Detection of bacterial virulence genes by subtractive hybridization: identification of capsular polysaccharide of Burkholderia pseudomallei as a major virulence determinant

Burkholderia pseudomallei, the etiologic agent of melioidosis, is responsible for a broad spectrum of illnesses in humans and animals particularly in Southeast Asia and northern Australia, where it is endemic. Burkholderia thailandensis is a nonpathogenic environmental organism closely related to B. pseudomallei. Subtractive hybridization was carried out between these two species to identify genes encoding virulence determinants in B. pseudomallei. Screening of the subtraction library revealed A-T-rich DNA sequences unique to B. pseudomallei, suggesting they may have been acquired by horizontal transfer. One of the subtraction clones, pDD1015, encoded a protein with homology to a glycosyltransferase from Pseudomonas aeruginosa. This gene was insertionally inactivated in wild-type B. pseudomallei to create SR1015. It was determined by enzyme-linked immunosorbent assay and immunoelectron microscopy that the inactivated gene was involved in the production of a major surface polysaccharide. The 50% lethal dose (LD(50)) for wild-type B. pseudomallei is <10 CFU; the LD(50) for SR1015 was determined to be 3.5 x 10(5) CFU, similar to that of B. thailandensis (6.8 x 10(5) CFU). DNA sequencing of the region flanking the glycosyltransferase gene revealed open reading frames similar to capsular polysaccharide genes in Haemophilus influenzae, Escherichia coli, and Neisseria meningitidis. In addition, DNA from Burkholderia mallei and Burkholderia stabilis hybridized to a glycosyltransferase fragment probe, and a capsular structure was identified on the surface of B. stabilis via immunoelectron microscopy. Thus, the combination of PCR-based subtractive hybridization, insertional inactivation, and animal virulence studies has facilitated the identification of an important virulence determinant in B. pseudomallei.     

Virulence of Burkholderia pseudomallei does not correlate with biofilm formation

Burkholderia pseudomallei is the causative agent of melioidosis but currently the pathogenesis of the disease is still poorly understood. One of the virulent factors of gram-negative bacteria is the ability to produce biofilm to evade host defense. As B. pseudomallei has also been reported to develop the biofilm [1], in the present study, we therefore, quantified the biofilm formation in 50 strains of B. pseudomallei and compared with 50 strains of its avirulent counterpart Burkholderia thailandensis using a modified microtiter-plate test. The results showed that the quantity of biofilm produced by B. pseudomallei was statistically higher (P< 0.01) than that of B. thailandensis (means and SEs of the corrected OD630 were 2.17+/-0.29 and 0.59+/-0.05, respectively). Transmission electron micrographs of the B. pseudomallei strain with high biofilm formation exhibited microcolonies of bacterial cells surrounded by dense extracellular slime matrix comparing with only trace quantity in the low biofilm-producing strain or the biofilm mutants generated by Tn5-OT182 mutagenesis. However, no correlation could be observed between the biofilm formation and virulence, judging from the LD50 values in BALB/c mice. The data obtained with these naturally occurring Burkholderia species and the biofilm mutants are incompatible with the possibility that the biofilm plays a role in the pathogenesis of B. pseudomallei infection.     

Comparative study of interleukin-1beta expression by peripheral blood mononuclear cells and purified monocytes experimentally infected with Burkholderia pseudomallei and Burkholderia thailandensis

Burkholderia pseudomallei is a causative agent of melioidosis. The present study investigated IL-1beta mRNA and protein expression by peripheral blood mononuclear cells and purified monocytes (n = 10) in response to infection with B. pseudomallei and B. thailandensis. Similarly increased IL-1beta mRNA and protein expression was found in both PBMC and purified monocytes stimulated with B. pseudomallei and B. thailandensis. Thus, this study suggests that IL-1beta response does not differ between infections with B. pseudomallei and its non-virulent counterpart and other mechanisms may be involved in their distinct virulence in causing the disease.     

