RESUMEN
OBJECTIVES: Rice (Oryza sativa) is the most important food for more than two thirds of the world's population. Bangladesh is the third largest producer and consumer of rice globally. Recently, several symptoms of Bacterial Panicle Blight (BPB) in rice, including seedling blight, sheath rot, floret sterility, and spotted grains, have been detected in the country. In addition, the presence of the most prevalent and virulent causative agent of BPB, Burkholderia glumae, has been confirmed in rice displaying symptoms of the disease. BPB could become one of the next emerging diseases of rice in Bangladesh, and a complete genome of a B. glumae strain from the country will help clarify its origin and devise proper management systems to continue sustainable rice production. DATA DESCRIPTION: We report the first complete genome sequence of a B. glumae strain (BD_21g) isolated from symptomatic rice grains in Bangladesh (Natore District). The genome contains 2 chromosomes (1 and 2, with 3,417,499 and 3,855,283 bp, respectively) and 4 plasmids (1-4, with 123,248, 46,628, 88,744 and 53,064 bp, respectively).
Asunto(s)
Burkholderia , Genoma Bacteriano , Oryza , Enfermedades de las Plantas , Oryza/microbiología , Burkholderia/genética , Burkholderia/aislamiento & purificación , Burkholderia/patogenicidad , Bangladesh , Genoma Bacteriano/genética , Enfermedades de las Plantas/microbiología , Secuenciación Completa del GenomaRESUMEN
Melioidosis caused by Burkholderia pseudomallei is an infectious disease with a high mortality rate. In acute melioidosis, sepsis is a major cause of death among patients. Once the bacterium enters the bloodstream, immune system dysregulation ensues, leading to cytokine storms. In contrast to B. pseudomallei, a closely related but non-virulent strain B. thailandensis has rarely been reported to cause cytokine storms or death in patients. However, the mechanisms in which the virulent B. pseudomallei causes sepsis are not fully elucidated. It is well-documented that monocytes play an essential role in cytokine production in the bloodstream. The present study, therefore, determined whether there is a difference in the innate immune response to B. pseudomallei and B. thailandensis during infection of primary human monocytes and THP-1 monocytic cells by investigating pyroptosis, an inflammatory death pathway known to play a pivotal role in sepsis. Our results showed that although both bacterial species exhibited a similar ability to invade human monocytes, only B. pseudomallei can significantly increase the release of cytosolic enzyme lactate dehydrogenase (LDH) as well as the increases in caspase-1 and gasdermin D activations in both cell types. The results were consistent with the significant increase in IL-1ß and IL-18 production, key cytokines involved in pyroptosis. Interestingly, there was no significant difference in other cytokine secretion, such as IL-1RA, IL-10, IL-12p70, IL-15, IL-8, and IL-23 in cells infected by both bacterial species. Furthermore, we also demonstrated that ROS production played a crucial role in controlling pyroptosis activation during B. pseudomallei infection in primary human monocytes. These findings suggested that pyroptosis induced by B. pseudomallei in the human monocytes may contribute to the pathogenesis of sepsis in acute melioidosis patients.
Asunto(s)
Burkholderia pseudomallei , Burkholderia , Melioidosis , Monocitos , Piroptosis , Sepsis , Humanos , Burkholderia pseudomallei/inmunología , Burkholderia pseudomallei/fisiología , Monocitos/inmunología , Monocitos/microbiología , Melioidosis/microbiología , Melioidosis/inmunología , Burkholderia/patogenicidad , Sepsis/microbiología , Sepsis/inmunología , Citocinas/metabolismo , Células THP-1 , Inmunidad Innata , Células CultivadasRESUMEN
OBJECTIVES: Burkholderia dolosa is a clinically important opportunistic pathogen in inpatients. Here we characterised an extensively drug-resistant and hypervirulent B. dolosa isolate from a patient hospitalised for stroke. METHODS: Resistance to 41 antibiotics was tested with the agar disc diffusion, minimum inhibitory concentration, or broth microdilution method. The complete genome was assembled using short-reads and long-reads and the hybrid de novo assembly method. Allelic profiles obtained by multilocus sequence typing were analysed using the PubMLST database. Antibiotic-resistance and virulence genes were predicted in silico using public databases and the 'baargin' workflow. B. dolosa N149 phylogenetic relationships with all available B. dolosa strains and Burkholderia cepacia complex strains were analysed using the pangenome obtained with Roary. RESULTS: B. dolosa N149 displayed extensive resistance to 31 antibiotics and intermediate resistance to 4 antibiotics. The complete genome included three circular chromosomes (6 338 630 bp in total) and one plasmid (167 591 bp). Genotypic analysis revealed various gene clusters (acr, amr, amp, emr, ade, bla and tet) associated with resistance to 35 antibiotic classes. The major intrinsic resistance mechanisms were multidrug efflux pump alterations, inactivation and reduced permeability of targeted antibiotics. Moreover, 91 virulence genes (encoding proteins involved in adherence, formation of capsule, biofilm and colony, motility, phagocytosis inhibition, secretion systems, protease secretion, transmission and quorum sensing) were identified. B. dolosa N149 was assigned to a novel sequence type (ST2237) and formed a mono-phylogenetic clade separated from other B. dolosa strains. CONCLUSIONS: This study provided insights into the antimicrobial resistance and virulence mechanisms of B. dolosa.
