Animales de Enfermedad , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/fisiología , Conducta Alimentaria , Guanosina Pentafosfato/metabolismo , Humanos , Ligasas/genética , Ligasas/metabolismo , Mutación , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/fisiología , Ratas , VirulenciaRESUMEN
Natural isolates of Burkholderia pseudomallei (Bp), the causative agent of melioidosis, can exhibit significant ecological flexibility that is likely reflective of a dynamic genome. Using whole-genome Bp microarrays, we examined patterns of gene presence and absence across 94 South East Asian strains isolated from a variety of clinical, environmental, or animal sources. 86% of the Bp K96243 reference genome was common to all the strains representing the Bp "core genome", comprising genes largely involved in essential functions (eg amino acid metabolism, protein translation). In contrast, 14% of the K96243 genome was variably present across the isolates. This Bp accessory genome encompassed multiple genomic islands (GIs), paralogous genes, and insertions/deletions, including three distinct lipopolysaccharide (LPS)-related gene clusters. Strikingly, strains recovered from cases of human melioidosis clustered on a tree based on accessory gene content, and were significantly more likely to harbor certain GIs compared to animal and environmental isolates. Consistent with the inference that the GIs may contribute to pathogenesis, experimental mutation of BPSS2053, a GI gene, reduced microbial adherence to human epithelial cells. Our results suggest that the Bp accessory genome is likely to play an important role in microbial adaptation and virulence.
Asunto(s)
Burkholderia pseudomallei/genética , Genoma Bacteriano , Islas Genómicas , Melioidosis/microbiología , Animales , Aves , Burkholderia pseudomallei/aislamiento & purificación , Burkholderia pseudomallei/patogenicidad , Análisis por Conglomerados , Perros , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Frecuencia de los Genes , Haplorrinos , Humanos , Mutación INDEL , Melioidosis/genética , Melioidosis/veterinaria , Análisis de Secuencia por Matrices de Oligonucleótidos , Filogenia , Porcinos , Factores de Virulencia/genéticaRESUMEN
BACKGROUND: Burkholderia pseudomallei and Burkholderia mallei cause the diseases melioidosis and glanders, respectively. A well-studied aspect of pathogenesis by these closely-related bacteria is their ability to invade and multiply within eukaryotic cells. In contrast, the means by which B. pseudomallei and B. mallei adhere to cells are poorly defined. The purpose of this study was to identify adherence factors expressed by these organisms. RESULTS: Comparative sequence analyses identified a gene product in the published genome of B. mallei strain ATCC23344 (locus # BMAA0649) that resembles the well-characterized Yersinia enterocolitica autotransporter adhesin YadA. The gene encoding this B. mallei protein, designated boaA, was expressed in Escherichia coli and shown to significantly increase adherence to human epithelial cell lines, specifically HEp2 (laryngeal cells) and A549 (type II pneumocytes), as well as to cultures of normal human bronchial epithelium (NHBE). Consistent with these findings, disruption of the boaA gene in B. mallei ATCC23344 reduced adherence to all three cell types by ~50%. The genomes of the B. pseudomallei strains K96243 and DD503 were also found to contain boaA and inactivation of the gene in DD503 considerably decreased binding to monolayers of HEp2 and A549 cells and to NHBE cultures.A second YadA-like gene product highly similar to BoaA (65% identity) was identified in the published genomic sequence of B. pseudomallei strain K96243 (locus # BPSL1705). The gene specifying this protein, termed boaB, appears to be B. pseudomallei-specific. Quantitative attachment assays demonstrated that recombinant E. coli expressing BoaB displayed greater binding to A549 pneumocytes, HEp2 cells and NHBE cultures. Moreover, a boaB mutant of B. pseudomallei DD503 showed decreased adherence to these respiratory cells. Additionally, a B. pseudomallei strain lacking expression of both boaA and boaB was impaired in its ability to thrive inside J774A.1 murine macrophages, suggesting a possible role for these proteins in survival within professional phagocytic cells. CONCLUSIONS: The boaA and boaB genes specify adhesins that mediate adherence to epithelial cells of the human respiratory tract. The boaA gene product is shared by B. pseudomallei and B. mallei whereas BoaB appears to be a B. pseudomallei-specific adherence factor.
