Invasion of spores of the arbuscular mycorrhizal fungus Gigaspora decipiens by Burkholderia spp

Burkholderia species are bacterial soil inhabitants that are capable of interacting with a variety of eukaryotes, in some cases occupying intracellular habitats. Pathogenic and nonpathogenic Burkholderia spp., including B. vietnamiensis, B. cepacia, and B. pseudomallei, were grown on germinating spores of the arbuscular mycorrhizal fungus Gigaspora decipiens. Spore lysis assays revealed that all Burkholderia spp. tested were able to colonize the interior of G. decipiens spores. Amplification of specific DNA sequences and transmission electron microscopy confirmed the intracellular presence of B. vietnamiensis. Twelve percent of all spores were invaded by B. vietnamiensis, with an average of 1.5 x 10(6) CFU recovered from individual infected spores. Of those spores inoculated with B. pseudomallei, 7% were invaded, with an average of 5.5 x 10(5) CFU recovered from individual infected spores. Scanning electron and fluorescence microscopy provided insights into the morphology of surfaces of spores and hyphae of G. decipiens and the attachment of bacteria. Burkholderia spp. colonized both hyphae and spores, attaching to surfaces in either an end-on or side-on fashion. Adherence of Burkholderia spp. to eukaryotic surfaces also involved the formation of numerous fibrillar structures.     

Stimulation of autophagy suppresses the intracellular survival of Burkholderia pseudomallei in mammalian cell lines

Burkholderia pseudomallei is the causative agent of melioidosis, a tropical infection of humans and other animals. The bacterium is an intracellular pathogen that can escape from endosomes into the host cytoplasm, where it replicates and infects adjacent cells. We investigated the role played by autophagy in the intracellular survival of B. pseudomallei in phagocytic and non-phagocytic cell lines. Autophagy was induced in response to B. pseudomallei invasion of murine macrophage (RAW 264.7) cells and a proportion of the bacteria co-localized with the autophagy effector protein LC3, a marker for autophagosome formation. Pharmacological stimulation of autophagy in RAW 264.7 and murine embryonic fibroblast (MEF) cell lines resulted in increased co-localization of B. pseudomallei with LC3 while basal levels of co-localization could be abrogated using inhibitors of the autophagic pathway. Furthermore, induction of autophagy decreased the intracellular survival of B. pseudomallei in these cell lines, but bacterial survival was not affected in MEF cell lines deficient in autophagy. Treatment of infected macrophages with chloramphenicol increased the proportion of bacteria within autophagosomes indicating that autophagic evasion is an active process relying on bacterial protein synthesis. Consistent with this hypothesis, we identified a B. pseudomallei type III secreted protein, BopA, which plays a role in mediating bacterial evasion of autophagy. We conclude that the autophagic pathway is a component of the innate defense system against invading B. pseudomallei, but which the bacteria can actively evade. However, when autophagy is pharmacologically induced using rapamycin, bacteria are actively sequestered in autophagosomes, ultimately decreasing their survival.     

Nitric oxide-dependent killing of aerobic, anaerobic and persistent Burkholderia pseudomallei

Burkholderia pseudomallei infections are fastidious to treat with conventional antibiotic therapy, often involving a combination of drugs and long-term regimes. Bacterial genetic determinants contribute to the resistance of B. pseudomallei to many classes of antibiotics. In addition, anaerobiosis and hypoxia in abscesses typical of melioidosis select for persistent populations of B. pseudomallei refractory to a broad spectrum of antibacterials. We tested the susceptibility of B. pseudomallei to the drugs hydroxyurea, spermine NONOate and DETA NONOate that release nitric oxide (NO). Our investigations indicate that B. pseudomallei are killed by NO in a concentration and time-dependent fashion. The cytoxicity of this diatomic radical against B. pseudomallei depends on both the culture medium and growth phase of the bacteria. Rapidly growing, but not stationary phase, B. pseudomallei are readily killed upon exposure to the NO donor spermine NONOate. NO also has excellent antimicrobial activity against anaerobic B. pseudomallei. In addition, persistent bacteria highly resistant to most conventional antibiotics are remarkably susceptible to NO. Sublethal concentrations of NO inhibited the enzymatic activity of [4Fe-4S]-cofactored aconitase of aerobic and anaerobic B. pseudomallei. The strong anti-B. pseudomallei activity of NO described herein merits further studies on the application of NO-based antibiotics for the treatment of melioidosis.     

