Burkholderia thailandensis strain E555 is a surrogate for the investigation of Burkholderia pseudomallei replication and survival in macrophages.

BACKGROUND: Burkholderia pseudomallei is a human pathogen causing severe infections in tropical and subtropical regions and is classified as a bio-threat agent. B. thailandensis strain E264 has been proposed as less pathogenic surrogate for understanding the interactions of B. pseudomallei with host cells. RESULTS: We show that, unlike B. thailandensis strain E264, the pattern of growth of B. thailandensis strain E555 in macrophages is similar to that of B. pseudomallei. We have genome sequenced B. thailandensis strain E555 and using the annotated sequence identified genes and proteins up-regulated during infection. Changes in gene expression identified more of the known B. pseudomallei virulence factors than changes in protein levels and used together we identified 16% of the currently known B. pseudomallei virulence factors. These findings demonstrate the utility of B. thailandensis strain E555 to study virulence of B. pseudomallei. CONCLUSIONS: A weakness of studies using B. thailandensis as a surrogate for B. pseudomallei is that the strains used replicate at a slower rate in infected cells. We show that the pattern of growth of B. thailandensis strain E555 in macrophages closely mirrors that of B. pseudomallei. Using this infection model we have shown that virulence factors of B. pseudomallei can be identified as genes or proteins whose expression is elevated on the infection of macrophages. This finding confirms the utility of B. thailandensis strain E555 as a surrogate for B. pseudomallei and this strain should be used for future studies on virulence mechanisms.

Burkholderia pseudomallei BimC Is Required for Actin-Based Motility, Intracellular Survival, and Virulence.

The intracellular pathogen Burkholderia pseudomallei, the etiological agent of melioidosis in humans and various animals, is capable of survival and movement within the cytoplasm of host cells by a process known as actin-based motility. The bacterial factor BimA is required for actin-based motility through its direct interaction with actin, and by mediating actin polymerization at a single pole of the bacterium to promote movement both within and between cells. However, little is known about the other bacterial proteins required for this process. Here, we have investigated the role of the bimC gene (bpss1491) which lies immediately upstream of the bimA gene (bpss1492) on the B. pseudomallei chromosome 2. Conserved amongst all B. pseudomallei, B. mallei and B. thailandensis strains sequenced to date, this gene encodes an iron-binding protein with homology to a group of proteins known as the bacterial autotransporter heptosyltransferase (BAHT) family. We have constructed a B. pseudomallei bimC deletion mutant and demonstrate that it is defective in intracellular survival in HeLa cells, but not in J774.1 macrophage-like cells. The bimC mutant is defective in cell to cell spread as demonstrated by ablation of plaque formation in HeLa cells, and by the inability to form multi-nucleated giant cells in J774.1 cells. These phenotypes in intracellular survival and cell to cell spread are not due to the loss of expression and polar localization of the BimA protein on the surface of intracellular bacteria, however they do correlate with an inability of the bacteria to recruit and polymerize actin. Furthermore, we also establish a role for bimC in virulence of B. pseudomallei using a Galleria mellonella larvae model of infection. Taken together, our findings indicate that B. pseudomallei BimC plays an important role in intracellular behavior and virulence of this emerging pathogen.

Lipid A Remodeling Is a Pathoadaptive Mechanism That Impacts Lipopolysaccharide Recognition and Intracellular Survival of Burkholderia pseudomallei.

Burkholderia pseudomallei causes the severe disease melioidosis. The bacterium subverts the host immune system and replicates inside cells, and host mortality results primarily from sepsis-related complications. Lipopolysaccharide (LPS) is a major virulence factor and mediator of sepsis that many pathogens capable of intracellular growth modify to reduce their immunological "footprint." The binding strength of B. pseudomallei LPS for human LPS binding protein (hLBP) was measured using surface plasmon resonance. The structures of lipid A isolated from B. pseudomallei under different temperatures were analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), and the gene expression of two lipid A remodeling genes, lpxO and pagL, was investigated. The LPS was characterized for its ability to trigger tumor necrosis factor alpha (TNF-α) release and to activate caspase-11-triggered pyroptosis by introduction of LPS into the cytosol. Lipid A from long-term chronic-infection isolates was isolated and characterized by MALDI-TOF MS and also by the ability to trigger caspase-11-mediated cell death. Lipid A from B. pseudomallei 1026b lpxO and pagL mutants were characterized by positive- and negative-mode MALDI-TOF MS to ultimately identify their role in lipid A structural modifications. Replication of lpxO and pagL mutants and their complements within macrophages showed that lipid A remodeling can effect growth in host cells and activation of caspase-11-mediated cytotoxicity.