Asunto(s)
Adhesinas Bacterianas/genética , Burkholderia mallei/genética , Burkholderia pseudomallei/genética , Células Epiteliales/microbiología , Secuencia de Aminoácidos , Animales , Burkholderia mallei/clasificación , Burkholderia mallei/patogenicidad , Burkholderia pseudomallei/clasificación , Burkholderia pseudomallei/patogenicidad , Línea Celular , Femenino , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Humanos , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Especificidad de la EspecieRESUMEN
Burkholderia pseudomallei, the causative agent of melioidosis, has often been called the great "mimicker," and clinical disease due to this organism may include acute, chronic, and latent pulmonary infections. Interestingly, chronic pulmonary melioidosis is often mistaken for tuberculosis, and this can have significant consequences, as the treatments for these two infections are radically different. The recurrent misdiagnosis of melioidosis for tuberculosis has caused many to speculate that these two bacterial pathogens use similar pathways to produce latent infections. Here we show that isocitrate lyase is a persistence factor for B. pseudomallei, and inhibiting the activity of this enzyme during experimental chronic B. pseudomallei lung infection forces the infection into an acute state, which can then be treated with antibiotics. We found that if antibiotics are not provided in combination with isocitrate lyase inhibitors, the resulting B. pseudomallei infection overwhelms the host, resulting in death. These results suggest that the inhibition of isocitrate lyase activity does not necessarily attenuate virulence as previously observed for Mycobacterium tuberculosis infections but does force the bacteria into a replicating state where antibiotics are effective. Therefore, isocitrate lyase inhibitors could be developed for chronic B. pseudomallei infections but only for use in combination with effective antibiotics.
Asunto(s)
Antibacterianos/farmacología , Burkholderia pseudomallei/enzimología , Inhibidores Enzimáticos/farmacología , Isocitratoliasa/antagonistas & inhibidores , Melioidosis/tratamiento farmacológico , Melioidosis/microbiología , Animales , Burkholderia pseudomallei/patogenicidad , Recuento de Colonia Microbiana , Isocitratoliasa/fisiología , Dosificación Letal Mediana , Pulmón/microbiología , Ratas , Análisis de SupervivenciaRESUMEN
BACKGROUND: Rhamnolipids are surface active molecules composed of rhamnose and beta-hydroxydecanoic acid. These biosurfactants are produced mainly by Pseudomonas aeruginosa and have been thoroughly investigated since their early discovery. Recently, they have attracted renewed attention because of their involvement in various multicellular behaviors. Despite this high interest, only very few studies have focused on the production of rhamnolipids by Burkholderia species. RESULTS: Orthologs of rhlA, rhlB and rhlC, which are responsible for the biosynthesis of rhamnolipids in P. aeruginosa, have been found in the non-infectious Burkholderia thailandensis, as well as in the genetically similar important pathogen B. pseudomallei. In contrast to P. aeruginosa, both Burkholderia species contain these three genes necessary for rhamnolipid production within a single gene cluster. Furthermore, two identical, paralogous copies of this gene cluster are found on the second chromosome of these bacteria. Both Burkholderia spp. produce rhamnolipids containing 3-hydroxy fatty acid moieties with longer side chains than those described for P. aeruginosa. Additionally, the rhamnolipids produced by B. thailandensis contain a much larger proportion of dirhamnolipids versus monorhamnolipids when compared to P. aeruginosa. The rhamnolipids produced by B. thailandensis reduce the surface tension of water to 42 mN/m while displaying a critical micelle concentration value of 225 mg/L. Separate mutations in both rhlA alleles, which are responsible for the synthesis of the rhamnolipid precursor 3-(3-hydroxyalkanoyloxy)alkanoic acid, prove that both copies of the rhl gene cluster are functional, but one contributes more to the total production than the other. Finally, a double DeltarhlA mutant that is completely devoid of rhamnolipid production is incapable of swarming motility, showing that both gene clusters contribute to this phenotype. CONCLUSIONS: Collectively, these results add another Burkholderia species to the list of bacteria able to produce rhamnolipids and this, by the means of two identical functional gene clusters. Our results also demonstrate the very impressive tensio-active properties these long-chain rhamnolipids possess in comparison to the well-studied short-chain ones from P. aeruginosa.
