Characterization of the murine macrophage response to infection with virulent and avirulent Burkholderia species.

BACKGROUND: Burkholderia pseudomallei (Bp) and Burkholderia mallei (Bm) are Gram-negative facultative intracellular pathogens, which are the causative agents of melioidosis and glanders, respectively. Depending on the route of exposure, aerosol or transcutaneous, infection by Bp or Bm can result in an extensive range of disease - from acute to chronic, relapsing illness to fatal septicemia. Both diseases are associated with difficult diagnosis and high fatality rates. About ninety five percent of patients succumb to untreated septicemic infections and the fatality rate is 50 % even when standard antibiotic treatments are administered. RESULTS: The goal of this study is to profile murine macrophage-mediated phenotypic and molecular responses that are characteristic to a collection of Bp, Bm, Burkholderia thailandensis (Bt) and Burkholderia oklahomensis (Bo) strains obtained from humans, animals, environment and geographically diverse locations. Burkholderia spp. (N = 21) were able to invade and replicate in macrophages, albeit to varying degrees. All Bp (N = 9) and four Bm strains were able to induce actin polymerization on the bacterial surface following infection. Several Bp and Bm strains showed reduced ability to induce multinucleated giant cell (MNGC) formation, while Bo and Bp 776 were unable to induce this phenotype. Measurement of host cytokine responses revealed a statistically significant Bm mediated IL-6 and IL-10 production compared to Bp strains. Hierarchical clustering of transcriptional data from 84 mouse cytokines, chemokines and their corresponding receptors identified 29 host genes as indicators of differential responses between the Burkholderia spp. Further validation confirmed Bm mediated Il-1b, Il-10, Tnfrsf1b and Il-36a mRNA expressions were significantly higher when compared to Bp and Bt. CONCLUSIONS: These results characterize the phenotypic and immunological differences in the host innate response to pathogenic and avirulent Burkholderia strains and provide insight into the phenotypic alterations and molecular targets underlying host-Burkholderia interactions.

A high-content imaging assay for the quantification of the Burkholderia pseudomallei induced multinucleated giant cell (MNGC) phenotype in murine macrophages.

BACKGROUND: Burkholderia pseudomallei (Bp), a Gram-negative, motile, facultative intracellular bacterium is the causative agent of melioidosis in humans and animals. The Bp genome encodes a repertoire of virulence factors, including the cluster 3 type III secretion system (T3SS-3), the cluster 1 type VI secretion system (T6SS-1), and the intracellular motility protein BimA, that enable the pathogen to invade both phagocytic and non-phagocytic cells. A unique hallmark of Bp infection both in vitro and in vivo is its ability to induce cell-to-cell fusion of macrophages to form multinucleated giant cells (MNGCs), which to date are semi-quantitatively reported following visual inspection. RESULTS: In this study we report the development of an automated high-content image acquisition and analysis assay to quantitate the Bp induced MNGC phenotype. Validation of the assay was performed using T6SS-1 (∆hcp1) and T3SS-3 (∆bsaZ) mutants of Bp that have been previously reported to exhibit defects in their ability to induce MNGCs. Finally, screening of a focused small molecule library identified several Histone Deacetylase (HDAC) inhibitors that inhibited Bp-induced MNGC formation of macrophages. CONCLUSIONS: We have successfully developed an automated HCI assay to quantitate MNGCs induced by Bp in macrophages. This assay was then used to characterize the phenotype of the Bp mutants for their ability to induce MNGC formation and identify small molecules that interfere with this process. Successful application of chemical genetics and functional reverse genetics siRNA approaches in the MNGC assay will help gain a better understanding of the molecular targets and cellular mechanisms responsible for the MNGC phenotype induced by Bp, by other bacteria such as Mycobacterium tuberculosis, or by exogenously added cytokines.

Characterization of the Burkholderia thailandensis SOS response by using whole-transcriptome shotgun sequencing.