Burkholderia pseudomallei interferes with inducible nitric oxide synthase (iNOS) production: a possible mechanism of evading macrophage killing

Burkholderia pseudomallei is a causative agent of melioidosis, a life threatening disease which affects humans and animals in tropical and subtropical areas. This bacterium is known to survive and multiply inside cells such as macrophages. The mechanism of host defense against this bacterium is still unknown. In this study, we demonstrated that B. pseudomallei exhibited unique macrophage activation activity compared with Escherichia coli and Salmonella typhi. The mouse macrophage cell line (RAW 264.7) infected with B. pseudomallei at MOI of 0.1:1, 1:1 and 10:1 did not express a detectable level of inducible nitric oxide synthase (iNOS). Moreover, the B. pseudomallei infected cells released TNF-alpha only when they were infected with high MOI (10:1). Unlike the cells infected with B. pseudomallei, the cells infected with E. coli, and S. typhi expressed iNOS even at MOI of 0.1:1. These infected cells also released a significantly higher level of TNF-alpha at the low MOI ratio. The cells that were preactivated with IFN-gamma prior to being infected with B. pseudomallei exhibited an enhanced production of iNOS and TNF-alpha release. The increased macrophage activation activity in the presence of IFN-gamma also correlated with the restriction of the intracellular bacteria survival. Moreover, IFN-gamma also prevented cell fusion and multinucleated cell formation induced by B. pseudomallei, a phenomenon recently described by our group. Altogether, these results indicate that internalization of B. pseudomallei failed to trigger substantial macrophage activation, a phenomenon which could prolong their survival inside the phagocytic cells and facilitate a direct cell to cell spreading of B. pseudomallei to neighboring cells.     

Comparative genomics and an insect model rapidly identify novel virulence genes of Burkholderia mallei

Burkholderia pseudomallei and its host-adapted deletion clone Burkholderia mallei cause the potentially fatal human diseases melioidosis and glanders, respectively. The antibiotic resistance profile and ability to infect via aerosol of these organisms and the absence of protective vaccines have led to their classification as major biothreats and select agents. Although documented infections by these bacteria date back over 100 years, relatively little is known about their virulence and pathogenicity mechanisms. We used in silico genomic subtraction to generate their virulome, a set of 650 putative virulence-related genes shared by B. pseudomallei and B. mallei but not present in five closely related nonpathogenic Burkholderia species. Although most of these genes are clustered in putative operons, the number of targets for mutant construction and verification of reduced virulence in animal models is formidable. Therefore, Galleria mellonella (wax moth) larvae were evaluated as a surrogate host; we found that B. pseudomallei and B. mallei, but not other phylogenetically related bacteria, were highly pathogenic for this insect. More importantly, four previously characterized B. mallei mutants with reduced virulence in hamsters or mice had similarly reduced virulence in G. mellonella larvae. Site-specific inactivation of selected genes in the computationally derived virulome identified three new potential virulence genes, each of which was required for rapid and efficient killing of larvae. Thus, this approach may provide a means to quickly identify high-probability virulence genes in B. pseudomallei, B. mallei, and other pathogens.     

Out of the ground: aerial and exotic habitats of the melioidosis bacterium Burkholderia pseudomallei in grasses in Australia

Melioidosis is an emerging infectious disease of humans and animals in the tropics caused by the soil bacterium Burkholderia pseudomallei. Despite high fatality rates, the ecology of B.pseudomallei remains unclear. We used a combination of field and laboratory studies to investigate B.pseudomallei colonization of native and exotic grasses in northern Australia. Multivariable and spatial analyses were performed to determine significant predictors for B.pseudomallei occurrence in plants and soil collected longitudinally from field sites. In plant inoculation experiments, the impact of B.pseudomallei upon these grasses was studied and the bacterial load semi-quantified. Fluorescence in situ hybridization and confocal laser scanning microscopy were performed to localize the bacteria in plants. Burkholderia pseudomallei was found to inhabit not only the rhizosphere and roots but also aerial parts of specific grasses. This raises questions about the potential spread of B.pseudomallei by grazing animals whose droppings were found to be positive for these bacteria. In particular, B.pseudomallei readily colonized exotic grasses introduced to Australia for pasture. The ongoing spread of these introduced grasses creates new habitats suitable for B.pseudomallei survival and may be an important factor in the evolving epidemiology of melioidosis seen both in northern Australia and elsewhere globally.     

