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TheBurkholderia pseudomalleicomplex (Bpc) includesB. pseudomallei, B. malleiandB. thailandensis. These species share conserved virulence determinants that facilitate survival in mammalian cells and can spread from cell to cell by a unique mechanism involving fusion of plasma membranes. The activity of a contractile type VI secretion system, T6SS-5, is a central requirement. Using fluorescence confocal microscopy, we found localization and dynamic turnover of fluorescently-labeled T6SS-5 components at the forward pole ofBurkholderiaresiding at the ends of actin protrusions. We identified the proline-alanine-alanine-arginine repeat protein of T6SS-5 (PAAR5), which forms the heteromeric tip of the T6SS-5 apparatus along with VgrG5. Mutational analysis revealed a unique N-terminal extension (NTE) of PAAR5 that is indispensable for cell fusion. Deletion ofpaar5allowed us to uncouple fusogenic activity from the functionality of T6SS-5 for exploring the role of cell fusion in pathogenesis.B. pseudomalleiΔpaar5deletion mutants retained a functional T6SS-5 apparatus and the ability to secrete the Hcp5 protein. In cellular and animal infection models, Δpaar5mutants mirrored the phenotype of a T6SS-5-defective ΔvgrG5strain, being defective for cell fusion and avirulent in hamsters. These results demonstrate concordance between the fusogenic andin vivovirulence phenotypes, suggesting that T6SS-5-mediated cell fusion may be a central feature ofB. pseudomalleipathogenesis and not anin vitroartifact.

Burkholderia pseudomallei, the causative agent of melioidosis, is classified by the CDC as a tier 1 select agent, and work involving it must be performed in a biosafety level 3 (BSL-3) laboratory. Three BSL-2 surrogate strains derived from B. pseudomallei 1026b, a virulent clinical isolate, have been removed from the CDC select agent list. These strains, Bp82, B0011, and JW270, are highly attenuated in rodent models of melioidosis and cannot be utilized to identify virulence determinants because of their high 50% lethal dose (LD(50)). We previously demonstrated that the Madagascar hissing cockroach (MHC) is a tractable surrogate host to study the innate immune response against Burkholderia. In this study, we found that JW270 maintains its virulence in MHCs. This surprising result indicates that it may be possible to identify potential virulence genes in JW270 by using MHCs at BSL-2. We tested this hypothesis by constructing JW270 mutations in genes that are required (hcp1) or dispensable (hcp2) for B. pseudomallei virulence in rodents. JW270 Δhcp1 was avirulent in MHCs and JW270 Δhcp2 was virulent, suggesting that MHCs can be used at BSL-2 for the discovery of important virulence factors. JW270 ΔBPSS2185, a strain harboring a mutation in a type IV pilin locus (TFP8) required for full virulence in BALB/c mice, was also found to be attenuated in MHCs. Finally, we demonstrate that the hmqA-G locus, which encodes the production of a family of secondary metabolites called 4-hydroxy-3-methyl-2-alkylquinolines, is important for JW270 virulence in MHCs and may represent a novel virulence determinant.

Type IV pili are involved in adhesion, twitching motility, aggregation, biofilm formation and virulence in a variety of Gram-negative bacteria.

Successful bacterial pathogens have evolved to avoid activating an innate immune system in the host that responds to the pathogen through distinct Toll-like receptors (TLRs). The general class of biochemical components that activate TLRs has been studied extensively, but less is known about how TLRs interact with the class of compounds that are still associated with the live pathogen. Accordingly, we examined the activation of surface assembled TLR 2, 4, and 5 with live Tier 1 Gram-negative pathogens that included Yersinia pestis (plague), Burkholderia mallei (glanders), Burkholderia pseudomallei (melioidosis), and Francisella tularensis (tularemia). We found that Y. pestis CO92 grown at 28°C activated TLR2 and TLR4, but at 37°C the pathogen activated primarily TLR2. Although B. mallei and B. pseudomallei are genetically related, the former microorganism activated predominately TLR4, while the latter activated predominately TLR2. The capsule of wild-type B. pseudomallei 1026b was found to mitigate the activation of TLR2 and TLR4 when compared to a capsule mutant. Live F. tularensis (Ft) Schu S4 did not activate TLR2 or 4, although the less virulent Ft LVS and F. novicida activated only TLR2. B. pseudomallei purified flagellin or flagella attached to the microorganism activated TLR5. Activation of TLR5 was abolished by an antibody to TLR5, or a mutation of fliC, or elimination of the pathogen by filtration. In conclusion, we have uncovered new properties of the Gram-negative pathogens, and their interaction with TLRs of the host. Further studies are needed to include other microorganism to extend our observations with their interaction with TLRs, and to the possibility of leading to new efforts in therapeutics against these pathogens.