The bacterial SOS response is a well-characterized regulatory network encoded by most prokaryotic bacterial species and is involved in DNA repair. In addition to nucleic acid repair, the SOS response is involved in pathogenicity, stress-induced mutagenesis, and the emergence and dissemination of antibiotic resistance. Using high-throughput sequencing technology (SOLiD RNA-Seq), we analyzed the Burkholderia thailandensis global SOS response to the fluoroquinolone antibiotic, ciprofloxacin (CIP), and the DNA-damaging chemical, mitomycin C (MMC). We demonstrate that a B. thailandensis recA mutant (RU0643) is ∼4-fold more sensitive to CIP in contrast to the parental strain B. thailandensis DW503. Our RNA-Seq results show that CIP and MMC treatment (P < 0.01) resulted in the differential expression of 344 genes in B. thailandensis and 210 genes in RU0643. Several genes associated with the SOS response were induced and include lexA, uvrA, dnaE, dinB, recX, and recA. At the genome-wide level, we found an overall decrease in gene expression, especially for genes involved in amino acid and carbohydrate transport and metabolism, following both CIP and MMC exposure. Interestingly, we observed the upregulation of several genes involved in bacterial motility and enhanced transcription of a B. thailandensis genomic island encoding a Siphoviridae bacteriophage designated E264. Using B. thailandensis plaque assays and PCR with B. mallei ATCC 23344 as the host, we demonstrate that CIP and MMC exposure in B. thailandensis DW503 induces the transcription and translation of viable bacteriophage in a RecA-dependent manner. This is the first report of the SOS response in Burkholderia spp. to DNA-damaging agents. We have identified both common and unique adaptive responses of B. thailandensis to chemical stress and DNA damage.

The early stage of bacterial genome-reductive evolution in the host.

The equine-associated obligate pathogen Burkholderia mallei was developed by reductive evolution involving a substantial portion of the genome from Burkholderia pseudomallei, a free-living opportunistic pathogen. With its short history of divergence (approximately 3.5 myr), B. mallei provides an excellent resource to study the early steps in bacterial genome reductive evolution in the host. By examining 20 genomes of B. mallei and B. pseudomallei, we found that stepwise massive expansion of IS (insertion sequence) elements ISBma1, ISBma2, and IS407A occurred during the evolution of B. mallei. Each element proliferated through the sites where its target selection preference was met. Then, ISBma1 and ISBma2 contributed to the further spread of IS407A by providing secondary insertion sites. This spread increased genomic deletions and rearrangements, which were predominantly mediated by IS407A. There were also nucleotide-level disruptions in a large number of genes. However, no significant signs of erosion were yet noted in these genes. Intriguingly, all these genomic modifications did not seriously alter the gene expression patterns inherited from B. pseudomallei. This efficient and elaborate genomic transition was enabled largely through the formation of the highly flexible IS-blended genome and the guidance by selective forces in the host. The detailed IS intervention, unveiled for the first time in this study, may represent the key component of a general mechanism for early bacterial evolution in the host.

Genetic and phenotypic diversity in Burkholderia: contributions by prophage and phage-like elements.

BACKGROUND: Burkholderia species exhibit enormous phenotypic diversity, ranging from the nonpathogenic, soil- and water-inhabiting Burkholderia thailandensis to the virulent, host-adapted mammalian pathogen B. mallei. Genomic diversity is evident within Burkholderia species as well. Individual isolates of Burkholderia pseudomallei and B. thailandensis, for example, carry a variety of strain-specific genomic islands (GIs), including putative pathogenicity and metabolic islands, prophage-like islands, and prophages. These GIs may provide some strains with a competitive advantage in the environment and/or in the host relative to other strains. RESULTS: Here we present the results of analysis of 37 prophages, putative prophages, and prophage-like elements from six different Burkholderia species. Five of these were spontaneously induced to form bacteriophage particles from B. pseudomallei and B. thailandensis strains and were isolated and fully sequenced; 24 were computationally predicted in sequenced Burkholderia genomes; and eight are previously characterized prophages or prophage-like elements. The results reveal numerous differences in both genome structure and gene content among elements derived from different species as well as from strains within species, due in part to the incorporation of additional DNA, or 'morons' into the prophage genomes. Implications for pathogenicity are also discussed. Lastly, RNAseq analysis of gene expression showed that many of the genes in varphi1026b that appear to contribute to phage and lysogen fitness were expressed independently of the phage structural and replication genes. CONCLUSIONS: This study provides the first estimate of the relative contribution of prophages to the vast phenotypic diversity found among the Burkholderiae.