Virulent Burkholderia pseudomallei is more efficient than avirulent Burkholderia thailandensis in invasion of and adherence to cultured human epithelial cells

Burkholderia pseudomallei, a causative agent of melioidosis, is a facultative intracellular gram-negative bacillus that is closely related to its avirulent counterpart, Burkholderia thailandensis. However, pathogenic mechanisms and virulence factors of B. pseudomallei remain elusive. In the present study, we compared the invasiveness, adherence, and replication of B. pseudomallei and B. thailandensis in human respiratory epithelial cells A549. Invasion was determined after 4 h of coculturing using antibiotic protection assay. Adherence was demonstrated by coculturing the cells with fluorescein-labeled bacteria for 1 h and the number of positive cells was analyzed by flow cytometry. The results obtained with this in vitro study demonstrated that compared with its avirulent counterpart, B. pseudomallei is significantly more efficient (P<0.01) in invasion, adherence and inducing cellular damage, as represented by plaque formation.     

Contribution of gene loss to the pathogenic evolution of Burkholderia pseudomallei and Burkholderia mallei

Burkholderia pseudomallei is the causative agent of melioidosis. Burkholderia thailandensis is a closely related species that can readily utilize l-arabinose as a sole carbon source, whereas B. pseudomallei cannot. We used Tn5-OT182 mutagenesis to isolate an arabinose-negative mutant of B. thailandensis. Sequence analysis of regions flanking the transposon insertion revealed the presence of an arabinose assimilation operon consisting of nine genes. Analysis of the B. pseudomallei chromosome showed a deletion of the operon from this organism. This deletion was detected in all B. pseudomallei and Burkholderia mallei strains investigated. We cloned the B. thailandensis E264 arabinose assimilation operon and introduced the entire operon into the chromosome of B. pseudomallei 406e via homologous recombination. The resultant strain, B. pseudomallei SZ5028, was able to utilize l-arabinose as a sole carbon source. Strain SZ5028 had a significantly higher 50% lethal dose for Syrian hamsters compared to the parent strain 406e. Microarray analysis revealed that a number of genes in a type III secretion system were down-regulated in strain SZ5028 when cells were grown in l-arabinose, suggesting a regulatory role for l-arabinose or a metabolite of l-arabinose. These results suggest that the ability to metabolize l-arabinose reduces the virulence of B. pseudomallei and that the genes encoding arabinose assimilation may be considered antivirulence genes. The increase in virulence associated with the loss of these genes may have provided a selective advantage for B. pseudomallei as these organisms adapted to survival in animal hosts.     

Use of antigens derived from Burkholderia pseudomallei, B. thailandensis, and B. cepacia in the indirect hemagglutination assay for melioidosis

The serological diagnosis of melioidosis is carried out using the indirect hemagglutination assay. We looked at the reactivity of sera from culture-proven cases of melioidosis from north Queensland against antigens derived from Burkholderia pseudomallei, B. thailandensis, and B. cepacia. Cross-reactivity between sera from culture-positive cases of melioidosis and B. thailandensis was demonstrated.     

Pneumonia and septicemia caused by Burkholderia thailandensis in the United States

Burkholderia thailandensis is closely related to Burkholderia pseudomallei, the causative agent of melioidosis. It is generally considered avirulent and previously has been reported to occur only in Southeast Asia. We report the first case of pneumonia and septicemia caused by B. thailandensis in the United States.     

Cellular fatty acid profile distinguishes Burkholderia pseudomallei from avirulent Burkholderia thailandensis

Burkholderia pseudomallei, the cause of melioidosis, can be distinguished from the closely related but nonpathogenic Burkholderia thailandensis by gas chromatography (GC) analysis of fatty acid derivatives. A 2-hydroxymyristic acid derivative (14:0 2OH) was present in 95% of B. pseudomallei isolates and no B. thailandensis isolates. GC mass spectrophotometry confirmed that 2-hydroxymyristic acid was present in B. pseudomallei. GC-fatty acid methyl ester analysis may be useful in distinguishing these two closely related species.     

Multilocus sequence typing and evolutionary relationships among the causative agents of melioidosis and glanders,Burkholderia pseudomallei and Burkholderia mallei