Burkholderia thailandensis has emerged as a model organism for investigating the production and regulation of diverse secondary metabolites. Most of the biosynthetic gene clusters encoded in B. thailandensis are silent, motivating the development of new methods for accessing their products. In the current work, we add to the canon of available approaches using phenotype-guided transposon mutagenesis to characterize a silent biosynthetic gene cluster. Because secondary metabolite biosynthesis is often associated with phenotypic changes, we carried out random transposon mutagenesis followed by phenotypic inspection of the resulting colonies. Several mutants exhibited intense pigmentation and enhanced expression of an iterative type I polyketide synthase cluster that we term org. Disruptions of orgA, orgB, and orgC abolished the biosynthesis of the diffusible pigment, thus linking it to the org operon. Isolation and structural elucidation by HR-MS and 1D/2D NMR spectroscopy revealed three novel, cryptic metabolites, thailandene A-C. Thailandenes are linear formylated or acidic polyenes containing a combination of cis and trans double bonds. Variants A and B exhibited potent antibiotic activity against Staphylococcus aureus and Saccharomyces cerevisiae but not against Escherichia coli. One of the transposon mutants that exhibited an enhanced expression of org contained an insertion upstream of a σ54-dependent transcription factor. Closer inspection of the org operon uncovered a σ54 promoter consensus sequence upstream of orgA, providing clues regarding its regulation. Our results showcase the utility of phenotype-guided transposon mutagenesis in uncovering cryptic metabolites encoded in bacterial genomes.

The Burkholderia genus encompasses many Gram-negative bacteria living in the rhizosphere. Some Burkholderia species can cause life-threatening human infections, highlighting the need for clinical interventions targeting specific lipopolysaccharide proteins. Burkholderia cenocepacia O-linked protein glycosylation has been reported, but the chemical structure of the O-glycan and the machinery required for its biosynthesis are unknown and could reveal potential therapeutic targets. Here, using bioinformatics approaches, gene-knockout mutants, purified recombinant proteins, LC-MS–based analyses of O-glycans, and NMR-based structural analyses, we identified a B. cenocepacia O-glycosylation (ogc) gene cluster necessary for synthesis, assembly, and membrane translocation of a lipid-linked O-glycan, as well as its structure, which consists of a β-Gal-(1,3)–α-GalNAc-(1,3)–β-GalNAc trisaccharide. We demonstrate that the ogc cluster is conserved in the Burkholderia genus, and we confirm the production of glycoproteins with similar glycans in the Burkholderia species: B. thailandensis, B. gladioli, and B. pseudomallei. Furthermore, we show that absence of protein O-glycosylation severely affects bacterial fitness and accelerates bacterial clearance in a Galleria mellonella larva infection model. Finally, our experiments revealed that patients infected with B. cenocepacia, Burkholderia multivorans, B. pseudomallei, or Burkholderia mallei develop O-glycan–specific antibodies. Together, these results highlight the importance of general protein O-glycosylation in the biology of the Burkholderia genus and its potential as a target for inhibition or immunotherapy approaches to control Burkholderia infections.

The Burkholderia pseudomallei complex consists of six phylogenetically related Gram-negative bacterial species that include environmental saprophytes and mammalian pathogens. These microbes possess multiple type VI secretion systems (T6SS) that provide a fitness advantage in diverse niches by translocating effector molecules into prokaryotic and eukaryotic cells in a contact-dependent manner. Several recent studies have elucidated the regulation and function of T6SS-2, a novel contact-independent member of the T6SS family. Expression of the T6SS-2 gene cluster is repressed by OxyR, Zur and TctR and is activated by GvmR and reactive oxygen species (ROS). The last two genes of the T6SS-2 gene cluster encode a zincophore (TseZ) and a manganeseophore (TseM) that are exported into the extracellular milieu in a contact-independent fashion when microbes encounter oxidative stress. TseZ and TseM bind Zn2+ and Mn2+, respectively, and deliver them to bacteria where they provide protection against the lethal effects of ROS. The TonB-dependent transporters that interact with TseZ and TseM, and actively transport Zn2+ and Mn2+ across the outer membrane, have also been identified. Finally, T6SS-2 provides a contact-independent growth advantage in nutrient limited environments and is critical for virulence in Galleria mellonella larvae, but is dispensable for virulence in rodent models of infection.