Virulence and cellular interactions of Burkholderia multivorans in chronic granulomatous disease.

Chronic granulomatous disease (CGD) patients are susceptible to life-threatening infections by the Burkholderia cepacia complex. We used leukocytes from CGD and healthy donors and compared cell association, invasion, and cytokine induction by Burkholderia multivorans strains. A CGD isolate, CGD1, showed higher cell association than that of an environmental isolate, Env1, which correlated with cell entry. All B. multivorans strains associated significantly more with cells from CGD patients than with those from healthy donors. Similar findings were observed with another CGD pathogen, Serratia marcescens, but not with Escherichia coli. In a mouse model of CGD, strain CGD1 was virulent while Env1 was avirulent. B. multivorans organisms were found in the spleens of CGD1-infected mice at levels that were 1,000 times higher than those found in Env1-infected mice, which was coincident with higher levels of the proinflammatory cytokine interleukin-1beta. Taken together, these results may shed light on the unique susceptibility of CGD patients to specific pathogens.

Defining the mode of action of tetramic acid antibacterials derived from Pseudomonas aeruginosa quorum sensing signals.

In nature, bacteria rarely exist as single, isolated entities, but rather as communities comprised of many other species including higher host organisms. To survive in these competitive environments, microorganisms have developed elaborate tactics such as the formation of biofilms and the production of antimicrobial toxins. Recently, it was discovered that the gram-negative bacterium Pseudomonas aeruginosa , an opportunistic human pathogen, produces an antibiotic, 3-(1-hydroxydecylidene)-5-(2-hydroxyethyl)pyrrolidine-2,4-dione (C(12)-TA), derived from one of its quorum sensing molecules. Here, we present a comprehensive study of the expanded spectrum of C(12)-TA antibacterial activity against microbial competitors encountered by P. aeruginosa in nature as well as significant human pathogens. The mechanism of action of C(12)-TA was also elucidated, and C(12)-TA was found to dissipate both the membrane potential and the pH gradient of Gram-positive bacteria, correlating well with cell death. Notably, in stark contrast to its parent molecule 3-oxo-dodecanoyl homoserine lactone (3-oxo-C(12)-HSL), neither activation of cellular stress pathways nor cytotoxicity was observed in human cells treated with C(12)-TA. Our results suggest that the QS machinery of P. aeruginosa has evolved for a dual-function, both to signal others of the same species and also to defend against host immunity and competing bacteria. Because of the broad-spectrum antibacterial activity, established mode of action, lack of rapid resistance development, and tolerance by human cells, the C(12)-TA scaffold may also serve as a new lead compound for the development of antimicrobial therapeutics.

Novel broad-spectrum bis-(imidazolinylindole) derivatives with potent antibacterial activities against antibiotic-resistant strains.

Given the limited number of structural classes of clinically available antimicrobial drugs, the discovery of antibacterials with novel chemical scaffolds is an important strategy in the development of effective therapeutics for both naturally occurring and engineered resistant strains of pathogenic bacteria. In this study, several diarylamidine derivatives were evaluated for their ability to protect macrophages from cell death following infection with Bacillus anthracis, a gram-positive spore-forming bacterium. Four bis-(imidazolinylindole) compounds were identified with potent antibacterial activity as measured by the protection of macrophages and by the inhibition of bacterial growth in vitro. These compounds were effective against a broad range of gram-positive and gram-negative bacterial species, including several antibiotic-resistant strains. Minor structural variations among the four compounds correlated with differences in their effects on bacterial macromolecular synthesis and mechanisms of resistance. In vivo studies revealed protection by two of the compounds of mice lethally infected with B. anthracis, Staphylococcus aureus, or Yersinia pestis. Taken together, these results indicate that the bis-(imidazolinylindole) compounds represent a new chemotype for the development of therapeutics for both gram-positive and gram-negative bacterial species as well as against antibiotic-resistant infections.