Asunto(s)
Antibacterianos , Infecciones por Burkholderia , Farmacorresistencia Bacteriana Múltiple , Genoma Bacteriano , Pruebas de Sensibilidad Microbiana , Tipificación de Secuencias Multilocus , Filogenia , Accidente Cerebrovascular , Humanos , Antibacterianos/farmacología , Vietnam , Infecciones por Burkholderia/microbiología , Accidente Cerebrovascular/microbiología , Burkholderia/genética , Burkholderia/efectos de los fármacos , Burkholderia/aislamiento & purificación , Burkholderia/clasificación , Burkholderia/patogenicidad , Virulencia/genética , Factores de Virulencia/genética , Secuenciación Completa del Genoma , Pueblos del Sudeste AsiáticoRESUMEN
The genus Burkholderia contains over 80 different Gram-negative species including both plant and human pathogens, the latter of which can be classified into one of two groups: the Burkholderia pseudomallei complex (Bpc) or the Burkholderia cepacia complex (Bcc). Bpc pathogens Burkholderia pseudomallei and Burkholderia mallei are highly virulent, and both have considerable potential for use as Tier 1 bioterrorism agents; thus there is great interest in the development of novel vaccines and therapeutics for the prevention and treatment of these infections. While Bcc pathogens Burkholderia cenocepacia, Burkholderia multivorans, and Burkholderia cepacia are not considered bioterror threats, the incredible impact these infections have on the cystic fibrosis community inspires a similar demand for vaccines and therapeutics for the prevention and treatment of these infections as well. Understanding how these pathogens interact with and evade the host immune system will help uncover novel therapeutic targets within these organisms. Given the important role of the complement system in the clearance of bacterial pathogens, this arm of the immune response must be efficiently evaded for successful infection to occur. In this review, we will introduce the Burkholderia species to be discussed, followed by a summary of the complement system and known mechanisms by which pathogens interact with this critical system to evade clearance within the host. We will conclude with a review of literature relating to the interactions between the herein discussed Burkholderia species and the host complement system, with the goal of highlighting areas in this field that warrant further investigation.
Asunto(s)
Infecciones por Burkholderia , Burkholderia , Proteínas del Sistema Complemento , Evasión Inmune , Melioidosis , Burkholderia/patogenicidad , Infecciones por Burkholderia/inmunología , Burkholderia pseudomallei , Proteínas del Sistema Complemento/inmunología , Humanos , Melioidosis/inmunologíaRESUMEN
Bacteria in the Burkholderia cepacia complex (BCC) are significant pathogens for people with cystic fibrosis (CF) and are often extensively antibiotic resistant. Here, we assess the impacts of clinically observed mutations in fixL, which encodes the sensor histidine kinase FixL. FixL along with FixJ compose a two-component system that regulates multiple phenotypes. Mutations in fixL across two species, B. dolosa and B. multivorans, have shown evidence of positive selection during chronic lung infection in CF. Herein, we find that BCC carrying the conserved, ancestral fixL sequence have lower survival in macrophages and in murine pneumonia models than mutants carrying evolved fixL sequences associated with clinical decline in CF patients. In vitro phosphotransfer experiments found that one evolved FixL protein, W439S, has a reduced ability to autophosphorylate and phosphorylate FixJ, while LacZ reporter experiments demonstrate that B. dolosa carrying evolved fixL alleles has reduced fix pathway activity. Interestingly, B. dolosa carrying evolved fixL alleles was less fit in a soil assay than those strains carrying the ancestral allele, demonstrating that increased survival of these variants in macrophages and the murine lung comes at a potential expense in their environmental reservoir. Thus, modulation of the two-component system encoded by fixLJ by point mutations is one mechanism that allows BCC to adapt to the host infection environment. IMPORTANCE Infections caused by members of the Burkholderia cepacia complex (BCC) are a serious concern for patients with cystic fibrosis (CF) as these bacteria are often resistant to many antibiotics. During long-term infection of CF patients with BCC, mutations in genes encoding the FixLJ system often become prevalent, suggesting that these changes may benefit the bacteria during infection. The system encoded by fixLJ is involved in sensing oxygen and regulating many genes in response and is required for full virulence of the bacteria in a murine pneumonia model. Evolved fixL mutations seen later in infection improve bacterial persistence within macrophages and enhance infection within mice. However, these adaptations are short sighted because they reduce bacterial fitness within their natural habitat, soil.