Purpose. Burkholderia pseudomallei , the tier 1 agent of melioidosis, is a saprophytic microbe that causes endemic infections in tropical regions such as South-East Asia and Northern Australia. It is globally distributed, challenging to diagnose and treat, infectious by several routes including inhalation, and has potential for adversarial use. B. pseudomallei strain MSHR5848 produces two colony variants, smooth (S) and rough (R), which exhibit a divergent range of morphological, biochemical and metabolic phenotypes, and differ in macrophage and animal infectivity. We aimed to characterize two major phenotypic differences, analyse gene expression and study the regulatory basis of the variation. Methodology. Phenotypic expression was characterized by DNA and RNA sequencing, microscopy, and differential bacteriology. Regulatory genes were identified by cloning and bioinformatics. Results/Key findings. Whereas S produced larger quantities of extracellular DNA, R was upregulated in the production of a unique chromosome 1-encoded Siphoviridae-like bacteriophage, φMSHR5848. Exploratory transcriptional analyses revealed significant differences in variant expression of genes encoding siderophores, pili assembly, type VI secretion system cluster 4 (T6SS-4) proteins, several exopolysaccharides and secondary metabolites. A single 3 base duplication in S was the only difference that separated the variants genetically. It occurred upstream of a cluster of bacteriophage-associated genes on chromosome 2 that were upregulated in S. The first two genes were involved in regulating expression of the multiple phenotypes distinguishing S and R. Conclusion. Bacteriophage-associated proteins have a major role in the phenotypic expression of MSHR5848. The goals are to determine the regulatory basis of this phenotypic variation and its role in pathogenesis and environmental persistence of B. pseudomallei .

Biofilm communities cause many infectious diseases. Biofilms are diverse microbial species found either attached to a surface or aggregated into an extracellular matrix. Bacteria form biofilms as a default mode of growth or as a response mechanism to environmental conditions like stress. As such, biofilm strains are increasingly virulent causing a wide variety of chronic persistent diseases, are typically antibiotic-resistant and known to improve host mortality rate. Most biofilms contain polysaccharides, proteins, extracellular DNA (eDNA), RNA, and water. Determining and quantifying the major components of a biofilm may indicate an appropriate treatment for biofilm eradication. Burkholderia pseudomallei is a Gram-negative, motile bacillus typically found in surface water and/or soil in endemic regions. It is the etiologic agent of melioidosis and is capable of forming both surface adherent and air-liquid interface biofilms (pellicle) in broth cultures. This study evaluates the components of established biofilms using B. pseudomallei and Burkholderia thailandensis, a closely related nonpathogenic species. Using assays, fluorescent dyes and microscopy, we quantified the major components of biofilms produced by five genetically related B. pseudomallei strains and compared them to B. thailandensis E264. Our data show that biofilm produced by the B. pseudomallei 1026b derivatives and B. thailandensis E264 significantly differ. The molecular composition of the surface adherent biofilm is similar to the molecular composition of the air-liquid pellicle. Finally, the eDNA quantity biofilm produced by JW270 which bears a CPS I deletion, is significantly increased in comparison to 1026 and Bp82 biofilm.

Burkholderia pseudomallei: is the etiological agent of the disease melioidosis and is a Tier 1 select agent. It survives and replicates inside phagocytic cells by escaping from the endocytic vacuole, replicating in the cytosol, spreading to other cells via actin polymerization and promoting the fusion of infected and uninfected host cells to form multinucleated giant cells. In this study, we utilized a proteomics approach to identify bacterial proteins produced inside RAW264.7 murine macrophages and host proteins produced in response to B. pseudomallei infection. Cells infected with B. pseudomallei strain K96243 were lysed and the lysate proteins digested and analyzed using nanoflow reversed-phase liquid chromatography and tandem mass spectrometry. Approximately 160 bacterial proteins were identified in the infected macrophages, including BimA, TssA, TssB, Hcp1 and TssM. Several previously uncharacterized B. pseudomallei proteins were also identified, including BPSS1996 and BPSL2748. Mutations were constructed in the genes encoding these novel proteins and their relative virulence was assessed in BALB/c mice. The 50% lethal dose for the BPSS1996 mutant was approximately 55-fold higher than that of the wild type, suggesting that BPSS1996 is required for full virulence. Sera from B. pseudomallei-infected animals reacted with BPSS1996 and it was found to localize to the bacterial surface using indirect immunofluorescence. Finally, we identified 274 host proteins that were exclusively present or absent in infected RAW264.7 cells, including chemokines and cytokines involved in controlling the initial stages of infection.

Development, testing, and evaluation of medical countermeasures for melioidosis are hampered by a lack of well-characterized and standardized animal models. Recent work has both refined existing animal models for this disease and identified new ones. Head-to-head comparisons of mouse strains with varying susceptibility to the organism and using different routes of infection highlighted and confirmed important similarities and differences between murine models and exposure routes. Diabetic mouse models provided insight into the disease process in humans having this major risk factor. Large animal models, both livestock and non-human primate, have been established. Alternative (non-mammalian) models have been useful in identification of virulence factors and screening of therapeutic candidates. They hold potential for large-scale screening that would not be appropriate or practical for mammalian species. Recent advances in animal and alternative modeling will enhance our understanding of the organism and the disease process, as well as accelerating the development of medical countermeasures.