Asunto(s)
Burkholderia/genética , Glucolípidos/biosíntesis , Familia de Multigenes , Alelos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Burkholderia/metabolismo , Burkholderia pseudomallei/genética , Burkholderia pseudomallei/metabolismo , Genes Bacterianos , MutaciónRESUMEN
Burkholderia pseudomallei is the causative agent of melioidosis, a fatal disease that is endemic to Southeast Asia and northern Australia. The clinical manifestations of melioidosis may range from an acute pneumonia or acute septicemia, to chronic and latent infections. B. pseudomallei is inherently resistant to a number of antibiotics, and even with aggressive antibiotic therapy, the mortality rate remains high, and the incidence of relapse is common. The resistance of this organism to a number of antibiotics has created a need for the development of other therapeutic strategies, including the identification of novel therapeutic targets. B. pseudomallei has been shown to produce a number of capsular polysaccharides, one of which has been shown to contribute to the virulence of the organism. The structures of these polysaccharides have been determined and the genes encoding for the biosynthesis of one of the capsular polysaccharides (CPS I) have been identified. Analysis of the genome sequence of this organism has revealed the presence of three other capsule gene clusters that may encode for the chemical structures previously identified. Since one of the capsules produced by B. pseudomallei has been shown to be important in virulence, the genes encoding for the proteins responsible for its biosynthesis may be considered as potential targets.
Asunto(s)
Burkholderia pseudomallei/efectos de los fármacos , Burkholderia pseudomallei/genética , Melioidosis/tratamiento farmacológico , Animales , Humanos , Melioidosis/microbiología , Polisacáridos/química , Polisacáridos/metabolismoRESUMEN
Burkholderia pseudomallei, which causes melioidosis, a severe, mainly pulmonary disease endemic in South-East Asia, is considered to be the most pathogenic of the Burkholderia genus. B. thailandensis, however, is considered avirulent. We determined differences in patterns of inflammation of B. pseudomallei 1026b (clinical virulent isolate), B. pseudomallei AJ1D8 (an in vitro invasion-deficient mutant generated from strain 1026b by Tn5-OT182 mutagenesis) and B. thailandensis by intranasally inoculating C57BL/6 mice with each strain. Mice infected with B. thailandensis showed a markedly decreased bacterial outgrowth from lungs, spleen and blood 24 h after inoculation, compared with infection with B. pseudomallei and the invasion mutant AJ1D8. Forty-eight hours after inoculation, B. thailandensis was no longer detectable. This was consistent with elevated pulmonary cytokine and chemokine concentrations after infection with B. pseudomallei 1026b and AJ1D8, and the absence of these mediators 48 h, but not 24 h, after inoculation with B. thailandensis. Histological examination, however, did show marked pulmonary inflammation in the mice infected with B. thailandensis, corresponding with substantial granulocyte influx and raised myeloperoxidase levels. Survival experiments showed that infection with 1 x 10(3) cfu B. thailandensis was not lethal, whereas inoculation with 1 x 10(6) cfu B. thailandensis was equally lethal as 1 x 10(3) cfu B. pseudomallei 1026b or AJ1D8. These data show that B. pseudomallei AJ1D8 is just as lethal as wild-type B. pseudomallei in an in vivo mouse model, and B. thailandensis is perhaps more virulent than is often recognized.