Mitogen-activated protein kinases (MAPKs) are modulated during in vitro and in vivo infection with the intracellular bacterium Burkholderia pseudomallei.

Burkholderia pseudomallei is a Gram-negative intracellular bacterium that causes the disease melioidosis. The disease can be fatal if left untreated or when antibiotic therapy is delayed and total clearance of the pathogen from the host is often not accomplished with current therapies. Thus, new therapeutic approaches for the treatment of infections caused by B. pseudomallei are required. To better understand host responses to B. pseudomallei infection, the activation of key proteins involved in the TLR inflammatory cascade was measured by western blotting. Activation of the mitogen-activated protein kinases (MAPKs) p38 and ERK were both significantly altered during both in vitro and in vivo infection. In considering an approach for therapy of B. pseudomallei infection the inhibition of ERK was achieved in vitro using the inhibitor PD0325901, along with decreased TNF-α production. However, the reduction in phosphorylated ERK and TNF-α release did not correspond with decreased bacterial replication or enhance clearance from infected macrophages. Despite this apparent lack of effect on the intracellular growth of B. pseudomallei in vitro, it is not clear what effect inhibition of ERK activation might have on outcome of disease in vivo. It may be that decreasing the levels of TNF-α in vivo could aid in reducing the overactive immune response that is known to ensue following B. pseudomallei infection, thereby increasing host survival.

Entry, Intracellular Survival, and Multinucleated-Giant-Cell-Forming Activity of Burkholderia pseudomallei in Human Primary Phagocytic and Nonphagocytic Cells.

The human pathogen Burkholderia pseudomallei and the related species Burkholderia thailandensis are facultative intracellular bacteria characterized by the ability to escape into the cytosol of the host cell and to stimulate the formation of multinucleated giant cells (MNGCs). MNGC formation is induced via an unknown mechanism by bacterial type VI secretion system 5 (T6SS-5), which is an essential virulence factor in both species. Despite the vital role of the intracellular life cycle in the pathogenesis of the bacteria, the range of host cell types permissive for initiation and completion of the intracellular cycle is poorly defined. In the present study, we used several different types of human primary cells to evaluate bacterial entry, intracellular survival, and MNGC formation. We report the capacity of B. pseudomallei to enter, efficiently replicate in, and mediate MNGC formation of vein endothelial and bronchial epithelial cells, indicating that the T6SS-5 is important in the host-pathogen interaction in these cells. Furthermore, we show that B. pseudomallei invades fibroblasts and keratinocytes and survives inside these cells as well as in monocyte-derived macrophages and neutrophils for at least 17 h postinfection; however, MNGC formation is not induced in these cells. In contrast, infection of mixed neutrophils and RAW264.7 macrophages with B. thailandensis stimulated the formation of heterotypic MNGCs in a T6SS-5-dependent manner. In summary, the ability of the bacteria to enter and survive as well as induce MNGC formation in certain host cells may contribute to the pathogenesis observed in B. pseudomallei infection.

A proteasome inhibitor produced by Burkholderia pseudomallei modulates intracellular growth.

The NRPS/PKS cluster encodes the enzymes necessary for glidobactin synthesis it is partially conserved in various members of the Burkholderia genus including B. pseudomallei. In this study we have shown that the insertional inactivation or deletion of glbC in this cluster in B. pseudomallei could reduce the ability of the bacterium to survive or grow in murine macrophages or in human neutrophils. Exogenously added proteasome inhibitors were able to chemically complement the mutation. The insertional inactivation or deletion of glbC increased virulence in an acute model of infection in Balb/c or C57BL/6 mice but virulence in a chronic model of infection was similar to that of the wild type. Our findings contrast with the previous finding that inactivation of the glb gene cluster in B. pseudomallei strain 1026b resulted in marked attenuation, and provides evidence of differential roles for some genes in virulence of different strains of B. pseudomallei.