Burkholderia pseudomallei infections are prevalent in Southeast Asia and northern Australia and often misdiagnosed. Diagnostics are often neither sensitive nor rapid, contributing up to 50% mortality rate. In this 2018 pilot study, we enrolled 100 patients aged 6 months–79 years from Kapit Hospital in Sarawak, Malaysia, with symptoms of B. pseudomallei infection. We used three different methods for the detection of B. pseudomallei: a real-time polymerase chain reaction (PCR) assay, a rapid lateral flow immunoassay, and the standard-of-care bacterial culture—the gold standard. Among the 100 participants, 24 (24%) were positive for B. pseudomallei by one or more of the detection methods. Comparing the two individual diagnostic methods against the gold standard—bacterial culture—of any positive test, there was low sensitivity for each test (25–44%) but high specificity (93–98%). It seems clear that more sensitive diagnostics or a sensitive screening diagnostic followed by specific confirmatory diagnostic is needed for this disease.

Melioidosis is an emerging disease that is caused by the facultative intracellular pathogen Burkholderia pseudomallei. It is intrinsically resistant to many antibiotics and host risk factors play a major role in susceptibility to infection. Currently, there is no human or animal vaccine against melioidosis. In this study, multiple B. pseudomallei MSHR668 deletion mutants were evaluated as live attenuated vaccines in the sensitive BALB/c mouse model of melioidosis. The most efficacious vaccines after an intraperitoneal challenge with 50-fold over the 50% median lethal dose (MLD50) with B. pseudomallei K96243 were 668 &Delta, hisF and 668 &Delta, ilvI. Both vaccines completely protected mice in the acute phase of infection and showed significant protection (50% survivors) during the chronic phase of infection. The spleens of the survivors that were examined were sterile. Splenocytes from mice vaccinated with 668 &Delta, ilvI expressed higher amounts of IFN-&gamma, after stimulation with B. pseudomallei antigens than splenocytes from mice vaccinated with less protective candidates. Finally, we demonstrate that 668 &Delta, hisF is nonlethal in immunocompromised NOD/SCID mice. Our results show that 668 &Delta, ilvI provide protective cell-mediated immune responses in the acute phase of infection and promote long term survival in the sensitive BALB/c mouse model of melioidosis.

The Burkholderia genus encompasses many Gram-negative bacteria living in the rhizosphere. Some Burkholderia species can cause life-threatening human infections, highlighting the need for clinical interventions targeting specific lipopolysaccharide proteins. Burkholderia cenocepacia O-linked protein glycosylation has been reported, but the chemical structure of the O-glycan and the machinery required for its biosynthesis are unknown and could reveal potential therapeutic targets. Here, using bioinformatics approaches, gene-knockout mutants, purified recombinant proteins, LC-MS–based analyses of O-glycans, and NMR-based structural analyses, we identified a B. cenocepacia O-glycosylation (ogc) gene cluster necessary for synthesis, assembly, and membrane translocation of a lipid-linked O-glycan, as well as its structure, which consists of a β-Gal-(1,3)–α-GalNAc-(1,3)–β-GalNAc trisaccharide. We demonstrate that the ogc cluster is conserved in the Burkholderia genus, and we confirm the production of glycoproteins with similar glycans in the Burkholderia species: B. thailandensis, B. gladioli, and B. pseudomallei. Furthermore, we show that absence of protein O-glycosylation severely affects bacterial fitness and accelerates bacterial clearance in a Galleria mellonella larva infection model. Finally, our experiments revealed that patients infected with B. cenocepacia, Burkholderia multivorans, B. pseudomallei, or Burkholderia mallei develop O-glycan–specific antibodies. Together, these results highlight the importance of general protein O-glycosylation in the biology of the Burkholderia genus and its potential as a target for inhibition or immunotherapy approaches to control Burkholderia infections.