Development of a diagnostic system for Burkholderia pseudomallei infections

Monoclonal antibodies were generated against whole cell lysate of Burkholderia pseudomallei. Two out of 6 monoclonal antibodies were found specific and exhibited high affinity against B. pseudomallei, one of which, was utilized to develop sandwich ELISA for detection of specific B. pseudomallei antigen. Immunoassays were found to be specific as no reaction was observed with closely related Burkholderia and Pseudomonas species. Blood samples from experimentally infected mice were found positive for isolation till 4 days post infection (DPI) and ELISA till 10 DPI. One out of 40 sick animal serum samples tested in Thailand was found positive by sandwich ELISA that was earlier confirmed by isolation of B. pseudomallei. The results indicate the potentiality of the assay for its applicability in specific diagnosis of septicaemic melioidosis.     

The use of nanoscale visible light-responsive photocatalyst TiO2-Pt for the elimination of soil-borne pathogens

Exposure to the soil-borne pathogens Burkholderia pseudomallei and Burkholderia cenocepacia can lead to severe infections and even mortality. These pathogens exhibit a high resistance to antibiotic treatments. In addition, no licensed vaccine is currently available. A nanoscale platinum-containing titania photocatalyst (TiO(2)-Pt) has been shown to have a superior visible light-responsive photocatalytic ability to degrade chemical contaminants like nitrogen oxides. The antibacterial activity of the catalyst and its potential use in soil pathogen control were evaluated. Using the plating method, we found that TiO(2)-Pt exerts superior antibacterial performance against Escherichia coli compared to other commercially available and laboratory prepared ultraviolet/visible light-responsive titania photocatalysts. TiO(2)-Pt-mediated photocatalysis also affectively eliminates the soil-borne bacteria B. pseudomallei and B. cenocepacia. An air pouch infection mouse model further revealed that TiO(2)-Pt-mediated photocatalysis could reduce the pathogenicity of both strains of bacteria. Unexpectedly, water containing up to 10% w/v dissolved soil particles did not reduce the antibacterial potency of TiO(2)-Pt, suggesting that the TiO(2)-Pt photocatalyst is suitable for use in soil-contaminated environments. The TiO(2)-Pt photocatalyst exerted superior antibacterial activity against a broad spectrum of human pathogens, including B. pseudomallei and B. cenocepacia. Soil particles (<10% w/v) did not significantly reduce the antibacterial activity of TiO(2)-Pt in water. These findings suggest that the TiO(2)-Pt photocatalyst may have potential applications in the development of bactericides for soil-borne pathogens.     

Detection and differentiation of Burkholderia pseudomallei, Burkholderia mallei and Burkholderia thailandensis by multiplex PCR

Burkholderia pseudomallei, a Gram-negative bacterium that causes melioidosis may be differentiated from closely related species of Burkholderia mallei that causes glanders and non-pathogenic species of Burkholderia thailandensis by multiplex PCR. The multiplex PCR consists of primers that flank a 10-bp repetitive element in B. pseudomallei and B. mallei amplifying PCR fragment of varying sizes between 400-700 bp, a unique sequence in B. thailandensis amplifying a PCR fragment of 308 bp and the metalloprotease gene amplifying a PCR fragment of 245 bp in B. pseudomallei and B. thailandensis. The multiplex PCR not only can differentiate the three Burkholderia species but can also be used for epidemiological typing of B. pseudomallei and B. mallei strains.     

Genomic diversity of Burkholderia pseudomallei clinical isolates: subtractive hybridization reveals a Burkholderia mallei-specific prophage in B. pseudomallei 1026b

Burkholderia pseudomallei is the etiologic agent of the disease melioidosis and is a category B biological threat agent. The genomic sequence of B. pseudomallei K96243 was recently determined, but little is known about the overall genetic diversity of this species. Suppression subtractive hybridization was employed to assess the genetic variability between two distinct clinical isolates of B. pseudomallei, 1026b and K96243. Numerous mobile genetic elements, including a temperate bacteriophage designated phi1026b, were identified among the 1026b-specific suppression subtractive hybridization products. Bacteriophage phi1026b was spontaneously produced by 1026b, and it had a restricted host range, infecting only Burkholderia mallei. It possessed a noncontractile tail, an isometric head, and a linear 54,865-bp genome. The mosaic nature of the phi1026b genome was revealed by comparison with bacteriophage phiE125, a B. mallei-specific bacteriophage produced by Burkholderia thailandensis. The phi1026b genes for DNA packaging, tail morphogenesis, host lysis, integration, and DNA replication were nearly identical to the corresponding genes in phiE125. On the other hand, phi1026b genes involved in head morphogenesis were similar to head morphogenesis genes encoded by Pseudomonas putida and Pseudomonas aeruginosa bacteriophages. Consistent with this observation, immunogold electron microscopy demonstrated that polyclonal antiserum against phiE125 reacted with the tail of phi1026b but not with the head. The results presented here suggest that B. pseudomallei strains are genetically heterogeneous and that bacteriophages are major contributors to the genomic diversity of this species. The bacteriophage characterized in this study may be a useful diagnostic tool for differentiating B. pseudomallei and B. mallei, two closely related biological threat agents.     