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Burkholderia/genética , Burkholderia/patogenicidad , Evolución Molecular , Mutación Puntual , Animales , Proteínas Bacterianas/genética , Infecciones por Burkholderia/microbiología , Complejo Burkholderia cepacia , Femenino , Histidina Quinasa/genética , Humanos , Pulmón/microbiología , Macrófagos/microbiología , Ratones , Ratones Endogámicos C57BL , Fenotipo , Neumonía/microbiología , Estudios Retrospectivos , Células THP-1 , VirulenciaRESUMEN
Rice is an important source of food for more than half of the world's population. Bacterial panicle blight (BPB) is a disease of rice characterized by grain discoloration or sheath rot caused mainly by Burkholderia glumae. B. glumae synthesizes toxoflavin, an essential virulence factor that is required for symptoms of the disease. The products of the tox operons, ToxABCDE and ToxFGHI, are responsible for the synthesis and the proton motive force (PMF)-dependent secretion of toxoflavin, respectively. The DedA family is a highly conserved membrane protein family found in most bacterial genomes that likely function as membrane transporters. Our previous work has demonstrated that absence of certain DedA family members results in pleiotropic effects, impacting multiple pathways that are energized by PMF. We have demonstrated that a member of the DedA family from Burkholderia thailandensis, named DbcA, is required for the extreme polymyxin resistance observed in this organism. B. glumae encodes a homolog of DbcA with 73% amino acid identity to Burkholderia thailandensis DbcA. Here, we created and characterized a B. glumae ΔdbcA strain. In addition to polymyxin sensitivity, the B. glumae ΔdbcA strain is compromised for virulence in several BPB infection models and secretes only low amounts of toxoflavin (â¼15% of wild-type levels). Changes in membrane potential in the B. glumae ΔdbcA strain were reproduced in the wild-type strain by the addition of subinhibitory concentrations of sodium bicarbonate, previously demonstrated to cause disruption of PMF. Sodium bicarbonate inhibited B. glumae virulence in rice, suggesting a possible non-toxic chemical intervention for bacterial panicle blight. IMPORTANCE Bacterial panicle blight (BPB) is a disease of rice characterized by grain discoloration or sheath rot caused mainly by Burkholderia glumae. The DedA family is a highly conserved membrane protein family found in most bacterial genomes that likely function as membrane transporters. Here, we constructed a B. glumae mutant with a deletion in a DedA family member named dbcA and report a loss of virulence in models of BPB. Physiological analysis of the mutant shows that the proton motive force is disrupted, leading to reduction of secretion of the essential virulence factor toxoflavin. The mutant phenotypes are reproduced in the virulent wild-type strain without an effect on growth using sodium bicarbonate, a nontoxic buffer that has been reported to disrupt the PMF. The results presented here suggest that bicarbonate may be an effective antivirulence agent capable of controlling BPB without imposing an undue burden on the environment.
Asunto(s)
Burkholderia , Oryza/microbiología , Enfermedades de las Plantas/microbiología , Fuerza Protón-Motriz , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Burkholderia/efectos de los fármacos , Burkholderia/genética , Burkholderia/metabolismo , Burkholderia/patogenicidad , Carbonil Cianuro m-Clorofenil Hidrazona/farmacología , Cebollas/microbiología , Pirimidinonas/metabolismo , Bicarbonato de Sodio/farmacología , Triazinas/metabolismo , Virulencia , Factores de Virulencia/metabolismoRESUMEN
Interbacterial antagonism and communication are driving forces behind microbial community development. In many Gram-negative bacteria, contact-dependent growth inhibition (CDI) systems contribute to these microbial interactions. CDI systems deliver the toxic C-terminus of a large surface exposed protein to the cytoplasm of neighboring bacteria upon cell-contact. Termed the BcpA-CT, import of this toxic effector domain is mediated by specific, yet largely unknown receptors on the recipient cell outer and inner membranes. In this study, we demonstrated that cytoplasmic membrane proteins GltJK, components of a predicted ABC-type transporter, are required for entry of CDI system protein BcpA-2 into Burkholderia multivorans recipient cells. Consistent with current CDI models, gltJK were also required for recipient cell susceptibility to a distinct BcpA-CT that shared sequences within the predicted "translocation domain" of BcpA-2. Strikingly, this translocation domain showed low sequence identity to the analogous region of an Escherichia coli GltJK-utilizing CDI system protein. Our results demonstrated that recipient bacteria expressing E. coli gltJK were resistant to BcpA-2-mediated interbacterial antagonism, suggesting that BcpA-2 specifically recognizes Burkholderia GltJK. Using a series of chimeric proteins, the specificity determinant was mapped to Burkholderia-specific sequences at the GltK C-terminus, providing insight into BcpA transport across the recipient cell cytoplasmic membrane.