Burkholderia pseudomallei, the aetiological agent of melioidosis, is an inhabitant of soil and water in many tropical and subtropical regions worldwide. It possesses six distinct type VI secretion systems (T6SS-1 to T6SS-6), but little is known about most of them, as they are poorly expressed in laboratory culture media. A genetic screen was devised to locate a putative repressor of the T6SS-2 gene cluster and a MarR family transcriptional regulator, termed TctR, was identified. The inactivation of tctR resulted in a 50-fold increase in the expression of an hcp2–lacZ transcriptional fusion, indicating that TctR is a negative regulator of the T6SS-2 gene cluster. Surprisingly, the tctR mutation resulted in a significant decrease in the expression of an hcp6–lacZ transcriptional fusion. B. pseudomallei K96243 and a tctR mutant were grown to logarithmic phase in rich culture medium and RNA was isolated and sequenced in order to identify other genes regulated by TctR. The results identified seven gene clusters that were repressed by TctR, including T6SS-2, and three gene clusters that were significantly activated. A small molecule library consisting of 1120 structurally defined compounds was screened to identify a putative ligand (or ligands) that might bind TctR and derepress transcription of the T6SS-2 gene cluster. Seven compounds, six fluoroquinolones and one quinolone, activated the expression of hcp2–lacZ. Subinhibitory ciprofloxacin also increased the expression of the T6SS-3, T6SS-4 and T6SS-6 gene clusters. This study highlights the complex layers of regulatory control that B. pseudomallei utilizes to ensure that T6SS expression only occurs under very defined environmental conditions.

Many aspects of innate immunity are conserved between mammals and insects. An insect, the Madagascar hissing cockroach from the genus Gromphadorhina, can be utilized as an alternative animal model for the study of virulence, host-pathogen interaction, innate immune response, and drug efficacy. Details for the rearing, care and breeding of the hissing cockroach are provided. We also illustrate how it can be infected with bacteria such as the intracellular pathogens Burkholderia mallei, B. pseudomallei, and B. thailandensis. Use of the hissing cockroach is inexpensive and overcomes regulatory issues dealing with the use of mammals in research. In addition, results found using the hissing cockroach model are reproducible and similar to those obtained using mammalian models. Thus, the Madagascar hissing cockroach represents an attractive surrogate host that should be explored when conducting animal studies.

Burkholderia pseudomallei , the etiologic agent of melioidosis, causes severe disease in humans and animals. Diagnosis and treatment of melioidosis can be challenging, and no licensed vaccines currently exist. Several studies have shown that this pathogen expresses a variety of structurally conserved protective antigens that include cell surface polysaccharides and cell-associated and cell-secreted proteins. Based on those findings, such antigens have become important components of the subunit vaccine candidates that we are currently developing. In the present study, the 6-deoxyheptan capsular polysaccharide (CPS) from B. pseudomallei was purified, chemically activated, and covalently linked to recombinant CRM197 diphtheria toxin mutant (CRM197) to produce CPS-CRM197. Additionally, tandem nickel-cobalt affinity chromatography was used to prepare highly purified recombinant B. pseudomallei Hcp1 and TssM proteins. Immunization of C57BL/6 mice with CPS-CRM197 produced high-titer IgG and opsonizing antibody responses against the CPS component of the glycoconjugate, while immunization with Hcp1 and TssM produced high-titer IgG and robust gamma interferon-secreting T cell responses against the proteins. Extending upon these studies, we found that when mice were vaccinated with a combination of CPS-CRM197 and Hcp1, 100% of the mice survived a lethal inhalational challenge with B. pseudomallei . Remarkably, 70% of the survivors had no culturable bacteria in their lungs, livers, or spleens, indicating that the vaccine formulation had generated sterilizing immune responses. Collectively, these studies help to better establish surrogates of antigen-induced immunity against B. pseudomallei as well as provide valuable insights toward the development of a safe, affordable, and effective melioidosis vaccine.

Burkholderia pseudomallei is a Gram-negative bacterium that causes melioidosis, a multifaceted disease that is highly endemic in Southeast Asia and northern Australia. This facultative intracellular pathogen possesses a large genome that encodes a wide array of virulence factors that promote survival in vivo by manipulating host cell processes and disarming elements of the host immune system. Antigens and systems that play key roles in B. pseudomallei virulence include capsular polysaccharide, lipopolysaccharide, adhesins, specialized secretion systems, actin-based motility and various secreted factors. This review provides an overview of the current and steadily expanding knowledge regarding the molecular mechanisms used by this organism to survive within a host and their contribution to the pathogenesis of melioidosis.

Burkholderia pseudomallei , the etiologic agent of melioidosis, is an opportunistic pathogen that harbors a wide array of secretion systems, including a type II secretion system (T2SS), three type III secretion systems (T3SS), and six type VI secretion systems (T6SS). The proteins exported by these systems provide B. pseudomallei with a growth advantage in vitro and in vivo , but relatively little is known about the full repertoire of exoproducts associated with each system. In this study, we constructed deletion mutations in gspD and gspE , T2SS genes encoding an outer membrane secretin and a cytoplasmic ATPase, respectively. The secretion profiles of B. pseudomallei MSHR668 and its T2SS mutants were noticeably different when analyzed by SDS-PAGE. We utilized liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify proteins present in the supernatants of B. pseudomallei MSHR668 and B. pseudomallei Δ gspD grown in rich and minimal media. The MSHR668 supernatants contained 48 proteins that were either absent or substantially reduced in the supernatants of Δ gspD strains. Many of these proteins were putative hydrolytic enzymes, including 12 proteases, two phospholipases, and a chitinase. Biochemical assays validated the LC-MS/MS results and demonstrated that the export of protease, phospholipase C, and chitinase activities is T2SS dependent. Previous studies had failed to identify the mechanism of secretion of TssM, a deubiquitinase that plays an integral role in regulating the innate immune response. Here we present evidence that TssM harbors an atypical signal sequence and that its secretion is mediated by the T2SS. This study provides the first in-depth characterization of the B. pseudomallei T2SS secretome.