Actin-binding proteins from Burkholderia mallei and Burkholderia thailandensis can functionally compensate for the actin-based motility defect of a Burkholderia pseudomallei bimA mutant

Recently we identified a bacterial factor (BimA) required for actin-based motility of Burkholderia pseudomallei. Here we report that Burkholderia mallei and Burkholderia thailandensis are capable of actin-based motility in J774.2 cells and that BimA homologs of these bacteria can restore the actin-based motility defect of a B. pseudomallei bimA mutant. While the BimA homologs differ in their amino-terminal sequence, they interact directly with actin in vitro and vary in their ability to bind Arp3.     

Alanine racemase mutants of Burkholderia pseudomallei and Burkholderia mallei and use of alanine racemase as a non-antibiotic-based selectable marker

Burkholderia pseudomallei and Burkholderia mallei are category B select agents and must be studied under BSL3 containment in the United States. They are typically resistant to multiple antibiotics, and the antibiotics used to treat B. pseudomallei or B. mallei infections may not be used as selective agents with the corresponding Burkholderia species. Here, we investigated alanine racemase deficient mutants of B. pseudomallei and B. mallei for development of non-antibiotic-based genetic selection methods and for attenuation of virulence. The genome of B. pseudomallei K96243 has two annotated alanine racemase genes (bpsl2179 and bpss0711), and B. mallei ATCC 23344 has one (bma1575). Each of these genes encodes a functional enzyme that can complement the alanine racemase deficiency of Escherichia coli strain ALA1. Herein, we show that B. pseudomallei with in-frame deletions in both bpsl2179 and bpss0711, or B. mallei with an in-frame deletion in bma1575, requires exogenous D-alanine for growth. Introduction of bpsl2179 on a multicopy plasmid into alanine racemase deficient variants of either Burkholderia species eliminated the requirement for D-alanine. During log phase growth without D-alanine, the viable counts of alanine racemase deficient mutants of B. pseudomallei and B. mallei decreased within 2 hours by about 1000-fold and 10-fold, respectively, and no viable bacteria were present at 24 hours. We constructed several genetic tools with bpsl2179 as a selectable genetic marker, and we used them without any antibiotic selection to construct an in-frame ΔflgK mutant in the alanine racemase deficient variant of B. pseudomallei K96243. In murine peritoneal macrophages, wild type B. mallei ATCC 23344 was killed much more rapidly than wild type B. pseudomallei K96243. In addition, the alanine racemase deficient mutant of B. pseudomallei K96243 exhibited attenuation versus its isogenic parental strain with respect to growth and survival in murine peritoneal macrophages.     

Identification of new transposable genetic elements in Burkholderia pseudomallei using subtractive hybridisation

A subtraction library of Burkholderia pseudomallei was constructed by subtractive hybridisation of B. pseudomallei genomic DNA with Burkholderia thailandensis genomic DNA. Two clones were found to have significant sequence similarity to insertion sequences which have previously not been found in B. pseudomallei (designated ISA and ISB); and two clones showed sequence similarity to different regions of Burkholderia cepacia IS407 that has recently been detected in B. pseudomallei. The former, though possibly non-functional, represents new transposable genetic elements of B. pseudomallei. All three sequences were found to be present in multi-copy in the genomes of a number of B. pseudomallei strains and in B. thailandensis, which are the first transposable elements identified in this species.     