Trehalase plays a role in macrophage colonization and virulence of Burkholderia pseudomallei in insect and mammalian hosts.

Trehalose is a disaccharide formed from two glucose molecules. This sugar molecule can be isolated from a range of organisms including bacteria, fungi, plants and invertebrates. Trehalose has a variety of functions including a role as an energy storage molecule, a structural component of glycolipids and plays a role in the virulence of some microorganisms. There are many metabolic pathways that control the biosynthesis and degradation of trehalose in different organisms. The enzyme trehalase forms part of a pathway that converts trehalose into glucose. In this study we set out to investigate whether trehalase plays a role in both stress adaptation and virulence of Burkholderia pseudomallei. We show that a trehalase deletion mutant (treA) had increased tolerance to thermal stress and produced less biofilm than the wild type B. pseudomallei K96243 strain. We also show that the ΔtreA mutant has reduced ability to survive in macrophages and that it is attenuated in both Galleria mellonella (wax moth larvae) and a mouse infection model. This is the first report that trehalase is important for bacterial virulence.

A predicted cation transporter protein, BPSS1228, is involved in intracellular behaviour of Burkholderia pseudomallei in a human lung epithelial cell line (A549).

Burkholderia pseudomallei causes melioidosis, a potentially fatal infectious disease in tropical and subtropical countries worldwide. The intracellular behaviour of this pathogen in host cells has been reported to impact the severity of melioidosis, including the development of septicaemia, a consequence of pneumonia melioidosis. We previously identified a predicted cation transporter protein, BPSS1228, that participates in the transitional stage of this intracellular pathogen. For further analysis, in this study B. pseudomallei bpss1228 mutant and complemented strains were constructed and bacterial infectivity on human lung epithelial cells, A549, investigated in vitro Burkholderia pseudomallei bpss1228 mutant showed impaired bacterial adhesion and invasion into A549 cells compared with wild-type strain, while the deficient phenotypes were restored to wild-type levels by the complemented strain. Additionally, the inactivation of bpss1228 in the mutant strain affected flagella-based swimming on a semi-solid surface and resistance to acid stresses simulating intracellular environments. These observations of BPSS1228 relating to B. pseudomallei infection strategies shed a new light on its association with intracellular B. pseudomallei during the interaction with host cells.

Antibiotic resistance in Burkholderia species.

The genus Burkholderia comprises metabolically diverse and adaptable Gram-negative bacteria, which thrive in often adversarial environments. A few members of the genus are prominent opportunistic pathogens. These include Burkholderia mallei and Burkholderia pseudomallei of the B. pseudomallei complex, which cause glanders and melioidosis, respectively. Burkholderia cenocepacia, Burkholderia multivorans, and Burkholderia vietnamiensis belong to the Burkholderia cepacia complex and affect mostly cystic fibrosis patients. Infections caused by these bacteria are difficult to treat because of significant antibiotic resistance. The first line of defense against antimicrobials in Burkholderia species is the outer membrane penetration barrier. Most Burkholderia contain a modified lipopolysaccharide that causes intrinsic polymyxin resistance. Contributing to reduced drug penetration are restrictive porin proteins. Efflux pumps of the resistance nodulation cell division family are major players in Burkholderia multidrug resistance. Third and fourth generation ß-lactam antibiotics are seminal for treatment of Burkholderia infections, but therapeutic efficacy is compromised by expression of several ß-lactamases and ceftazidime target mutations. Altered DNA gyrase and dihydrofolate reductase targets cause fluoroquinolone and trimethoprim resistance, respectively. Although antibiotic resistance hampers therapy of Burkholderia infections, the characterization of resistance mechanisms lags behind other non-enteric Gram-negative pathogens, especially ESKAPE bacteria such as Acinetobacter baumannii, Klebsiella pneumoniae and Pseudomonas aeruginosa.

Mapping epigenetic changes to the host cell genome induced by Burkholderia pseudomallei reveals pathogen-specific and pathogen-generic signatures of infection.