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Proteínas Bacterianas/fisiología , Burkholderia/fisiología , Proteínas de la Membrana/fisiología , Interacciones Microbianas , Adhesión Bacteriana , Fenómenos Fisiológicos Bacterianos , Biopelículas/crecimiento & desarrollo , Burkholderia/patogenicidad , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Humanos , Mutagénesis Insercional/métodos , Dominios Proteicos , Especificidad de la EspecieRESUMEN
Burkholderia infections can result in serious diseases with high mortality, such as melioidosis, and they are difficult to treat with antibiotics. Innate immunity is critical for cell-autonomous clearance of intracellular pathogens like Burkholderia by regulating programmed cell death. Inflammasome-dependent inflammatory cytokine release and cell death contribute to host protection against Burkholderia pseudomallei and Burkholderia thailandensis; however, the contribution of apoptosis and necroptosis to protection is not known. Here, we found that bone marrow-derived macrophages (BMDMs) lacking key components of pyroptosis died via apoptosis during infection. BMDMs lacking molecules required for pyroptosis, apoptosis, and necroptosis (PANoptosis), however, were significantly resistant to B. thailandensis-induced cell death until later stages of infection. Consequently, PANoptosis-deficient BMDMs failed to limit B. thailandensis-induced cell-cell fusion, which permits increased intercellular spread and replication compared to wild-type or pyroptosis-deficient BMDMs. Respiratory B. thailandensis infection resulted in higher mortality in PANoptosis-deficient mice than in pyroptosis-deficient mice, indicating that, in the absence of pyroptosis, apoptosis is essential for efficient control of infection in vivo. Together, these findings suggest both pyroptosis and apoptosis are necessary for host-mediated control of Burkholderia infection. IMPORTANCEBurkholderia infections result in a high degree of mortality when left untreated; therefore, understanding the host immune response required to control infection is critical. In this study, we found a hierarchical cell death program utilized by infected cells to disrupt the intracellular niche of Burkholderia thailandensis, which limits bacterial intercellular spread, host cell-cell fusion, and bacterial replication. In macrophages, combined loss of key PANoptosis components results in extensive B. thailandensis infection-induced cell-cell fusion, bacterial replication, and increased cell death at later stages of infection compared with both wild-type (WT) and pyroptosis-deficient cells. During respiratory infection, mortality was increased in PANoptosis-deficient mice compared to pyroptosis-deficient mice, identifying an essential role for multiple cell death pathways in controlling B. thailandensis infection. These findings advance our understanding of the physiological role of programmed cell death in controlling Burkholderia infection.
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Apoptosis/inmunología , Infecciones por Burkholderia/inmunología , Burkholderia/patogenicidad , Inmunidad Innata , Macrófagos/microbiología , Macrófagos/patología , Animales , Burkholderia/inmunología , Caspasas/clasificación , Caspasas/genética , Caspasas/inmunología , Femenino , Masculino , Ratones , Necroptosis/inmunología , Piroptosis/inmunologíaRESUMEN
Burkholderia cepacia complex bacteria comprise opportunistic pathogens causing chronic respiratory infections in cystic fibrosis (CF) patients. These microorganisms produce an exopolysaccharide named cepacian, which is considered a virulence determinant. To find genes implicated in the regulation of cepacian biosynthesis, we characterized an evolved nonmucoid variant (17616nmv) derived from the ancestor, Burkholderia multivorans ATCC 17616, after prolonged stationary phase. Lack of cepacian biosynthesis was correlated with downregulation of the expression of bce genes implicated in its biosynthesis. Furthermore, genome sequencing of the variant identified the transposition of the mobile element IS406 upstream of the coding sequence of an hns-like gene (Bmul_0158) encoding a histone-like nucleoid structuring (H-NS) protein, a known global transcriptional repressor. This insertion sequence (IS) element upregulated the expression of Bmul_0158 by 4-fold. Transcriptome analysis identified the global effects of this mutation on gene expression, with major changes in genes implicated in motility, pilus synthesis, type VI secretion, and chromosome-associated functions. Concomitant with these differences, the nonmucoid variant displays reduced adherence to a CF lung bronchial cell line and reduced surface hydrophobicity and forms smaller cellular aggregates but has an increase in swimming and swarming motilities. Finally, analysis of the GC content of the upstream region of differentially expressed genes led to the identification of various genomic regions, possibly acquired by horizontal gene transfer, which were transcriptionally repressed by the increased expression of the Bmul_0158 gene in the 17616nmv strain. Taken together, the results revealed a significant role for this H-NS protein in the regulation of B. multivorans persistence- and virulence-associated genes. IMPORTANCE Members of the histone-like nucleoid structuring (H-NS) family of proteins, present in many bacteria, are important global regulators of gene expression. Many of the regulated genes were acquired horizontally and include pathogenicity islands and prophages, among others. Additionally, H-NS can play a structural role by bridging and compacting DNA, fulfilling a crucial role in cell physiology. Several virulence phenotypes have been frequently identified in several bacteria as dependent on H-NS activity. Here, we describe an H-NS-like protein of the opportunistic pathogen Burkholderia multivorans, a species commonly infecting the respiratory tract of cystic fibrosis patients. Our results indicate that this protein is involved in regulating virulence traits such as exopolysaccharide biosynthesis, adhesion to biotic surfaces, cellular aggregation, and motility. Furthermore, this H-NS-like protein is one out of eight orthologs present in the B. multivorans ATCC 17616 genome, posing relevant questions to be investigated on how these proteins coordinate the expression of virulence traits.