Antigenic relatedness between Burkholderia pseudomallei and Burkholderia mallei

Burkholderia pseudomallei and Burkholderia mallei are causative agents of distinct diseases, namely, melioidosis and glanders, respectively. The two species are very closely related, based on DNA-DNA homology, base sequence of the 16S rRNA, and phenotypic characteristics. Based on the use of polyclonal antisera, B. pseudomallei and B. mallei are also found to be antigenically closely related to one another. We previously reported the production of monoclonal antibodies (MAbs) against B. pseudomallei antigens; one group was specific for the 200-kDa exopolysaccharide present on the surface of all B. pseudomallei isolates, and the other was specific for the lipopolysaccharide (LPS) structure present on more than 95% of the B. pseudomallei tested. In the present study, we showed that the MAbs against 200-kDa antigen of B. pseudomallei cross-reacted with a component present also in some B. mallei isolates (3/6), but the positive immunoblot reaction was noted below the 200-kDa position. On the other hand, none of the six B. mallei isolates reacted with the MAb specific for B. pseudomallei LPS. It was of interest to observe that only the 3 exopolysaccharide-positive B. mallei isolates reacted with a commercial MAb against B. mallei LPS. The data presented suggest that B. mallei can be classified antigenically into two types based on their reactivities with different MAbs, i.e., the presence or absence of exopolysaccharide and the types of lipopolysaccharide. The heterogeneity of the LPS from these two closely related organisms is most likely related to the differences in its O-polysaccharide side chain.     

Effect of Chemical Factors on Natural Biocontrol of the Melioidosis Agent by AMP1-Like Bacteriophages in Agricultural Ecosystems

Microbiology - The causative agent of melioidosis, Burkholderia pseudomallei, as well as closely related nonpathogenic bacteria of the B. pseudomallei/thailandensis complex of species, are found in...     

Human single-chain Fv antibodies against Burkholderia mallei and Burkholderia pseudomallei

Much effort has been devoted to the development of mouse monoclonal antibodies that react specifically with Burkholderia mallei and Burkholderia pseudomallei for diagnostic and/or therapeutic purposes. Our present study focused on the screening of a phage-displayed nonimmune human single-chain Fv (scFv) antibody library against heat-killed B. mallei and B. pseudomallei for the generation of human scFv antibodies specific to the two pathogenic species of bacteria. Using two different panning procedures, we obtained seven different scFv phage antibodies that interacted with the heat-killed whole bacterial cells of B. mallei and B. pseudomallei. Our results demonstrate that panning of a human scFv antibody library against heat-killed whole bacterial cells may provide a valuable strategy for developing human monoclonal antibodies against the highly pathogenic bacteria.     

Inflammation patterns induced by different Burkholderia species in mice

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 × 10³ cfu B. thailandensis was not lethal, whereas inoculation with 1 × 10⁶ cfu B. thailandensis was equally lethal as 1 × 10³ 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.     

Epitope mapping of Burkholderia pseudomallei serine metalloprotease: identification of serine protease epitope mimics

Filamentous phage random peptide libraries were used to identify the epitopes of Burkholderia pseudomallei protease by panning against IgG polyclonal sera that exhibited protease neutralizing properties. The isolated fusion peptides presented a consensus peptide sequence, TKSMALSG, which closely resembles part of the active site sequence, 435GTSMATPHVAG445, of B. pseudomallei serine metalloprotease. By comparing the consensus sequence, TKSMALSG, with the predicted three-dimensional molecular model of B. pseudomallei serine metalloprotease, it appears that the potential antibody binding epitope was buried within the molecule. This active site was conformational whereby one continuous sub-region (SMA) was located between two discontinuous sub-regions, supplied by the flanking residues in the same polypeptide. All phages selected from the biopanning with IgG polyclonal sera showed good binding towards the polyclonal antibodies when compared to the negative control. In addition, these peptide-bearing phages showed competitive inhibition of B. pseudomallei serine metalloprotease binding to the polyclonal IgG.     

The wbiA locus is required for the 2-O-acetylation of lipopolysaccharides expressed by Burkholderia pseudomallei and Burkholderia thailandensis

Burkholderia pseudomallei and Burkholderia thailandensis express similar O-antigens (O-PS II) in which their 6-deoxy-alpha-L-talopyranosyl (L-6dTalp) residues are variably substituted with O-acetyl groups at the O-2 or O-4 positions. In previous studies we demonstrated that the protective monoclonal antibody, Pp-PS-W, reacted with O-PS II expressed by wild-type B. pseudomallei strains but not by a B. pseudomallei wbiA null mutant. In the present study we demonstrate that WbiA activity is required for the acetylation of the L-6dTalp residues at the O-2 position and that structural modification of O-PS II molecules at this site is critical for recognition by Pp-PS-W.