Asunto(s)
Burkholderia/inmunología , Burkholderia/patogenicidad , Inflamación/patología , Melioidosis/microbiología , Melioidosis/patología , Animales , Sangre/microbiología , Burkholderia/genética , Recuento de Colonia Microbiana , Citocinas/análisis , Elementos Transponibles de ADN/genética , Granulocitos/inmunología , Pulmón/química , Pulmón/microbiología , Pulmón/patología , Melioidosis/inmunología , Ratones , Ratones Endogámicos C57BL , Mutagénesis Insercional , Peroxidasa/análisis , Bazo/microbiología , Análisis de SupervivenciaRESUMEN
4-Hydroxy-2-alkylquinolines (HAQs), especially 3,4-dihydroxy-2-heptylquinoline (Pseudomonas quinolone signal) and its precursor, 4-hydroxy-2-heptylquinoline, are attracting much attention, mainly because of their role as signaling molecules in Pseudomonas aeruginosa. The pqsABCDE operon is centrally involved in their biosynthesis. The presence of a homologous operon in Burkholderia pseudomallei and B. thailandensis was recently reported. Thus, we have investigated the abilities of 11 Burkholderia species to produce HAQ-like molecules by liquid chromatography/mass spectrometry. We have identified 29 different HAQ derivatives produced by the only three Burkholderia species where a pqsABCDE homologue was found among available sequenced Burkholderia species genomes, including B. ambifaria, a member of the Burkholderia cepacia complex. In contrast with those of P. aeruginosa, Burkholderia HAQs typically bear a methyl group, hence their designation as 4-hydroxy-3-methyl-2-alkylquinolines (HMAQs). We identified three families of HMAQs with a saturated or unsaturated alkyl chain at the 2' position, in contrast with the 1' position of P. aeruginosa, including one with an N-oxide group. Furthermore, the operon in these species contains two more genes downstream of the pqsE homologue, resulting in the hmqABCDEFG operon. While the inactivation of hmqA inhibits the production of HMAQs, the methylation of the quinoline ring requires a putative methyltransferase encoded by hmqG. Interestingly, hmqA or hmqG mutations increase the production of acyl homoserine lactones and, consequently, phenotypes under the control of quorum sensing in B. ambifaria: antifungal activity, siderophore production, and proteolytic activity. These results indicate that only HAQs bearing a methyl group (HMAQs) are involved in quorum-sensing regulation.
Asunto(s)
Burkholderia/fisiología , Quinolinas/metabolismo , Percepción de Quorum , Acil-Butirolactonas/metabolismo , Proteínas Bacterianas/genética , Burkholderia/química , Burkholderia/genética , Burkholderia/metabolismo , Cromatografía Liquida , Eliminación de Gen , Genes Bacterianos , Espectrometría de Masas , Redes y Vías Metabólicas , Metiltransferasas/genética , Estructura Molecular , Mutagénesis Insercional , Operón , Quinolinas/química , Quinolinas/aislamiento & purificaciónRESUMEN
Burkholderia pseudomallei is a facultative intracellular pathogen capable of surviving and replicating within eukaryotic cells. Recent studies have shown that B. pseudomallei Bsa type III secretion system 3 (T3SS-3) mutants exhibit vacuolar escape and replication defects in J774.2 murine macrophages. In the present study, we characterized the interactions of a B. pseudomallei bsaZ mutant with RAW 264.7 murine macrophages. Following uptake, the mutant was found to survive and replicate within infected RAW 264.7 cells over an 18-h period. In addition, high levels of tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), granulocyte-macrophage colony-stimulating factor (GM-CSF), and RANTES, but not IL-1alpha and IL-1beta, were detected in culture supernatants harvested from infected monolayers. The subcellular location of B. pseudomallei within infected RAW 264.7 cells was determined, and as expected, the bsaZ mutant demonstrated early-vacuolar-escape defects. Interestingly, however, experiments also indicated that this mutant was capable of delayed vacuolar escape. Consistent with this finding, evidence of actin-based motility and multinucleated giant cell formation were observed between 12 and 18 h postinfection. Further studies demonstrated that a triple mutant defective in all three B. pseudomallei T3SSs exhibited the same phenotype as the bsaZ mutant, indicating that functional T3SS-1 and T3SS-2 did not appear to be responsible for the delayed escape phenotype in RAW 264.7 cells. Based upon these findings, it appears that B. pseudomallei may not require T3SS-1, -2, and -3 to facilitate survival, delayed vacuolar escape, and actin-based motility in activated RAW 264.7 macrophages.