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Proteínas Bacterianas/genética , Burkholderia/genética , Burkholderia/patogenicidad , Virulencia/genética , Adhesión Bacteriana , Burkholderia/fisiología , Agregación Celular , Línea Celular , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Genoma Bacteriano , Histonas , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Fenotipo , Polisacáridos Bacterianos/biosíntesisRESUMEN
Caspase-11 sensing of intracellular lipopolysaccharide (LPS) plays critical roles during infections and sepsis. However, the key cell types that sense intracellular LPS and their contributions to the host responses at the organismal level are not completely clear. Here, we show that macrophage/monocyte-specific caspase-11 plays a dominant role in mediating the pathological manifestations of endotoxemia, including gasdermin D (GSDMD) activation, interleukin (IL)-1ß, IL-18, and damage-associated molecular pattern (DAMP) release, tissue damage, and death. Surprisingly, caspase-11 expression in CD11c+ cells and intestinal epithelial cells (IECs) plays minor detrimental roles in LPS shock. In contrast, caspase-11 expression in neutrophils is dispensable for LPS-induced lethality. Importantly, caspase-11 sensing of intracellular LPS in LyzM+ myeloid cells and MRP8+ neutrophils, but not CD11c+ cells and IECs, is necessary for bacterial clearance and host survival during intracellular bacterial infection. Thus, we reveal hierarchical cell-type-specific roles of caspase-11 that govern the host-protective and host-detrimental functions of the cytosolic LPS surveillance.
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Caspasas Iniciadoras/genética , Lipopolisacáridos/toxicidad , Macrófagos Peritoneales/inmunología , Neutrófilos/inmunología , Choque Séptico/inmunología , Bazo/inmunología , Animales , Burkholderia/crecimiento & desarrollo , Burkholderia/patogenicidad , Antígenos CD11/genética , Antígenos CD11/inmunología , Calgranulina A/genética , Calgranulina A/inmunología , Caspasas Iniciadoras/inmunología , Células Dendríticas/inmunología , Células Dendríticas/microbiología , Células Epiteliales/inmunología , Células Epiteliales/microbiología , Femenino , Regulación de la Expresión Génica , Interleucina-18/genética , Interleucina-18/inmunología , Interleucina-1beta/genética , Interleucina-1beta/inmunología , Hígado/inmunología , Hígado/microbiología , Macrófagos Peritoneales/microbiología , Masculino , Ratones , Ratones Transgénicos , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/inmunología , Monocitos/inmunología , Monocitos/microbiología , Neutrófilos/microbiología , Proteínas de Unión a Fosfato/genética , Proteínas de Unión a Fosfato/inmunología , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/inmunología , Choque Séptico/genética , Choque Séptico/microbiología , Choque Séptico/mortalidad , Transducción de Señal , Bazo/microbiología , Análisis de SupervivenciaRESUMEN
Jumbo phages have DNA genomes larger than 200 kbp in large virions composed of an icosahedral head, tail, and other adsorption structures, and they are known to be abundant biological substances in nature. In this study, phages in leaf litter compost were screened for their potential to suppress rice seedling rot disease caused by the bacterium Burkholderia glumae, and a novel phage was identified in a filtrate-enriched suspension of leaf litter compost. The phage particles consisted of a rigid tailed icosahedral head and contained a DNA genome of 227,105 bp. The phage could lyse five strains of B. glumae and six strains of Burkholderia plantarii. The phage was named jumbo Burkholderia phage FLC6. Proteomic tree analysis revealed that phage FLC6 belongs to the same clade as two jumbo Ralstonia phages, namely RSF1 and RSL2, which are members of the genus Chiangmaivirus (family: Myoviridae; order: Caudovirales). Interestingly, FLC6 could also lyse two strains of Ralstonia pseudosolanacearum, the causal agent of bacterial wilt, suggesting that FLC6 has a broad host range that may make it especially advantageous as a bio-control agent for several bacterial diseases in economically important crops. The novel jumbo phage FLC6 may enable leaf litter compost to suppress several bacterial diseases and may itself be useful for controlling plant diseases in crop cultivation.
Asunto(s)
Bacteriófagos/genética , Bacteriófagos/aislamiento & purificación , Agentes de Control Biológico/aislamiento & purificación , Burkholderia/virología , Compostaje , Hojas de la Planta/virología , Plantones/microbiología , Bacteriófagos/química , Agentes de Control Biológico/farmacología , Burkholderia/patogenicidad , Genoma Viral/genética , Especificidad del Huésped , Oryza/microbiología , Terapia de Fagos , Enfermedades de las Plantas/terapia , Hojas de la Planta/microbiología , Proteómica , Ralstonia/patogenicidad , Ralstonia/virologíaRESUMEN
The Burkholderia pseudomallei phylogenetic cluster includes B. pseudomallei, B. mallei, B. thailandensis, B. oklahomensis, B. humptydooensis and B. singularis. Regarded as the only pathogenic members of this group, B. pseudomallei and B. mallei cause the diseases melioidosis and glanders, respectively. Additionally, variant strains of B. pseudomallei and B. thailandensis exist that include the geographically restricted B. pseudomallei that express a B. mallei-like BimA protein (BPBM), and B. thailandensis that express a B. pseudomallei-like capsular polysaccharide (BTCV). To establish a PCR-based assay for the detection of pathogenic Burkholderia species or their variants, five PCR primers were designed to amplify species-specific sequences within the bimA (Burkholderia intracellular motility A) gene. Our multiplex PCR assay could distinguish pathogenic B. pseudomallei and BPBM from the non-pathogenic B. thailandensis and the BTCV strains. A second singleplex PCR successfully discriminated the BTCV from B. thailandensis. Apart from B. humptydooensis, specificity testing against other Burkholderia spp., as well as other Gram-negative and Gram-positive bacteria produced a negative result. The detection limit of the multiplex PCR in soil samples artificially spiked with known quantities of B. pseudomallei and B. thailandensis were 5 and 6 CFU/g soil, respectively. Furthermore, comparison between standard bacterial culture and the multiplex PCR to detect B. pseudomallei from 34 soil samples, collected from an endemic area of melioidosis, showed high sensitivity and specificity. This robust, sensitive, and specific PCR assay will be a useful tool for epidemiological study of B. pseudomallei and closely related members with pathogenic potential in soil.