Exogenous Yersinia pestis quorum sensing molecules N-octanoyl-homoserine lactone and N-(3-oxooctanoyl)-homoserine lactone regulate the LcrV virulence factor.

LcrV is a key Yersinia pestis antigen, immune regulator, and component of the type III secretion system (T3SS). Researchers have shown that N-acyl-homoserine lactones (AHLs) can down-regulate the expression of the LcrV homolog, PcrV, in Pseudomonas aeruginosa. Using ELISA, western blot, DNA microarray analysis, and real time PCR we demonstrate that the addition of AHL molecules N-octanoyl-homoserine lactone (C8) or N-(3-oxooctanoyl)-homoserine lactone (oxo-C8) to Y. pestis cultures down-regulates LcrV protein expression. DNA microarray analysis shows 10 additional T3SS genes are consistently down-regulated by C8 or oxo-C8. This is the first report demonstrating that AHLs regulate Y. pestis virulence factor expression.

The Burkholderia mallei BmaR3-BmaI3 quorum-sensing system produces and responds to N-3-hydroxy-octanoyl homoserine lactone.

Burkholderia mallei has two acyl-homoserine lactone (acyl-HSL) signal generator-receptor pairs and two additional signal receptors, all of which contribute to virulence. We show that B. mallei produces N-3-hydroxy-octanoyl HSL (3OHC8-HSL) but a bmaI3 mutant does not. Recombinant Escherichia coli expressing BmaI3 produces hydroxylated acyl-HSLs, with 3OHC8-HSL being the most abundant compound. In recombinant E. coli, BmaR3 responds to 3OHC8-HSL but not to other acyl-HSLs. These data indicate that the signal for BmaR3-BmaI3 quorum sensing is 3OHC8-HSL.

Development of a polymerase chain reaction assay for the specific identification of Burkholderia mallei and differentiation from Burkholderia pseudomallei and other closely related Burkholderiaceae.

Burkholderia mallei and Burkholderia pseudomallei, the etiologic agents responsible for glanders and melioidosis, respectively, are genetically and phenotypically similar and are category B biothreat agents. We used an in silico approach to compare the B. mallei ATCC 23344 and B. pseudomallei K96243 genomes to identify nucleotide sequences unique to B. mallei. Five distinct B. mallei DNA sequences and/or genes were identified and evaluated for polymerase chain reaction (PCR) assay development. Genomic DNAs from a collection of 31 B. mallei and 34 B. pseudomallei isolates, obtained from various geographic, clinical, and environmental sources over a 70-year period, were tested with PCR primers targeted for each of the B. mallei ATCC 23344-specific nucleotide sequences. Of the 5 chromosomal targets analyzed, only PCR primers designed to bimA(Bm) were specific for B. mallei. These primers were used to develop a rapid PCR assay for the definitive identification of B. mallei and differentiation from all other bacteria.

Using real-time PCR to specifically detect Burkholderia mallei.

Burkholderia mallei is the causative agent of human and animal glanders and is a category B biothreat agent. Rapid diagnosis of B. mallei and immediate prophylactic treatment are essential for patient survival. The majority of current bacteriological and immunological techniques for identifying B. mallei from clinical samples are time-consuming, and cross-reactivity with closely related organisms (i.e. Burkholderia pseudomallei) is a problem. In this investigation, two B. mallei-specific real-time PCR assays targeting the B. mallei bimA(ma) gene (Burkholderia intracellular motility A; BMAA0749), which encodes a protein involved in actin polymerization, were developed. The PCR primer and probe sets were tested for specificity against a collection of B. mallei and B. pseudomallei isolates obtained from numerous clinical and environmental (B. pseudomallei only) sources. The assays were also tested for cross-reactivity using template DNA from 14 closely related Burkholderia species. The relative limit of detection for the assays was found to be 1 pg or 424 genome equivalents. The authors also analysed the applicability of assays to detect B. mallei within infected BALB/c mouse tissues. Beginning 1 h post aerosol exposure, B. mallei was successfully identified within the lungs, and starting at 24 h post exposure, in the spleen and liver. Surprisingly, B. mallei was not detected in the blood of acutely infected animals. This investigation provides two real-time PCR assays for the rapid and specific identification of B. mallei.