The potential for epigenetic changes in host cells following microbial infection has been widely suggested, but few examples have been reported. We assessed genome-wide patterns of DNA methylation in human macrophage-like U937 cells following infection with Burkholderia pseudomallei, an intracellular bacterial pathogen and the causative agent of human melioidosis. Our analyses revealed significant changes in host cell DNA methylation, at multiple CpG sites in the host cell genome, following infection. Infection induced differentially methylated probes (iDMPs) showing the greatest changes in DNA methylation were found to be in the vicinity of genes involved in inflammatory responses, intracellular signalling, apoptosis and pathogen-induced signalling. A comparison of our data with reported methylome changes in cells infected with M. tuberculosis revealed commonality of differentially methylated genes, including genes involved in T cell responses (BCL11B, FOXO1, KIF13B, PAWR, SOX4, SYK), actin cytoskeleton organisation (ACTR3, CDC42BPA, DTNBP1, FERMT2, PRKCZ, RAC1), and cytokine production (FOXP1, IRF8, MR1). Overall our findings show that pathogenic-specific and pathogen-common changes in the methylome occur following infection.

Burkholderia pseudomallei Differentially Regulates Host Innate Immune Response Genes for Intracellular Survival in Lung Epithelial Cells.

BACKGROUND: Burkholderia pseudomallei, the causative agent of melioidosis poses a serious threat to humankind. B. pseudomallei secretes numerous virulence proteins that alter host cell functions to escape from intracellular immune sensors. However, the events underlying disease pathogenesis are poorly understood. METHODS: We determined the ability of B. pseudomallei to invade and survive intracellularly in A549 human lung epithelial cells, and also investigated the early transcriptional responses using an Illumina HumanHT-12 v4 microarray platform, after three hours of exposure to live B. pseudomallei (BCMS) and its secreted proteins (CCMS). RESULTS: We found that the ability of B. pseudomallei to invade and survive intracellularly correlated with increase of multiplicity of infection and duration of contact. Activation of host carbohydrate metabolism and apoptosis as well as suppression of amino acid metabolism and innate immune responses both by live bacteria and its secreted proteins were evident. These early events might be linked to initial activation of host genes directed towards bacterial dissemination from lungs to target organs (via proposed in vivo mechanisms) or to escape potential sensing by macrophages. CONCLUSION: Understanding the early responses of A549 cells toward B. pseudomallei infection provide preliminary insights into the likely pathogenesis mechanisms underlying melioidosis, and could contribute to development of novel intervention strategies to combat B. pseudomallei infections.

Burkholderia pseudomallei Colony Morphotypes Show a Synchronized Metabolic Pattern after Acute Infection.

BACKGROUND: Burkholderia pseudomallei is a water and soil bacterium and the causative agent of melioidosis. A characteristic feature of this bacterium is the formation of different colony morphologies which can be isolated from environmental samples as well as from clinical samples, but can also be induced in vitro. Previous studies indicate that morphotypes can differ in a number of characteristics such as resistance to oxidative stress, cellular adhesion and intracellular replication. Yet the metabolic features of B. pseudomallei and its different morphotypes have not been examined in detail so far. Therefore, this study aimed to characterize the exometabolome of B. pseudomallei morphotypes and the impact of acute infection on their metabolic characteristics. METHODS AND PRINCIPAL FINDINGS: We applied nuclear magnetic resonance spectroscopy (1H-NMR) in a metabolic footprint approach to compare nutrition uptake and metabolite secretion of starvation induced morphotypes of the B. pseudomallei strains K96243 and E8. We observed gluconate production and uptake in all morphotype cultures. Our study also revealed that among all morphotypes amino acids could be classified with regard to their fast and slow consumption. In addition to these shared metabolic features, the morphotypes varied highly in amino acid uptake profiles, secretion of branched chain amino acid metabolites and carbon utilization. After intracellular passage in vitro or murine acute infection in vivo, we observed a switch of the various morphotypes towards a single morphotype and a synchronization of nutrient uptake and metabolite secretion. CONCLUSION: To our knowledge, this study provides first insights into the basic metabolism of B. pseudomallei and its colony morphotypes. Furthermore, our data suggest, that acute infection leads to the synchronization of B. pseudomallei colony morphology and metabolism through yet unknown host signals and bacterial mechanisms.