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Burkholderia/aislamiento & purificación , Código de Barras del ADN Taxonómico/métodos , Microbiología del Suelo , Burkholderia/genética , Burkholderia/patogenicidad , Microbiota , Reacción en Cadena de la Polimerasa/métodosRESUMEN
One of the most commonly prescribed antibiotics against Burkholderia infections is co-trimoxazole, a cocktail of trimethoprim and sulfamethoxazole. Trimethoprim elicits an upregulation of the mal gene cluster, which encodes proteins involved in synthesis of the cytotoxic polyketide malleilactone; trimethoprim does so by increasing expression of the malR gene, which encodes the activator MalR. We report that B. thailandensis grown on trimethoprim exhibited increased virulence against Caenorhabditis elegans. This enhanced virulence correlated with an increase in expression of the mal gene cluster. Notably, inhibition of xanthine dehydrogenase by addition of allopurinol led to similar upregulation of malA and malR, with addition of trimethoprim or allopurinol also resulting in an equivalent intracellular accumulation of xanthine. Xanthine is a ligand for the transcription factor MftR that leads to attenuated DNA binding, and we show using chromatin immunoprecipitation that MftR binds directly to malR. Our gene expression data suggest that malR expression is repressed by both MftR and by a separate transcription factor, which also responds to a metabolite that accumulates on exposure to trimethoprim. Since allopurinol elicits a similar increase in malR/malA expression as trimethoprim, we suggest that impaired purine homeostasis plays a primary role in trimethoprim-mediated induction of malR and in turn malA.
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Proteínas Bacterianas/fisiología , Burkholderia/efectos de los fármacos , Burkholderia/fisiología , Caenorhabditis elegans/microbiología , Regulación Bacteriana de la Expresión Génica , Purinas/metabolismo , Proteínas Represoras/fisiología , Trimetoprim/farmacología , Animales , Antibacterianos/farmacología , Burkholderia/patogenicidad , Infecciones por Burkholderia/microbiología , Homeostasis , Familia de Multigenes , Sulfametoxazol/farmacología , Factores de Transcripción/metabolismo , Combinación Trimetoprim y Sulfametoxazol/farmacología , Virulencia , Xantina/metabolismoRESUMEN
Rice is often infected by bacterial panicle blight disease caused by Burkholderia glumae. Since most studies have assessed the transcriptome of the plant when it is exposed to bacteria, the gene expression of the phytopathogenic bacteria have not been well elaborated during the infection process or in the host cell. Recently, a few researches were conducted to evaluate the in vivo transcriptome of bacteria during the infective process. Most bacterial cells do not express genes involved in pathogenicity in culture medium making it difficult to investigate gene expression of bacterial cells in plant cells. Here, we sought a simulated patho-system that would allow bacterial cells to express their pathogenic genes. Thus, rice root exudates (RE) and bacterial N-acyl homoserine lactone (AHL) were used and their effects on bacterial gene expression were assessed. Transcription patterns of B. glumae virulence determinants showed that enrichment medium (LB + RE + C8-HSL) could significantly induce virulence factor genes compared with Luria Bertani (LB; control) medium. The data indicate that the artificial environment is similar to the real patho-system, and that this induced maximum relevant gene expression. In this model system, bacterial gene expression changes are traceable in the infection process. Bacterial cells exposed to either an artificial environment or LB + RE + C8-HSL behaved similarly to the natural environment in situ.