Bacterial genome adaptation to niches: divergence of the potential virulence genes in three Burkholderia species of different survival strategies.

BACKGROUND: Two closely related species Burkholderia mallei (Bm) and Burkholderia pseudomallei (Bp) are serious human health hazards and are potential bio-warfare agents, whereas another closely related species Burkholderia thailandensis (Bt) is a non-pathogenic saprophyte. To investigate the genomic factors resulting in such a dramatic difference, we first identified the Bm genes responsive to the mouse environment, and then examined the divergence of these genes in Bp and Bt. RESULTS: The genes down-expressed, which largely encode cell growth-related proteins, are conserved well in all three species, whereas those up-expressed, which include potential virulence genes, are less well conserved or absent notably in Bt. However, a substantial number of up-expressed genes is still conserved in Bt. Bm and Bp further diverged from each other in a small number of genes resulting from unit number changes in simple sequence repeats (ssr) in the homologs. CONCLUSION: Our data suggest that divergent evolution of a small set of genes, rather than acquisition or loss of pathogenic islands, is associated with the development of different life styles in these bacteria of similar genomic contents. Further divergence between Bm and Bp mediated by ssr changes may reflect different adaptive processes of Bm and Bp fine-tuning into their host environments.

Role of quorum sensing in the pathogenicity of Burkholderia pseudomallei.

Burkholderia pseudomallei is the causative agent of human and animal melioidosis. The role of quorum sensing (QS) in the in vivo pathogenicity of B. pseudomallei via inhalational exposure of BALB/c mice and intraperitoneal challenge of Syrian hamsters has not been reported. This investigation demonstrates that B. pseudomallei encodes a minimum of three luxI and five luxR homologues that are involved in animal pathogenicity. Mass spectrometry analysis of culture supernatants revealed that wild-type B. pseudomallei and the luxI mutants synthesized numerous signalling molecules, including N-octanoyl-homoserine lactone, N-decanoyl-homoserine lactone, N-(3-hydroxyoctanoyl)-L-homoserine lactone, N-(3-hydroxydecanoyl)-L-homoserine lactone and N-(3-oxotetradecanoyl)-L-homoserine lactone, which was further confirmed by heterologous expression of the B. pseudomallei luxI alleles in Escherichia coli. Mutagenesis of the B. pseudomallei QS system increased the time to death and reduced organ colonization of aerosolized BALB/c mice. Further, intraperitoneal challenge of Syrian hamsters with the B. pseudomallei QS mutants resulted in a significant increase in the LD50. Using semi-quantitative plate assays, preliminary analysis suggests that QS does not affect lipase, protease and phospholipase C biosynthesis/secretion in B. pseudomallei. The findings of the investigation demonstrate that B. pseudomallei encodes multiple luxIR genes, and disruption of the QS alleles reduces animal pathogenicity, but does not affect exoproduct secretion.

Integrating high-content imaging and chemical genetics to probe host cellular pathways critical for Yersinia pestis infection.

The molecular machinery that regulates the entry and survival of Yersinia pestis in host macrophages is poorly understood. Here, we report the development of automated high-content imaging assays to quantitate the internalization of virulent Y. pestis CO92 by macrophages and the subsequent activation of host NF-κB. Implementation of these assays in a focused chemical screen identified kinase inhibitors that inhibited both of these processes. Rac-2-ethoxy-3 octadecanamido-1-propylphosphocholine (a protein Kinase C inhibitor), wortmannin (a PI3K inhibitor), and parthenolide (an IκB kinase inhibitor), inhibited pathogen-induced NF-κB activation and reduced bacterial entry and survival within macrophages. Parthenolide inhibited NF-κB activation in response to stimulation with Pam3CSK4 (a TLR2 agonist), E. coli LPS (a TLR4 agonist) or Y. pestis infection, while the PI3K and PKC inhibitors were selective only for Y. pestis infection. Together, our results suggest that phagocytosis is the major stimulus for NF-κB activation in response to Y. pestis infection, and that Y. pestis entry into macrophages may involve the participation of protein kinases such as PI3K and PKC. More importantly, the automated image-based screening platform described here can be applied to the study of other bacteria in general and, in combination with chemical genetic screening, can be used to identify host cell functions facilitating the identification of novel antibacterial therapeutics.