Asunto(s)
Proteínas Bacterianas/metabolismo , Burkholderia pseudomallei/fisiología , Macrófagos/microbiología , Mutación , Vacuolas/microbiología , Actinas/metabolismo , Animales , Proteínas Bacterianas/genética , Burkholderia pseudomallei/clasificación , Burkholderia pseudomallei/genética , Citocinas/metabolismo , Macrófagos/inmunología , Macrófagos/ultraestructura , Ratones , FenotipoRESUMEN
Burkholderia pseudomallei causes the disease melioidosis. The most common clinical presentation of melioidosis is pneumonia which can occur in acute and chronic forms. The tsunami of 2004 demonstrated a new risk factor for the acquisition of melioidosis and resulted in the proposal that direct delivery of B. pseudomallei into the lungs may result in the enhanced ability of this pathogen to cause disease. In the present studies, we present the development and characterization of rat models of acute and chronic pulmonary melioidosis, and we have utilized these models to demonstrate that direct delivery of B. pseudomallei into the lungs does indeed result in the enhanced ability of this pathogen to cause disease. Importantly, the rat lung infection models for melioidosis can quantify differences in virulence between individual B. pseudomallei wild type strains during both acute and chronic infections. Further, the histopathology associated with pulmonary melioidosis in the rat resembles that seen in tuberculosis. B. pseudomallei microarrays were used to characterize gene expression patterns during chronic pulmonary infections. Transcriptional profiling at several time points during chronic infection revealed that a wide range of genes associated with virulence and metabolic functions are differentially regulated in vivo during chronic infections.
Asunto(s)
Proteínas Bacterianas/metabolismo , Burkholderia pseudomallei/patogenicidad , Modelos Animales de Enfermedad , Regulación Bacteriana de la Expresión Génica , Pulmón/patología , Melioidosis/patología , Neumonía Bacteriana/patología , Enfermedad Aguda , Animales , Proteínas Bacterianas/genética , Burkholderia pseudomallei/genética , Burkholderia pseudomallei/metabolismo , Enfermedad Crónica , Perfilación de la Expresión Génica , Humanos , Pulmón/microbiología , Masculino , Melioidosis/microbiología , Análisis de Secuencia por Matrices de Oligonucleótidos , Neumonía Bacteriana/microbiología , Ratas , Ratas Sprague-Dawley , VirulenciaRESUMEN
BACKGROUND: More than 12,000 simple sequence repeats (SSRs) have been identified in the genome of Burkholderia mallei ATCC 23344. As a demonstrated mechanism of phase variation in other pathogenic bacteria, these may function as mutable loci leading to altered protein expression or structure variation. To determine if such alterations are occurring in vivo, the genomes of various single-colony passaged B. mallei ATCC 23344 isolates, one from each source, were sequenced from culture, a mouse, a horse, and two isolates from a single human patient, and the sequence compared to the published B. mallei ATCC 23344 genome sequence. RESULTS: Forty-nine insertions and deletions (indels) were detected at SSRs in the five passaged strains, a majority of which (67.3%) were located within noncoding areas, suggesting that such regions are more tolerant of sequence alterations. Expression profiling of the two human passaged isolates compared to the strain before passage revealed alterations in the mRNA levels of multiple genes when grown in culture. CONCLUSION: These data support the notion that genome variability upon passage is a feature of B. mallei ATCC23344, and that within a host B. mallei generates a diverse population of clones that accumulate genome sequence variation at SSR and other loci.
Asunto(s)
Burkholderia mallei/genética , Muermo/microbiología , Mutación/genética , Animales , Burkholderia mallei/crecimiento & desarrollo , Eliminación de Gen , Perfilación de la Expresión Génica , Genoma Bacteriano/genética , Caballos , Humanos , Ratones , Ratones Endogámicos BALB C , Repeticiones de Minisatélite/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Polimorfismo de Nucleótido Simple/genética , Análisis de Secuencia de ADN/métodosRESUMEN
The use of animal infection models is central to the study of microbial pathogenesis. In combination with genetic, immunological and antigen purification techniques, much can be learned regarding the pathogenesis of diseases caused by microorganisms. This update focuses on the recent use of animal infection models to study the pathogenesis of melioidosis and glanders.