A collection of 147 isolates of Burkholderia pseudomallei, B. mallei, and B. thailandensis was characterized by multilocus sequence typing (MLST). The 128 isolates of B. pseudomallei, the causative agent of melioidosis, were obtained from diverse geographic locations, from humans and animals with disease, and from the environment and were resolved into 71 sequence types. The utility of the MLST scheme for epidemiological investigations was established by analyzing isolates from captive marine mammals and birds and from humans in Hong Kong with melioidosis. MLST gave a level of resolution similar to that given by pulsed-field gel electrophoresis and identified the same three clones causing disease in animals, each of which was also associated with disease in humans. The average divergence between the alleles of B. thailandensis and B. pseudomallei was 3.2%, and there was no sharing of alleles between these species. Trees constructed from differences in the allelic profiles of the isolates and from the concatenated sequences of the seven loci showed that the B. pseudomallei isolates formed a cluster of closely related lineages that were fully resolved from the cluster of B. thailandensis isolates, confirming their separate species status. However, isolates of B. mallei, the causative agent of glanders, recovered from three continents over a 30-year period had identical allelic profiles, and the B. mallei isolates clustered within the B. pseudomallei group of isolates. Alleles at six of the seven loci in B. mallei were also present within B. pseudomallei isolates, and B. mallei is a clone of B. pseudomallei that, on population genetics grounds, should not be given separate species status.     

Patterns of large-scale genomic variation in virulent and avirulent Burkholderia species

The human diseases melioidosis and glanders are caused by the bacteria Burkholderia pseudomallei and B. mallei respectively, and both species are regarded as potential biowarfare agents. We used B. pseudomallei DNA microarrays to compare the genomes of several clinical and environmental isolates of B. pseudomallei, B. mallei, and B. thailandensis, a closely related but avirulent species. Open reading frames (ORFs) deleted between the three species were associated with diverse cellular functions, including nitrogen and iron metabolism, quorum sensing, and polysaccharide production. Deleted ORFs in B. mallei exhibited significant genomic clustering, whereas deletions in B. thailandensis were more uniformly dispersed, suggesting that B. mallei and B. thailandensis may have diverged from B. pseudomallei and each other via distinct mechanisms. The genomes of independent B. pseudomallei isolates were highly conserved with a large-scale variance of less than 3% between isolates, and at least three distinct molecular subtypes could be defined. An analysis of subtype-specific genomic regions suggests that DNA loss has played an important role in the evolutionary radiation of B. pseudomallei in the natural environment. Our results raise several hypotheses concerning the possible mechanisms underlying the diverse biological properties exhibited by members of the Burkholderia family.     

Comparison of diagnostic laboratory methods for identification of Burkholderia pseudomallei

Limited experience and a lack of validated diagnostic reagents make Burkholderia pseudomallei, the cause of melioidosis, difficult to recognize in the diagnostic microbiology laboratory. We compared three methods of confirming the identity of presumptive B. pseudomallei strains using a collection of Burkholderia species drawn from diverse geographic, clinical, and environmental sources. The 95 isolates studied included 71 B. pseudomallei and 3 B. thailandensis isolates. The API 20NE method identified only 37% of the B. pseudomallei isolates. The agglutinating antibody test identified 82% at first the attempt and 90% including results of a repeat test with previously negative isolates. Gas-liquid chromatography analysis of bacterial fatty acid methyl esters (GLC-FAME) identified 98% of the B. pseudomallei isolates. The agglutination test produced four false positive results, one B. cepacia, one B. multivorans, and two B. thailandensis. API produced three false positive results, one positive B. cepacia and two positive B. thailandensis. GLC-FAME analysis was positive for one B. cepacia isolate. On the basis of these results, the most robust B. pseudomallei discovery pathway combines the previously recommended isolate screening tests (Gram stain, oxidase test, gentamicin and polymyxin susceptibility) with monoclonal antibody agglutination on primary culture, followed by a repeat after 24 h incubation on agglutination-negative isolates and GLC-FAME analysis. Incorporation of PCR-based identification within this schema may improve percentages of recognition further but requires more detailed evaluation.     

Characterization of the Burkholderia pseudomallei K96243 capsular polysaccharide I coding region

Burkholderia pseudomallei is the causative agent of melioidosis, a disease endemic to regions of Southeast Asia and Northern Australia. Both humans and a range of other animal species are susceptible to melioidosis, and the production of a group 3 polysaccharide capsule in B. pseudomallei is essential for virulence. B. pseudomallei capsular polysaccharide (CPS) I comprises unbranched manno-heptopyranose residues and is encoded by a 34.5-kb locus on chromosome 1. Despite the importance of this locus, the role of all of the genes within this region is unclear. We inactivated 18 of these genes and analyzed their phenotype using Western blotting and immunofluorescence staining. Furthermore, by combining this approach with bioinformatic analysis, we were able to develop a model for CPS I biosynthesis and export. We report that inactivating gmhA, wcbJ, and wcbN in B. pseudomallei K96243 retains the immunogenic integrity of the polysaccharide despite causing attenuation in the BALB/c murine infection model. Mice immunized with the B. pseudomallei K96243 mutants lacking a functional copy of either gmhA or wcbJ were afforded significant levels of protection against a wild-type B. pseudomallei K96243 challenge.     