Increasing evidence has shown that small-molecule chemistry in microbes (i.e., secondary metabolism) can modulate the microbe-host response in infection and pathogenicity. The bacterial disease melioidosis is conferred by the highly virulent, antibiotic-resistant pathogen Burkholderia pseudomallei (BP). Whereas some macromolecular structures have been shown to influence BP virulence (e.g., secretion systems, cellular capsule, pili), the role of the large cryptic secondary metabolome encoded within its genome has been largely unexplored for its importance to virulence. Herein we demonstrate that BP-encoded small-molecule biosynthesis is indispensible for in vivo BP pathogenicity. Promoter exchange experiments were used to induce high-level molecule production from two gene clusters (MPN and SYR) found to be essential for in vivo virulence. NMR structural characterization of these metabolites identified a new class of lipopeptide biosurfactants/biofilm modulators (the malleipeptins) and syrbactin-type proteasome inhibitors, both of which represent overlooked small-molecule virulence factors for BP. Disruption of Burkholderia virulence by inhibiting the biosynthesis of these small-molecule biosynthetic pathways may prove to be an effective strategy for developing novel melioidosis-specific therapeutics.

Burkholderia mallei is an infectious intracellular pathogen whose virulence and resistance to antibiotics makes it a potential bioterrorism agent. Given its genetic origin as a commensal soil organism, it is equipped with an extensive and varied set of adapted mechanisms to cope with and modulate host-cell environments. One essential virulence mechanism constitutes the specialized secretion systems that are designed to penetrate host-cell membranes and insert pathogen proteins directly into the host cell's cytosol. However, the secretion systems' proteins and, in particular, their host targets are largely uncharacterized. Here, we used a combined in silico, in vitro, and in vivo approach to identify B. mallei proteins required for pathogenicity. We used bioinformatics tools, including orthology detection and ab initio predictions of secretion system proteins, as well as published experimental Burkholderia data to initially select a small number of proteins as putative virulence factors. We then used yeast two-hybrid assays against normalized whole human and whole murine proteome libraries to detect and identify interactions among each of these bacterial proteins and host proteins. Analysis of such interactions provided both verification of known virulence factors and identification of three new putative virulence proteins. We successfully created insertion mutants for each of these three proteins using the virulent B. mallei ATCC 23344 strain. We exposed BALB/c mice to mutant strains and the wild-type strain in an aerosol challenge model using lethal B. mallei doses. In each set of experiments, mice exposed to mutant strains survived for the 21-day duration of the experiment, whereas mice exposed to the wild-type strain rapidly died. Given their in vivo role in pathogenicity, and based on the yeast two-hybrid interaction data, these results point to the importance of these pathogen proteins in modulating host ubiquitination pathways, phagosomal escape, and actin-cytoskeleton rearrangement processes.

Burkholderia mallei and B. pseudomallei are the causative agents of glanders and melioidosis, respectively, diseases with high morbidity and mortality rates. B. mallei and B. pseudomallei are closely related genetically; B. mallei evolved from an ancestral strain of B. pseudomallei by genome reduction and adaptation to an obligate intracellular lifestyle. Although these two bacteria cause different diseases, they share multiple virulence factors, including bacterial secretion systems, which represent key components of bacterial pathogenicity. Despite recent progress, the secretion system proteins for B. mallei and B. pseudomallei, their pathogenic mechanisms of action, and host factors are not well characterized. We previously developed a manually curated database, DBSecSys, of bacterial secretion system proteins for B. mallei. Here, we report an expansion of the database with corresponding information about B. pseudomallei. DBSecSys 2.0 contains comprehensive literature-based and computationally derived information about B. mallei ATCC 23344 and literature-based and computationally derived information about B. pseudomallei K96243. The database contains updated information for 163 B. mallei proteins from the previous database and 61 additional B. mallei proteins, and new information for 281 B. pseudomallei proteins associated with 5 secretion systems, their 1,633 human- and murine-interacting targets, and 2,400 host-B. mallei interactions and 2,286 host-B. pseudomallei interactions. The database also includes information about 13 pathogenic mechanisms of action for B. mallei and B. pseudomallei secretion system proteins inferred from the available literature or computationally. Additionally, DBSecSys 2.0 provides details about 82 virulence attenuation experiments for 52 B. mallei secretion system proteins and 98 virulence attenuation experiments for 61 B. pseudomallei secretion system proteins. We updated the Web interface and data access layer to speed-up users’ search of detailed information for orthologous proteins related to secretion systems of the two pathogens. The updates of DBSecSys 2.0 provide unique capabilities to access comprehensive information about secretion systems of B. mallei and B. pseudomallei. They enable studies and comparisons of corresponding proteins of these two closely related pathogens and their host-interacting partners. The database is available at http://dbsecsys.bhsai.org .