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Burkholderia/genética , Burkholderia/patogenicidad , Oryza/microbiología , Transcriptoma , Factores de Virulencia/genética , Acil-Butirolactonas , Técnicas de Cultivo , Genes Bacterianos/genética , Exudados de PlantasRESUMEN
The genus Burkholderia includes a wide range of Gram-negative bacterial species some of which are pathogenic to humans and other vertebrates. The most pathogenic species are Burkholderia mallei, Burkholderia pseudomallei, and the members of the Burkholderia cepacia complex (Bcc). B. mallei and B. pseudomallei, the cause of glanders and melioidosis, respectively, are considered potential bioweapons. The Bcc comprises a subset of Burkholderia species associated with respiratory infections in people with chronic granulomatous disease and cystic fibrosis. Antimicrobial treatment of Burkholderia infections is difficult due to the intrinsic multidrug antibiotic resistance of these bacteria; prophylactic vaccines provide an attractive alternative to counteract these infections. Although commercial vaccines against Burkholderia infections are still unavailable, substantial progress has been made over recent years in the development of vaccines against B. pseudomallei and B. mallei. This review critically discusses the current advances in vaccine development against B. mallei, B. pseudomallei, and the Bcc.
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Vacunas Bacterianas/administración & dosificación , Infecciones por Burkholderia/prevención & control , Burkholderia/inmunología , Animales , Vacunas Bacterianas/inmunología , Burkholderia/genética , Burkholderia/patogenicidad , Infecciones por Burkholderia/inmunología , Infecciones por Burkholderia/microbiología , Humanos , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/inmunología , Vacunas de ADN/administración & dosificación , Vacunas de ADN/inmunología , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/inmunologíaRESUMEN
Bacterial bloodstream infections (BSI) are a major health concern and can cause up to 40% mortality. Pseudomonas aeruginosa BSI is often of nosocomial origin and is associated with a particularly poor prognosis. The mechanism of bacterial persistence in blood is still largely unknown. Here, we analyzed the behavior of a cohort of clinical and laboratory Pseudomonas aeruginosa strains in human blood. In this specific environment, complement was the main defensive mechanism, acting either by direct bacterial lysis or by opsonophagocytosis, which required recognition by immune cells. We found highly variable survival rates for different strains in blood, whatever their origin, serotype, or the nature of their secreted toxins (ExoS, ExoU or ExlA) and despite their detection by immune cells. We identified and characterized a complement-tolerant subpopulation of bacterial cells that we named "evaders". Evaders shared some features with bacterial persisters, which tolerate antibiotic treatment. Notably, in bi-phasic killing curves, the evaders represented 0.1-0.001% of the initial bacterial load and displayed transient tolerance. However, the evaders are not dormant and require active metabolism to persist in blood. We detected the evaders for five other major human pathogens: Acinetobacter baumannii, Burkholderia multivorans, enteroaggregative Escherichia coli, Klebsiella pneumoniae, and Yersinia enterocolitica. Thus, the evaders could allow the pathogen to persist within the bloodstream, and may be the cause of fatal bacteremia or dissemination, in particular in the absence of effective antibiotic treatments.
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Infecciones Bacterianas/sangre , Infecciones Bacterianas/inmunología , Activación de Complemento/inmunología , Acinetobacter baumannii/crecimiento & desarrollo , Acinetobacter baumannii/patogenicidad , Bacteriemia/sangre , Bacteriemia/inmunología , Bacteriemia/microbiología , Bacterias , Burkholderia/crecimiento & desarrollo , Burkholderia/patogenicidad , Proteínas del Sistema Complemento/inmunología , Escherichia coli/crecimiento & desarrollo , Escherichia coli/patogenicidad , Humanos , Klebsiella pneumoniae/crecimiento & desarrollo , Klebsiella pneumoniae/patogenicidad , Pruebas de Sensibilidad Microbiana , Infecciones por Pseudomonas/sangre , Infecciones por Pseudomonas/inmunología , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/crecimiento & desarrollo , Pseudomonas aeruginosa/patogenicidad , Yersinia enterocolitica/crecimiento & desarrollo , Yersinia enterocolitica/patogenicidadRESUMEN
Melioidosis is a neglected tropical disease caused by the Gram-negative soil bacterium Burkholderia pseudomallei. Current antibiotic regimens used to treat melioidosis are prolonged and expensive, and often ineffective because of intrinsic and acquired antimicrobial resistance. Efforts to develop new treatments for melioidosis are limited by the risks associated with handling pathogenic B. pseudomallei, which restricts research to facilities with biosafety level three containment. Closely related nonpathogenic Burkholderia can be investigated under less stringent biosafety level two containment, and we hypothesized that they could be used as model organisms for developing therapies that would also be effective against B. pseudomallei. We used microbroth dilution assays to compare drug susceptibility profiles of three B. pseudomallei strains and five nonpathogenic Burkholderia strains. Burkholderia humptydooensis, Burkholderia thailandensis, and Burkholderia territorii had similar susceptibility profiles to pathogenic B. pseudomallei that support their potential as safer in vitro models for developing new melioidosis therapies.