Identification of a Burkholderia mallei polysaccharide gene cluster by subtractive hybridization and demonstration that the encoded capsule is an essential virulence determinant

Little is known about the virulence factors of Burkholderia mallei, the etiologic agent of glanders. We employed subtractive hybridization to identify genetic determinants present in B. mallei but not in Burkholderia thailandensis, a non-pathogenic soil microbe. Three subtractive hybridization products were mapped to a genetic locus encoding proteins involved in the biosynthesis, export and translocation of a capsular polysaccharide. We identified an insertion sequence (IS 407 A) at one end of the capsule gene cluster and demonstrated that it was functional in B. mallei. Mutations were introduced in the B. mallei capsular gene cluster and the corresponding mutants were examined for their reactivity with antibodies raised against Burkholderia pseudomallei surface polysaccharides by immunoblotting and ELISA. Immunogold electron microscopy demonstrated the presence of a capsule on the surface of B. mallei ATCC 23344 (parental strain) but not on B. mallei DD3008 (capsule mutant) or B. thailandensis. Surprisingly, B. thailandensis also harboured a portion of the capsule gene cluster. ATCC 23344 was highly virulent in hamsters and mice, but DD3008 was avirulent in both animal models. The results presented here demonstrate that the capsular polysaccharide of B. mallei is required for production of disease in two animal models of glanders infection and is a major virulence factor.     

Identification and characterization of a two-component regulatory system involved in invasion of eukaryotic cells and heavy-metal resistance in Burkholderia pseudomallei.

Burkholderia pseudomallei is the causative agent of melioidosis, a disease increasingly recognized as an important cause of morbidity and mortality in many regions of the world. B. pseudomallei is a facultative intracellular pathogen capable of invading eukaryotic cells. We used Tn5-OT182 mutagenesis to generate mutants deficient in the ability to invade a human type II pneumocyte cell line (A549 cells). One of these mutants, AJ1D8, exhibited approximately 10% of the ability of the parental strain, 1026b, to invade A549 cells. There was no difference in the abilities of 1026b and AJ1D8 to resist killing by RAW macrophages or the human defensin HNP-1. The nucleotide sequence flanking the Tn5-OT182 integration in AJ1D8 was determined, and two open reading frames were identified. The predicted proteins shared considerable homology with two-component regulatory systems involved in the regulation of heavy-metal resistance in other organisms. AJ1D8 was 16-fold more sensitive to Cd2+ and twofold more sensitive to Zn2+ than was 1026b but was not sensitive to any of the other heavy metals examined. The B. pseudomallei two-component regulatory system, termed irlRS, complemented the invasion-deficient and heavy-metal-sensitive phenotype of AJ1D8 in trans. There was no significant difference between the virulence of AJ1D8 and that of 1026b in infant diabetic rats and Syrian hamsters, suggesting that the irlRS locus is probably not a virulence determinant in these animal models of acute B. pseudomallei infection.     

Identification and discrimination of Burkholderia pseudomallei, B. mallei, and B. thailandensis by real-time PCR targeting type III secretion system genes

Burkholderia pseudomallei and B. mallei are two highly pathogenic bacteria, responsible for melioidosis and glanders, respectively. The two are closely related and can also be mistaken for B. thailandensis, a nonpathogenic species. To improve their differential identification, we describe a hydrolysis probe-based real-time PCR method using the uneven distribution of type III secretion system genes among these three species.     