Gene Microarray Analyses of Daboia russelli russelli Daboiatoxin Treatment of THP-1 Human Macrophages Infected with Burkholderia pseudomallei.

Burkholderia pseudomallei is the causative agent of melioidosis and represents a potential bioterrorism threat. In this study, the transcriptomic responses of B. pseudomallei infection of a human macrophage cell model were investigated using whole-genome microarrays. Gene expression profiles were compared between infected THP-1 human monocytic leukemia cells with or without treatment with Daboia russelli russelli daboiatoxin (DRRDbTx) or ceftazidime (antibiotic control). Microarray analyses of infected and treated cells revealed differential upregulation of various inflammatory genes such as interleukin-1 (IL-1), IL-6, tumor necrosis factor-alpha (TNF-α), cyclooxygenase (COX-2), vascular endothelial growth factor (VEGF), chemokine C-X-C motif ligand 4 (CXCL4), transcription factor p65 (NF-kB); and several genes involved in immune and stress responses, cell cycle, and lipid metabolism. Moreover, following DRR-DbTx treatment of infected cells, there was enhanced expression of the tolllike receptor 2 (TLR-2) mediated signaling pathway involved in recognition and initiation of acute inflammatory responses. Importantly, we observed that highly inflammatory cytokine gene responses were similar in infected cells exposed to DRR-DbTx or ceftazidime after 24 h. Additionally, there were increased transcripts associated with cell death by caspase activation that can promote host tissue injury. In summary, the transcriptional responses during B. pseudomallei infection of macrophages highlight a broad range of innate immune mechanisms that are activated within 24 h post-infection. These data provide insights into the transcriptomic kinetics following DRR-DbTx treatment of human macrophages infected with B. pseudomallei.

Host Cytosolic Glutathione Sensing by a Membrane Histidine Kinase Activates the Type VI Secretion System in an Intracellular Bacterium.

Type VI secretion systems (T6SSs) are major virulence mechanisms in many Gram-negative bacteria, but the physiological signals that activate them are not well understood. The T6SS1 of Burkholderia pseudomallei is essential for pathogenesis in mammalian hosts and is only expressed when the bacterium is intracellular. We found that signals for T6SS1 activation reside in the host cytosol. Through site-directed mutagenesis and biochemical studies, we identified low molecular weight thiols, particularly glutathione, as the signal sensed by a periplasmic cysteine residue (C62) on the histidine kinase sensor VirA. Upon glutathione exposure, dimeric VirA is converted to monomers via reduction at C62. When glutathione in the host was depleted, T6SS1 expression was abrogated, and bacteria could no longer induce multinucleate giant cell formation, the hallmark of T6SS1 function. Therefore, intracellular bacteria exploit the abundance of glutathione in host cytosol as a signal for expression of virulence at the appropriate time and place.

Transcription level analysis of intracellular Burkholderia pseudomallei illustrates the role of BPSL1502 during bacterial interaction with human lung epithelial cells.

Melioidosis caused by Burkholderia pseudomallei is a globally important disease of increasing concern according to high case-fatality rate and epidemic spreading. The ability of B. pseudomallei to attach and invade host cells and subsequently survive intracellularly has stimulated many questions concerning the comprehension of bacterial pathogenesis progression. Transcription levels of intracellular B. pseudomallei genes in human lung epithelial cells were therefore analyzed using bioinformatic tools, RT-PCR and real time RT-PCR. Here, it is reported that the identification of bpsl1502, encoding B. pseudomallei SurE (stationary phase survival protein E) located in a global transcriptional regulation operon was accomplished. The up-regulation of B. pseudomallei SurE was demonstrated during intracellular survival of A549 cells at 12, 18, and 24 h post-infection. To investigate the role of this protein, a B. pseudomallei SurE defective mutant was constructed. The invasion and initial survival of the SurE mutants within the A549 cells were impaired. There was no difference, however, between the growth of B. pseudomallei SurE mutant as compared to the wild type in Luria-Bertani culture. These data suggest that SurE may assist B. pseudomallei host cells invade and facilitate early intracellular infection but is not crucial during the stationary growth phase. The identification of B. pseudomallei SurE provides more information of bacterial strategy during an early step of the pathogenesis process of melioidosis.