Asunto(s)
Burkholderia/patogenicidad , Modelos Animales de Enfermedad , Muermo/etiología , Melioidosis/etiología , Animales , Burkholderia/genética , Burkholderia pseudomallei/genética , Burkholderia pseudomallei/patogenicidad , Cricetinae , Genes Bacterianos , Muermo/microbiología , Ratones , Virulencia/genéticaRESUMEN
Burkholderia pseudomallei, the etiological agent of melioidosis, is an animal pathogen capable of inducing a highly fatal septicemia. B. pseudomallei possesses three type III secretion system (TTSS) clusters, two of which (TTSS1 and TTSS2) are homologous to the TTSS of the plant pathogen Ralstonia solanacearum, and one (TTSS3) is homologous to the Salmonella SPI-1 mammalian pathogenicity island. We have demonstrated that TTSS3 is required for the full virulence of B. pseudomallei in a hamster model of infection. We have also examined the virulence of B. pseudomallei mutants deficient in several putative TTSS3 effector molecules, and found no significant attenuation of B. pseudomallei virulence in the hamster model.
Asunto(s)
Burkholderia pseudomallei/metabolismo , Burkholderia pseudomallei/patogenicidad , Melioidosis/microbiología , Animales , Transporte Biológico/genética , Burkholderia pseudomallei/genética , Cricetinae , Modelos Animales de Enfermedad , Eliminación de Gen , Genes Bacterianos , Prueba de Complementación Genética , Mesocricetus , Mutación , Ralstonia solanacearum/genética , Salmonella/genética , Virulencia/genética , Factores de Virulencia/genética , Factores de Virulencia/fisiologíaRESUMEN
Burkholderia pseudomallei and B. mallei are the causative agents of melioidosis and glanders, respectively. As iron regulation of gene expression is common in bacteria, in the present studies, we have used microarray analysis to examine the effects of growth in different iron concentrations on the regulation of gene expression in B. pseudomallei and B. mallei. Gene expression profiles for these two bacterial species were similar under high and low iron growth conditions irrespective of growth phase. Growth in low iron led to reduced expression of genes encoding most respiratory metabolic systems and proteins of putative function, such as NADH-dehydrogenases, cytochrome oxidases, and ATP-synthases. In contrast, genes encoding siderophore-mediated iron transport, heme-hemin receptors, and a variety of metabolic enzymes for alternative metabolism were induced under low iron conditions. The overall gene expression profiles suggest that B. pseudomallei and B. mallei are able to adapt to the iron-restricted conditions in the host environment by up-regulating an iron-acquisition system and by using alternative metabolic pathways for energy production. The observations relative to the induction of specific metabolic enzymes during bacterial growth under low iron conditions warrants further experimentation.
Asunto(s)
Burkholderia mallei/genética , Burkholderia pseudomallei/genética , Regulación Bacteriana de la Expresión Génica , Hierro/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Adaptación Fisiológica , Animales , Burkholderia mallei/metabolismo , Burkholderia pseudomallei/metabolismo , Cricetinae , Femenino , Genes Bacterianos , Genoma Bacteriano , ARN Bacteriano/análisis , ARN Mensajero/análisisRESUMEN
Neutrophil elastase is present at high levels in airway fluid of patients with cystic fibrosis (CF), and is responsible for considerable inflammatory damage. Human monocyte/neutrophil elastase inhibitor (MNEI), a 42-kDa serpin protein, is an effective inhibitor of neutrophil elastase, cathepsin G, and proteinase-3, related proteases released from inflammatory neutrophils. We hypothesized that recombinant MNEI would reduce inflammatory damage and enhance bacterial clearance from the lung in an animal model of chronic Pseudomonas aeruginosa infection. In vitro studies showed that MNEI causes dose-dependent inhibition of the activity of rat neutrophil elastase. Recombinant MNEI was administered daily by aerosolization to rats previously inoculated with agar beads containing P. aeruginosa to initiate chronic infection. Administered MNEI was partially recovered in lavage fluid of treated rats as a 66-kDa complex with protease indicative of in vivo inhibition of elastase or a related protease. Aerosol treatment with MNEI significantly decreased the extent of inflammatory injury, quantified as the histopathology score. MNEI, which had no bactericidal effect on P. aeruginosa in vitro, significantly enhanced clearance of bacteria from infected rat lungs. The reduction of histopathology scores and enhancement of bacterial killing were evident 6 hr after a single aerosol treatment with MNEI. These findings indicate an important function of MNEI in protecting innate antimicrobial defense. Similar results were previously obtained for aerosolized prolastin (alpha1-antitrypsin), indicating that enhanced bacterial clearance by MNEI is due to inhibition of neutrophil protease. These findings demonstrate the value of this nonantibiotic protease inhibitor as an adjunct for the treatment and prevention of the infection component of CF lung disease.