Burkholderia mallei and B. pseudomallei cause glanders and melioidosis, respectively, in humans and animals. A hallmark of pathogenesis is the formation of granulomas containing multinucleated giant cells (MNGCs) and cell death. These processes depend on type 6 secretion system 1 (T6SS-1), which is required for virulence in animals. We examined the cell biology of MNGC formation and cell death. We found that chloroquine diphosphate (CLQ), an antimalarial drug, inhibits Burkholderia growth, phagosomal escape, and subsequent MNGC formation. This depends on CLQ's ability to neutralize the acid pH because other alkalinizing compounds similarly inhibit escape and MNGC formation. CLQ inhibits bacterial virulence protein expression because T6SS-1 and some effectors of type 3 secretion system 3 (T3SS-3), which is also required for virulence, are expressed at acid pH. We show that acid pH upregulates the expression of Hcp1 of T6SS-1 and TssM, a protein coregulated with T6SS-1. Finally, we demonstrate that CLQ treatment of Burkholderia -infected Madagascar hissing cockroaches (HCs) increases their survival. This study highlights the multiple mechanisms by which CLQ inhibits growth and virulence and suggests that CLQ be further tested and considered, in conjunction with antibiotic use, for the treatment of diseases caused by Burkholderia .

The genetic similarity between Burkholderia mallei (glanders) and Burkholderia pseudomallei (melioidosis) had led to the general assumption that pathogenesis of each bacterium would be similar. In 2000, the first human case of glanders in North America since 1945 was reported in a microbiology laboratory worker. Leveraging the availability of pre-exposure sera for this individual and employing the same well-characterized protein array platform that has been previously used to study a large cohort of melioidosis patients in southeast Asia, we describe the antibody response in a human with glanders. Analysis of 156 peptides present on the array revealed antibodies against 17 peptides with a > 2-fold increase in this infection. Unexpectedly, when the glanders data were compared with a previous data set from B. pseudomallei infections, there were only two highly increased antibodies shared between these two infections. These findings have implications in the diagnosis and treatment of B. mallei and B. pseudomallei infections.

Previous studies have shown that the O polysaccharides (OPS) expressed by Burkholderia mallei are similar to those produced by Burkholderia thailandensis except that they lack the 4- O -acetyl modifications on their 6-deoxy-α- l -talopyranosyl residues. In the present study, we describe the identification and characterization of an open reading frame, designated oacA , expressed by B. thailandensis that accounts for this phenomenon. Utilizing the B. thailandensis and B. mallei lipopolysaccharide (LPS)-specific monoclonal antibodies Pp-PS-W and 3D11, Western immunoblot analyses demonstrated that the LPS antigens expressed by the oacA mutant, B. thailandensis ZT0715, were antigenically similar to those produced by B. mallei ATCC 23344. In addition, immunoblot analyses demonstrated that when B. mallei ATCC 23344 was complemented in trans with oacA , it synthesized B. thailandensis -like LPS antigens. To elucidate the structure of the OPS moieties expressed by ZT0715, purified samples were analyzed via nuclear magnetic resonance spectroscopy. As predicted, these studies demonstrated that the loss of OacA activity influenced the O acetylation phenotype of the OPS moieties. Unexpectedly, however, the results indicated that the O methylation status of the OPS antigens was also affected by the loss of OacA activity. Nonetheless, it was revealed that the LPS moieties expressed by the oacA mutant reacted strongly with the B. mallei LPS-specific protective monoclonal antibody 9C1-2. Based on these findings, it appears that OacA is required for the 4- O acetylation and 2- O methylation of B. thailandensis OPS antigens and that ZT0715 may provide a safe and cost-effective source of B. mallei -like OPS to facilitate the synthesis of glanders subunit vaccine candidates.

We collected epidemiologic and molecular data from Burkholderia mallei isolates from equines in Punjab, Pakistan from 1999 through 2007. We show that recent outbreaks are genetically distinct from available whole genome sequences and that these genotypes are persistent and ubiquitous in Punjab, probably due to human-mediated movement of equines.