Transcriptome analysis of Burkholderia pseudomallei T6SS identifies Hcp1 as a potential serodiagnostic marker.

Burkholderia pseudomallei, the causative agent of melioidosis, is able to survive extreme environments and utilizes various virulence factors for survival and pathogenicity. To compete and survive within these different ecological niches, B. pseudomallei has evolved specialized pathways, including the Type VI secretion systems (T6SSs), that have a role in pathogenesis as well as interbacterial interactions. We examined the expression profile of B. pseudomallei T6SS six gene clusters during infection of U937 macrophage cells. T6SS-5 was robustly transcribed while the other five clusters were not significantly regulated proposing the utility of T6SS-5 as a potential biomarker of exposure to B. pseudomallei. Transcription of T6SS regulators VirAG and BprB was also not significant during infection when compared to bacteria grown in culture. Guided by these findings, three highly expressed T6SS genes, tssJ-4, hcp1 and tssE-5, were expressed as recombinant proteins and screened against melioidosis patient sera by western analysis and ELISA. Only Hcp1 was reactive by both types of analysis. The recombinant Hcp1 protein was further evaluated against a cohort of melioidosis patients (n = 32) and non-melioidosis individuals (n = 20) sera and the data clearly indicates a higher sensitivity (93.7%) and specificity (100%) for Hcp1 compared to bacterial lysate. The detection of anti-Hcp1 antibodies in patients' sera indicating the presence of B. pseudomallei highlights the potential of Hcp1 to be further developed as a serodiagnostic marker for melioidosis.

Characterization of a novel two-component system response regulator involved in biofilm formation and a low-iron response of Burkholderia pseudomallei.

Two-component systems (TCSs) regulate an adaptive response to environmental conditions, leading to changes in bacterial cellular processes. In this study, we identified a novel TCS response regulator gene, designated as bfmR (biofilm formation-associated regulator) that regulates biofilm formation by Burkholderia pseudomallei (Bp). An insertion mutant of the Bp bfmR gene resulted in a significant decrease in expression of fimbriae chaperone-usher assembly genes (BPSL2O28 and BPSL22 7), leading to suppression of assembly of fimbriae on the cell surface and reduced biofilm formation. The defective phenotypes of the mutant strain were restored by introducing a complementing plasmid having an intact bfmR gene. Using RT-PCR analyses, we found that bfmR gene expression was upregulated under low-iron growth conditions. In addition, the bfmR mutant strain showed retarded growth in low-iron medium and in phagocytic cells compared to the wild-type strain. These results indicate that bfmR is a novel positive regulator for controlling assembly of fimbriae and biofilm formation, and is upregulated under low-iron conditions.

Burkholderia mallei and Burkholderia pseudomallei cluster 1 type VI secretion system gene expression is negatively regulated by iron and zinc.

Burkholderia mallei is a facultative intracellular pathogen that causes glanders in humans and animals. Previous studies have demonstrated that the cluster 1 type VI secretion system (T6SS-1) expressed by this organism is essential for virulence in hamsters and is positively regulated by the VirAG two-component system. Recently, we have shown that T6SS-1 gene expression is up-regulated following internalization of this pathogen into phagocytic cells and that this system promotes multinucleated giant cell formation in infected tissue culture monolayers. In the present study, we further investigated the complex regulation of this important virulence factor. To assess T6SS-1 expression, B. mallei strains were cultured in various media conditions and Hcp1 production was analyzed by Western immunoblotting. Transcript levels of several VirAG-regulated genes (bimA, tssA, hcp1 and tssM) were also determined using quantitative real time PCR. Consistent with previous observations, T6SS-1 was not expressed during growth of B. mallei in rich media. Curiously, growth of the organism in minimal media (M9G) or minimal media plus casamino acids (M9CG) facilitated robust expression of T6SS-1 genes whereas growth in minimal media plus tryptone (M9TG) did not. Investigation of this phenomenon confirmed a regulatory role for VirAG in this process. Additionally, T6SS-1 gene expression was significantly down-regulated by the addition of iron and zinc to M9CG. Other genes under the control of VirAG did not appear to be as tightly regulated by these divalent metals. Similar results were observed for B. pseudomallei, but not for B. thailandensis. Collectively, our findings indicate that in addition to being positively regulated by VirAG, B. mallei and B. pseudomallei T6SS-1 gene expression is negatively regulated by iron and zinc.