Development and evaluation of a real-time PCR assay targeting the type III secretion system of Burkholderia pseudomallei

Here we report on the development of a discriminatory real-time assay for the rapid identification of Burkholderia pseudomallei isolates and the evaluation of this assay for sensitivity against related species and detection in spiked human blood samples. The assay targets a 115-base-pair region within orf2 of the B. pseudomallei type III secretion system gene cluster and distinguishes B. pseudomallei from other microbial species. Assay performance was evaluated with 224 geographically, temporally, and clinically diverse B. pseudomallei isolates from the Centers for Disease Control and Prevention strain collection. This represents the first real-time PCR for rapid and sensitive identification of B. pseudomallei that has been tested for cross-reactivity with 23 Burkholderia mallei, 5 Burkholderia thailandensis, and 35 Burkholderia and 76 non-Burkholderia organisms which have historically presented diagnostic challenges. The assay performed with 100% specificity. The limit of detection was found to be 76 femtograms of DNA (equivalent to 5.2 x 10(3) genome equivalents per ml) in a single PCR. In spiked human blood, the assay could detect as few as 8.4 x 10(3) CFU per ml. This rapid assay is a valuable tool for identification of B. pseudomallei and may improve diagnosis in regions endemic for melioidosis.     

Intracellular survival of Burkholderia pseudomallei.

Burkholderia pseudomallei is the causative agent of melioidosis, a disease being increasingly recognized as an important cause of morbidity and mortality in many regions of the world. Several features of melioidosis suggest that B. pseudomallei is a facultative intracellular pathogen. This study was designed to assess the ability of B. pseudomallei to invade and survive in eukaryotic cells. We have shown that B. pseudomallei has the capacity to invade cultured cell lines, including HeLa, CHO, A549, and Vero cells. We have demonstrated intracellular survival of B. pseudomallei in professional phagocytic cells, including rat alveolar macrophages. B pseudomallei was localized inside vacuoles in human monocyte-like U937 cells, a histiocytic lymphoma cell line with phagocytic properties. Additionally, electron microscopic visualization of B. pseudomallei-infected HeLa cells and polymorphonuclear leukocytes confirmed the presence of intracellular bacteria within membrane-bound vacuoles. B. pseudomallei was found to be resistant to the cationic peptide protamine and to purified human defensin HNP-1.     

Cloning and analysis of genomic differences unique to Burkholderia pseudomallei by comparison with B. thailandensis

Melioidosis is an infectious disease caused by Burkholderia pseudomallei. Genomic subtractive hybridisation was performed with the closely related avirulent species B. thailandensis to identify virulence genes of B. pseudomallei. The subtractive hybridisation products were highly specific for B. pseudomallei. Sequence analysis revealed a number of putative virulence factors, as well as apparently novel sequences of unknown function. The subtracted library contained DNA regions of relatively low G + C mol% content, which were distributed throughout the B. pseudomallei genome. The distribution of subtracted sequences amongst a collection of 22 B. pseudomallei isolates was found to be variable, with the exception of three strains which almost universally lacked the subtracted sequences. These three strains also differed in that they were highly haemolytic, indicating a possible separate virotype.     

Use of 16S rRNA gene sequencing for rapid identification and differentiation of Burkholderia pseudomallei and B. mallei

Burkholderia pseudomallei and B. mallei, the causative agents of melioidosis and glanders, respectively, are designated category B biothreat agents. Current methods for identifying these organisms rely on their phenotypic characteristics and an extensive set of biochemical reactions. We evaluated the use of 16S rRNA gene sequencing to rapidly identify these two species and differentiate them from each other as well as from closely related species and genera such as Pandoraea spp., Ralstonia spp., Burkholderia gladioli, Burkholderia cepacia, Burkholderia thailandensis, and Pseudomonas aeruginosa. We sequenced the 1.5-kb 16S rRNA gene of 56 B. pseudomallei and 23 B. mallei isolates selected to represent a wide range of temporal, geographic, and origin diversity. Among all 79 isolates, a total of 11 16S types were found based on eight positions of difference. Nine 16S types were identified in B. pseudomallei isolates based on six positions of difference, with differences ranging from 0.5 to 1.5 bp. Twenty-two of 23 B. mallei isolates showed 16S rRNA gene sequence identity and were designated 16S type 10, whereas the remaining isolate was designated type 11. This report provides a basis for rapidly identifying and differentiating B. pseudomallei and B. mallei by molecular methods.     

A type IV pilin, PilA, Contributes To Adherence of Burkholderia pseudomallei and virulence in vivo

The Burkholderia pseudomallei K96243 genome contains multiple type IV pilin-associated loci, including one encoding a putative pilus structural protein (pilA). A pilA deletion mutant has reduced adherence to human epithelial cells and is less virulent in the nematode model of virulence and the murine model of melioidosis, suggesting a role for type IV pili in B. pseudomallei virulence.