Identification of in vivo-induced conserved sequences from Yersinia pestis during experimental plague infection in the rabbit.

In an effort to identify the novel virulence determinants of Yersinia pestis, we applied the gene "discovery" methodology, in vivo-induced (IVI) antigen technology, to detect genes upregulated during infection in a laboratory rabbit model for bubonic plague. After screening over 70,000 Escherichia coli clones of Y. pestis DNA expression libraries, products from 25 loci were identified as being seroreactive to reductively adsorbed, pooled immune serum. Upon sequence analysis of the predicted IVI gene products, more frequently encountered conserved protein functional categories have emerged, to include type-V autotransporters and components of more complex secretion systems including types III and VI. The recombinant products from eight selected clones were subsequently immunoblotted against pooled immune serum from two naturally infected host species: the prairie dog, and a species refractory to lethal disease, the coyote. Immune prairie dog serum recognized 2-3 of the rabbit-reactive antigens, suggesting at least some overlap in the pathogen's in vivo survival mechanisms between these two hosts. Although the coyote serum failed to recognize most of the IVI antigens, LepA was universally reactive with all three host sera. Collectively, the profiles/patterns of IVI conserved sequences (IVICS) may represent immune "signatures" among different host species, possessing the potential for use as a diagnostic tool for plague. Further, the antigenic nature of IVICS makes them ideal for further evaluation as novel subunit vaccine candidates. The gathering of additional data and analysis of the intact IVI genes and the expressed IVICS products should provide insight into the unique biologic processes of Y. pestis during infection and reveal the genetic patterns of the pathogen's survival strategy in different hosts.

Reduced expression of CD45 protein-tyrosine phosphatase provides protection against anthrax pathogenesis.

The modulation of cellular processes by small molecule inhibitors, gene inactivation, or targeted knockdown strategies combined with phenotypic screens are powerful approaches to delineate complex cellular pathways and to identify key players involved in disease pathogenesis. Using chemical genetic screening, we tested a library of known phosphatase inhibitors and identified several compounds that protected Bacillus anthracis infected macrophages from cell death. The most potent compound was assayed against a panel of sixteen different phosphatases of which CD45 was found to be most sensitive to inhibition. Testing of a known CD45 inhibitor and antisense phosphorodiamidate morpholino oligomers targeting CD45 also protected B. anthracis-infected macrophages from cell death. However, reduced CD45 expression did not protect anthrax lethal toxin (LT) treated macrophages, suggesting that the pathogen and independently added LT may signal through distinct pathways. Subsequent, in vivo studies with both gene-targeted knockdown of CD45 and genetically engineered mice expressing reduced levels of CD45 resulted in protection of mice after infection with the virulent Ames B. anthracis. Intermediate levels of CD45 expression were critical for the protection, as mice expressing normal levels of CD45 or disrupted CD45 phosphatase activity or no CD45 all succumbed to this pathogen. Mechanism-based studies suggest that the protection provided by reduced CD45 levels results from regulated immune cell homeostasis that may diminish the impact of apoptosis during the infection. To date, this is the first report demonstrating that reduced levels of host phosphatase CD45 modulate anthrax pathogenesis.

Octanoyl-homoserine lactone is the cognate signal for Burkholderia mallei BmaR1-BmaI1 quorum sensing.

Acyl-homoserine lactones (HSLs) serve as quorum-sensing signals for many Proteobacteria. Members of the LuxI family of signal generators catalyze the production of acyl-HSLs, which bind to a cognate receptor in the LuxR family of transcription factors. The obligate animal pathogen Burkholderia mallei produces several acyl-HSLs, and the B. mallei genome has four luxR and two luxI homologs, each of which has been established as a virulence factor. To begin to delineate the relevant acyl-HSL signals for B. mallei LuxR homologs, we analyzed the BmaR1-BmaI1 system. A comparison of acyl-HSL profiles from B. mallei ATCC 23344 and a B. mallei bmaI1 mutant indicates that octanoyl-HSL synthesis is BmaI1 dependent. Furthermore, octanoyl-HSL is the predominant acyl-HSL produced by BmaI1 in recombinant Escherichia coli. The synthesis of soluble BmaR1 in recombinant E. coli requires octanoyl-HSL or decanoyl-HSL. Insoluble aggregates of BmaR1 are produced in the presence of other acyl-HSLs and in the absence of acyl-HSLs. The bmaI1 promoter is activated by BmaR1 and octanoyl-HSL, and a 20-bp inverted repeat in the bmaI1 promoter is required for bmaI1 activation. Purified BmaR1 binds to this promoter region. These findings implicate octanoyl-HSL as the signal for BmaR1-BmaI1 quorum sensing and show that octanoyl-HSL and BmaR1 activate bmaI1 transcription.