Quorum sensing negatively regulates multinucleate cell formation during intracellular growth of Burkholderia pseudomallei in macrophage-like cells.

Burkholderia pseudomallei is a Gram-negative environmental bacterium and the causative agent of melioidosis, a potentially fatal, acute or chronic disease endemic in the tropics. Acyl homoserine lactone (AHL)-mediated quorum sensing and signalling have been associated with virulence and biofilm formation in numerous bacterial pathogens. In the canonical acyl-homoserine lactone signalling paradigm, AHLs are detected by a response regulator. B. pseudomallei encodes three AHL synthases, encoded by bpsI1, bpsI2 and bpsI3, and five regulator genes. In this study, we mutated the B. pseudomallei AHL synthases individually and in double and triple combination. Five AHLs were detected and quantified by tandem liquid chromatography-mass spectroscopy. The major AHLs produced were N-octanoylhomoserine lactone and N-(3-hydroxy-decanoyl)homoserine lactone, the expression of which depended on bpsI1 and bpsI2, respectively. B. pseudomallei infection of macrophage cells causes cell fusion, leading to multinucleated cells (3 or more nuclei per cell). A triple mutant defective in production of all three AHL synthases was associated with a striking phenotype of massively enhanced host cellular fusion in macrophages. However, neither abrogation of host cell fusion, achieved by mutation of bimA or hcp1, nor enhancement of fusion altered intracellular replication of B. pseudomallei. Furthermore, when tested in murine models of acute melioidosis the AHL synthase mutants were not attenuated for virulence. Collectively, this study identifies important new aspects of the genetic basis of AHL synthesis in B. pseudomallei and the roles of these AHLs in systemic infection and in cell fusion in macrophages for this important human pathogen.

Involvement of signal regulatory protein α, a negative regulator of Toll-like receptor signaling, in impairing the MyD88-independent pathway and intracellular killing of Burkholderia pseudomallei-infected mouse macrophages.

The facultative intracellular gram-negative bacterium Burkholderia pseudomallei is the causative agent of melioidosis and is known for its ability to evade the Toll-like receptor (TLR)-mediated innate immune response. Previously it has been demonstrated that this bacterium was able to suppress the MyD88-independent pathway and can survive macrophage intracellular killing. However, the underlying mechanisms responsible for the suppression of this pathway are not fully understood. In the present study, we showed that both living and heat-killed B. pseudomallei bacteria restrict the TLR signaling response, particularly macrophage inducible nitric oxide synthase (iNOS) expression, by preventing downregulation of constitutively expressed signal regulatory protein α (SIRPα) molecule, a known negative regulator of TLR signaling. In contrast, a lipopolysaccharide (LPS) mutant of B. pseudomallei, a less virulent strain, was able to downregulate SIRPα expression in mouse macrophages. However, depletion of constitutively expressed SIRPα was able to induce the gene expression downstream of TLR signaling pathways (particularly the MyD88-independent pathway), such as that of the iNOS gene, leading to enhanced macrophage intracellular killing of B. pseudomallei. Induction of gene expression was consistent with the enhanced degradation pattern of IκBα with SIRPα depletion. Additionally, the downregulation of SIRPα expression with upregulation of iNOS was observed when the macrophages were pretreated with gamma interferon (IFN-γ) prior to the infection, suggesting that the enhanced intracellular killing of bacteria by IFN-γ is associated with the decreased SIRPα expression. Altogether our findings demonstrate that B. pseudomallei evades macrophage intracellular killing by preventing the downregulation of SIRPα that results in the inhibition of gene expression downstream of the MyD88-independent pathway.