Burkholderia mallei, a category B biothreat agent, is a facultative intracellular pathogen that causes the zoonotic disease glanders. TheB. malleiVirAG two-component regulatory system activates the transcription of ∼60 genes, including a large virulence gene cluster encoding a type VI secretion system (T6SS). TheB. mallei tssMgene encodes a putative ubiquitin-specific protease that is physically linked to, and transcriptionally coregulated with, the T6SS gene cluster. Mass spectrometry and immunoblot analysis demonstrated that TssM was secreted in avirAG-dependent manner in vitro. Surprisingly, the T6SS was found to be dispensable for the secretion of TssM. The C-terminal half of TssM, which contains Cys and His box motifs conserved in eukaryotic deubiquitinases, was purified and biochemically characterized. Recombinant TssM hydrolyzed multiple ubiquitinated substrates and the cysteine at position 102 was critical for enzymatic activity. ThetssMgene was expressed within 1 h after uptake ofB. malleiinto RAW 264.7 murine macrophages, suggesting that the TssM deubiquitinase is produced in this intracellular niche. Although the physiological substrate(s) is currently unknown, the TssM deubiquitinase may provideB. malleia selective advantage in the intracellular environment during infection.

Burkholderia mallei is the etiologic agent of glanders in solipeds (horses, mules and donkeys), and incidentally in carnivores and humans. Little is known about the molecular mechanisms of B. mallei pathogenesis. The putative carboxy-terminal processing protease (CtpA) of B. mallei is a member of a novel family of endoproteases involved in the maturation of proteins destined for the cell envelope. All species and isolates of Burkholderia carry a highly conserved copy of ctpA. We studied the involvement of CtpA on growth, cell morphology, persistence, and pathogenicity of B. mallei. A sucrose-resistant strain of B. mallei was constructed by deleting a major portion of the sacB gene of the wild type strain ATCC 23344 by gene replacement, and designated as strain 23344DeltasacB. A portion of the ctpA gene (encoding CtpA) of strain 23344DeltasacB was deleted by gene replacement to generate strain 23344DeltasacBDeltactpA. In contrast to the wild type ATCC 23344 or the sacB mutant 23344DeltasacB, the ctpA mutant 23344DeltasacBDeltactpA displayed altered cell morphologies with partially or fully disintegrated cell envelopes. Furthermore, relative to the wild type, the ctpA mutant displayed slower growth in vitro and less ability to survive in J774.2 murine macrophages. The expression of mRNA of adtA, the gene downstream of ctpA was similar among the three strains suggesting that disruption of ctpA did not induce any polar effects. As with the wild type or the sacB mutant, the ctpA mutant exhibited a dose-dependent lethality when inoculated intraperitoneally into CD1 mice. The CD1 mice inoculated with a non-lethal dose of the ctpA mutant produced specific serum immunoglobulins IgG1 and IgG2a and were partially protected against challenge with wild type B. mallei ATCC 23344. These findings suggest that CtpA regulates in vitro growth, cell morphology and intracellular survival of B. mallei, and a ctpA mutant protects CD1 mice against glanders.

Burkholderia pseudomallei is the causative agent of melioidosis, a bacterial infection endemic in tropical regions including southeast Asia and northern Australia. B. pseudomallei contains structurally unique polysaccharides (capsular polysaccharide and O−antigen saccharides of lipopolysaccharide). A polysaccharide microarray platform was developed by immobilizing these polysaccharides onto glass slides. Employing this microarray, we were able to demonstrate the presence of antibodies to these polysaccharide antigens in the sera of melioidosis patients, but not in serum from nonmelioidosis human subjects. The advantages of this polysaccharide microarray technology over the conventional tests for the serodiagnosis of melioidosis are discussed.

Burkholderia pseudomallei is the etiologic agent of the tropical disease melioidosis and is considered to be a potential biological weapon. Two B. pseudomallei-like species, Burkholderia oklahomensis and Burkholderia thailandensis, have been described in the literature. Infections with both of these microorganisms have occurred in the United States, but little is known about the relative virulence of these isolates in animal models of infection. In this study, B. oklahomensis and B. thailandensis CDC2721121 were determined to be avirulent in hamsters and mice at all challenge doses examined. The virulence of B. thailandensis CDC3015869, on the other hand, was more similar to the virulence of isolates of B. thailandensis from Southeast Asia.

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.

A polysaccharide microarray platform was prepared by immobilizing Burkholderia pseudomallei and Burkholderia mallei polysaccharides. This polysaccharide array was tested with success for detecting B. pseudomallei and B. mallei serum (human and animal) antibodies. The advantages of this microarray technology over the current serodiagnosis of the above bacterial infections were discussed.