The bacterial gene lfpA influences the potent induction of calcitonin receptor and osteoclast-related genes in Burkholderia pseudomallei-induced TRAP-positive multinucleated giant cells.

Burkholderia pseudomallei is a facultative intracellular pathogen and the causative agent of melioidosis, a spectrum of potentially fatal diseases endemic in Northern Australia and South-East Asia. We demonstrate that B. pseudomallei rapidly modifies infected macrophage-like cells in a manner analagous to osteoclastogenesis. These alterations include multinucleation and the expression by infected cells of mRNA for factors required for osteoclastogenesis: the chemokines monocyte chemotactic protein 1 (MCP-1), macrophage inflammatory protein 1 gamma (MIP-1gamma), 'regulated on activation normal T cell expressed and secreted' (RANTES) and the transcription factor 'nuclear factor of activated T-cells cytoplasmic 1' (NFATc1). An increase in expression of these factors was also observed after infection with Burkholderia thailandensis. Expression of genes for the osteoclast markers calcitonin receptor (CTR), cathepsin K (CTSK) and tartrate-resistant acid phosphatase (TRAP) was also increased by B. pseudomallei-infected, but not by B. thailandensis-infected cells. The expression by B. pseudomallei-infected cells of these chemokine and osteoclast marker genes was remarkably similar to cells treated with RANKL, a stimulator of osteoclastogenesis. Analysis of dentine resorption by B. pseudomallei-induced osteoclast-like cells revealed that demineralization may occur but that authentic excavation does not take place under the tested conditions. Furthermore, we identified and characterized lfpA (for lactonase family protein A) in B. pseudomallei, which shares significant sequence similarity with the eukaryotic protein 'regucalcin', also known as 'senescence marker protein-30' (SMP-30). LfpA orthologues are widespread in prokaryotes and are well conserved, but are phylogenetically distinct from eukaryotic regucalcin orthologues. We demonstrate that lfpA mRNA expression is dramatically increased in association with macrophage-like cells. Mutation of lfpA significantly reduced expression of the tested host genes, relative to the response to wild-type B. pseudomallei. We also show that lfpA is required for optimal virulence in vivo.

Type VI secretion is a major virulence determinant in Burkholderia mallei.

Burkholderia mallei is a host-adapted pathogen and a category B biothreat agent. Although the B. mallei VirAG two-component regulatory system is required for virulence in hamsters, the virulence genes it regulates are unknown. Here we show with expression profiling that overexpression of virAG resulted in transcriptional activation of approximately 60 genes, including some involved in capsule production, actin-based intracellular motility, and type VI secretion (T6S). The 15 genes encoding the major sugar component of the homopolymeric capsule were up-expressed > 2.5-fold, but capsule was still produced in the absence of virAG. Actin tail formation required virAG as well as bimB, bimC and bimE, three previously uncharacterized genes that were activated four- to 15-fold when VirAG was overproduced. Surprisingly, actin polymerization was found to be dispensable for virulence in hamsters. In contrast, genes encoding a T6S system were up-expressed as much as 30-fold and mutations in this T6S gene cluster resulted in strains that were avirulent in hamsters. SDS-PAGE and mass spectrometry demonstrated that BMAA0742 was secreted by the T6S system when virAG was overexpressed. Purified His-tagged BMAA0742 was recognized by glanders antiserum from a horse, a human and mice, indicating that this Hcp-family protein is produced in vivo during infection.