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Aerosoles/administración & dosificación , Neumonía Bacteriana/tratamiento farmacológico , Proteínas/administración & dosificación , Infecciones por Pseudomonas/tratamiento farmacológico , Pseudomonas aeruginosa/crecimiento & desarrollo , Animales , Western Blotting , Líquido del Lavado Bronquioalveolar/microbiología , Enfermedad Crónica , Recuento de Colonia Microbiana , Modelos Animales de Enfermedad , Técnicas In Vitro , Recuento de Leucocitos , Elastasa de Leucocito/antagonistas & inhibidores , Pulmón/microbiología , Pulmón/patología , Masculino , Neutrófilos/enzimología , Neutrófilos/patología , Neumonía Bacteriana/enzimología , Neumonía Bacteriana/microbiología , Infecciones por Pseudomonas/enzimología , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/aislamiento & purificación , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes , SerpinasRESUMEN
Gram-negative infections can cause overwhelming inflammatory responses. Although factors other than LPS are clearly involved, these factors and their mechanisms of action have been poorly defined. During studies of LPS-independent inflammatory responses of the gram-negative pathogen Pseudomonas aeruginosa, an important virulence factor (exoenzyme S) was shown to be a potent mitogen for T cells. The current work demonstrates that exoenzyme S selectively induced transcription and secretion of biologically active cytokines and chemokines (chemotactic for neutrophils and T cells) from monocytes. Exoenzyme S stimulated highly purified monocytes independent of T cells. In addition, exoenzyme S stimulated T cells directly; neither T-cell activation (CD69) nor apoptosis (hypodiploidy) required the presence of monocytes. However, T-cell activation was enhanced via a noncontact-dependent mechanism as a result of the secretion of TNF-alpha and IL-6. This study identifies a unique property of a gram-negative-derived microbial product capable of activating multiple cell types and suggests a mechanism by which exoenzyme S contributes to the immunopathogenesis of cystic fibrosis and sepsis in patients infected with P. aeruginosa.
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ADP Ribosa Transferasas/inmunología , Toxinas Bacterianas , Monocitos/inmunología , Linfocitos T/inmunología , ADP Ribosa Transferasas/farmacología , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/farmacología , Células Cultivadas , Técnicas de Cocultivo , Humanos , Interleucina-6/metabolismo , Activación de Linfocitos/efectos de los fármacos , Monocitos/efectos de los fármacos , Monocitos/microbiología , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/inmunología , Linfocitos T/efectos de los fármacos , Linfocitos T/microbiología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Considerable advances in understanding of the disease caused by Burkholderia mallei have been made employing a combination of tools including genetic techniques and animal infection models. The development of small animal models has allowed us to assess the role of a number of putative virulence determinants in the pathogenesis of disease due to B. mallei. Due to the difficulties in performing active immunization studies in small animals, and due to the fact that the horse is the target mammalian species for glanders, we have initiated experimental studies on glanders in horses. Intratracheal deposition of B. mallei produced clinical glanders with organisms being recovered from tissues of infected horses. The model should prove to be of considerable value in our ongoing studies on the pathogenesis and vaccine development for glanders.
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Burkholderia/aislamiento & purificación , Muermo/microbiología , Enfermedades de los Caballos/microbiología , Animales , Transmisión de Enfermedad Infecciosa/veterinaria , Muermo/epidemiología , CaballosRESUMEN
Melioidosis is a disease caused by the facultative intracellular pathogen Burkholderia pseudomallei iand is associated with a high mortality rate. Melioidosis is endemic in the tropics of southeast Asia and northern Australia and is of worldwide concern, particularly as it is a potential agent of bioterrorism or biological warfare. Also of concern is the lack of a fully effective antibiotic regime, as cases of bacteremia have unacceptably high mortality rates and relapse of melioidosis is common. This review focuses on the approaches that have been undertaken towards the development of an effective vaccine against this disease and highlights current strategies being used to move towards finalizing such a vaccine.