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Antibacterianos/farmacología , Burkholderia/efectos de los fármacos , Farmacorresistencia Bacteriana/genética , Melioidosis/tratamiento farmacológico , Burkholderia/genética , Burkholderia/patogenicidad , Humanos , Especificidad de la EspecieRESUMEN
Bacteria often possess relatively flexible genome structures and adaptive genetic variants that allow survival in unfavorable growth conditions. Bacterial survival tactics in disadvantageous microenvironments include mutations that are beneficial against threats in their niche. Here, we report that the aerobic rice bacterial pathogen Burkholderia glumae BGR1 changes a specific gene for improved survival in static culture conditions. Static culture triggered formation of colony variants with deletions or point mutations in the gene bspP (BGLU_RS28885), which putatively encodes a protein that contains PDC2, PAS-9, SpoIIE, and HATPase domains. The null mutant of bspP survived longer in static culture conditions and produced a higher level of bis-(3'-5')-cyclic dimeric guanosine monophosphate than the wild type. Expression of the bacterial cellulose synthase regulator (bcsB) gene was upregulated in the mutant, consistent with the observation that the mutant formed pellicles faster than the wild type. Mature pellicle formation was observed in the bspP mutant before pellicle formation in wild-type BGR1. However, the population density of the bspP null mutant decreased substantially when grown in Luria-Bertani medium with vigorous agitation due to failure of oxalate-mediated detoxification of the alkaline environment. The bspP null mutant was less virulent and exhibited less effective colonization of rice plants than the wild type. All phenotypes caused by mutations in bspP were recovered to those of the wild type by genetic complementation. Thus, although wild-type B. glumae BGR1 prolonged viability by spontaneous mutation under static culture conditions, such genetic changes negatively affected colonization in rice plants. These results suggest that adaptive gene sacrifice of B. glumae to survive unfavorable growth conditions is not always desirable as it can adversely affect adaptability in the host.
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Adaptación Biológica/genética , Burkholderia/genética , Burkholderia/metabolismo , Burkholderia/patogenicidad , Regulación Bacteriana de la Expresión Génica/genética , Genoma Bacteriano/genética , Genómica/métodos , Mutación , Oryza/microbiología , Enfermedades de las Plantas/microbiología , Percepción de Quorum/genética , Virulencia/genéticaRESUMEN
Stink bugs of the superfamilies Coreoidea and Lygaeoidea establish gut symbioses with environmentally acquired bacteria of the genus Burkholderia sensu lato. In the genus Burkholderia, the stink bug-associated strains form a monophyletic clade, named stink bug-associated beneficial and environmental (SBE) clade (or Caballeronia). Recently, we revealed that members of the family Largidae of the superfamily Pyrrhocoroidea are associated with Burkholderia but not specifically with the SBE Burkholderia; largid bugs harbor symbionts that belong to a clade of plant-associated group of Burkholderia, called plant-associated beneficial and environmental (PBE) clade (or Paraburkholderia). To understand the genomic features of Burkholderia symbionts of stink bugs, we isolated two symbiotic Burkholderia strains from a bordered plant bug Physopellta gutta (Pyrrhocoroidea: Largidae) and determined their complete genomes. The genome sizes of the insect-associated PBE (iPBE) are 9.5 Mb and 11.2 Mb, both of which are larger than the genomes of the SBE Burkholderia symbionts. A whole-genome comparison between two iPBE symbionts and three SBE symbionts highlighted that all previously reported symbiosis factors are shared and that 282 genes are specifically conserved in the five stink bug symbionts, over one-third of which have unknown function. Among the symbiont-specific genes, about 40 genes formed a cluster in all five symbionts; this suggests a "symbiotic island" in the genome of stink bug-associated Burkholderia.
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Burkholderia/genética , Genoma Bacteriano , Hemípteros/microbiología , Filogenia , Animales , Burkholderia/patogenicidad , Evolución Molecular , Especificidad del Huésped , Intestinos/microbiología , SimbiosisRESUMEN
The pathogenesis of the rice pathogenic bacterium Burkholderia glumae is under the tight regulation of the tofI/tofR quorum-sensing (QS) system. tepR, encoding a group I bacterial enhancer-binding protein, negatively regulates the production of toxoflavin, the phytotoxin acting as a major virulence factor in B. glumae. In this study, through a transcriptomic analysis, we identified the genes that were modulated by tepR and/or the tofI/tofR QS system. More than half of the differentially expressed genes, including the genes for the biosynthesis and transport of toxoflavin, were significantly more highly expressed in the ΔtepR mutant but less expressed in the ΔtofI-tofR (tofI/tofR QS-defective) mutant. In consonance with the transcriptome data, other virulence-related functions of B. glumae, extracellular protease activity and flagellum-dependent motility, were also negatively regulated by tepR, and this negative regulatory function of tepR was dependent on the IclR-type transcriptional regulator gene qsmR. Likewise, the ΔtepR mutant exhibited a higher level of heat tolerance in congruence with the higher transcription levels of heat shock protein genes in the mutant. Interestingly, tepR also exhibited its positive regulatory function on a previously uncharacterized type VI secretion system (denoted as BgT6SS-1). The survival of the both ΔtepR and ΔtssD (BgT6SS-1-defective) mutants was significantly compromised compared to the wild-type parent strain 336gr-1 in the presence of the natural rice-inhabiting bacterium, Pantoea sp. RSPAM1. Taken together, this study revealed pivotal regulatory roles of tepR in orchestrating multiple biological functions of B. glumae, including pathogenesis, heat tolerance, and bacterial interspecies competition.