Evaluation of recombinant antigens for diagnosis of melioidosis

Burkholderia pseudomallei, the causative agent of melioidosis, is endemic to Southeast Asia and northern Australia. Clinical manifestations of the disease are diverse, ranging from chronic localized infection to acute septicaemia, with death occurring within 24-48 h after the onset of symptoms. Definitive diagnosis of melioidosis involves bacterial culture and identification, with results obtained within 3-4 days. This delayed diagnosis is a major contributing factor to high mortality rates. Rapid diagnosis is vital for successful management of the disease. This study describes the purification and evaluation of three recombinant antigenic proteins, BPSL0972, BipD and OmpA from B. pseudomallei 08, for their potential in the serodiagnosis of melioidosis using an indirect enzyme-linked immunosorbent assay (ELISA) method. The recombinant proteins were evaluated using 74 serum samples from culture-confirmed melioidosis patients from Malaysia, Thailand and Australia. In addition, 62 nonmelioidosis controls consisting of serum samples from clinically suspected melioidosis patients (n=20) and from healthy blood donors from an endemic region (n=18) and a nonendemic region (n=24) were included. The indirect ELISAs using BipD and BPSL0972 as antigens demonstrated poor to moderate sensitivities (42% and 51%, respectively) but good specificity (both 100%). In contrast, the indirect ELISA using OmpA as an antigen achieved 95% sensitivity and 98% specificity. These results highlight the potential for OmpA to be used in the serodiagnosis of melioidosis in an endemic area.     

Overexpression of the Endothelial Protein C Receptor Is Detrimental during Pneumonia-Derived Gram-negative Sepsis (Melioidosis)

Background

The endothelial protein C receptor (EPCR) enhances anticoagulation by accelerating activation of protein C to activated protein C (APC) and mediates anti-inflammatory effects by facilitating APC-mediated signaling via protease activated receptor-1. We studied the role of EPCR in the host response during pneumonia-derived sepsis instigated by Burkholderia (B.) pseudomallei, the causative agent of melioidosis, a common form of community-acquired Gram-negative (pneumo)sepsis in South-East Asia.

Methodology/Principal Findings

Soluble EPCR was measured in plasma of patients with septic culture-proven melioidosis and healthy controls. Experimental melioidosis was induced by intranasal inoculation of B. pseudomallei in wild-type (WT) mice and mice with either EPCR-overexpression (Tie2-EPCR) or EPCR-deficiency (EPCR^−/−). Mice were sacrificed after 24, 48 or 72 hours. Organs and plasma were harvested to measure colony forming units, cellular influxes, cytokine levels and coagulation parameters. Plasma EPCR-levels were higher in melioidosis patients than in healthy controls and associated with an increased mortality. Tie2-EPCR mice demonstrated enhanced bacterial growth and dissemination to distant organs during experimental melioidosis, accompanied by increased lung damage, neutrophil influx and cytokine production, and attenuated coagulation activation. EPCR^−/− mice had an unremarkable response to B. pseudomallei infection as compared to WT mice, except for a difference in coagulation activation in plasma.

Conclusion/Significance

Increased EPCR-levels correlate with accelerated mortality in patients with melioidosis. In mice, transgenic overexpression of EPCR aggravates outcome during Gram-negative pneumonia-derived sepsis caused by B. pseudomallei, while endogenous EPCR does not impact on the host response. These results add to a better understanding of the regulation of coagulation during severe (pneumo)sepsis.     

Development of a Prototype Lateral Flow Immunoassay (LFI) for the Rapid Diagnosis of Melioidosis

Burkholderia pseudomallei is a soil-dwelling bacterium and the causative agent of melioidosis. Isolation of B. pseudomallei from clinical samples is the “gold standard” for the diagnosis of melioidosis; results can take 3–7 days to produce. Alternatively, antibody-based tests have low specificity due to a high percentage of seropositive individuals in endemic areas. There is a clear need to develop a rapid point-of-care antigen detection assay for the diagnosis of melioidosis. Previously, we employed In vivo Microbial Antigen Discovery (InMAD) to identify potential B. pseudomallei diagnostic biomarkers. The B. pseudomallei capsular polysaccharide (CPS) and numerous protein antigens were identified as potential candidates. Here, we describe the development of a diagnostic immunoassay based on the detection of CPS. Following production of a CPS-specific monoclonal antibody (mAb), an antigen-capture immunoassay was developed to determine the concentration of CPS within a panel of melioidosis patient serum and urine samples. The same mAb was used to produce a prototype Active Melioidosis Detect Lateral Flow Immunoassay (AMD LFI); the limit of detection of the LFI for CPS is comparable to the antigen-capture immunoassay (∼0.2 ng/ml). The analytical reactivity (inclusivity) of the AMD LFI was 98.7% (76/77) when tested against a large panel of B. pseudomallei isolates. Analytical specificity (cross-reactivity) testing determined that 97.2% of B. pseudomallei near neighbor species (35/36) were not reactive. The non-reactive B. pseudomallei strain and the reactive near neighbor strain can be explained through genetic sequence analysis. Importantly, we show the AMD LFI is capable of detecting CPS in a variety of patient samples. The LFI is currently being evaluated in Thailand and Australia; the focus is to optimize and validate testing procedures on melioidosis patient samples prior to initiation of a large, multisite pre-clinical evaluation.     

NLRC4 and TLR5 Each Contribute to Host Defense in Respiratory Melioidosis

Burkholderia pseudomallei causes the tropical infection melioidosis. Pneumonia is a common manifestation of melioidosis and is associated with high mortality. Understanding the key elements of host defense is essential to developing new therapeutics for melioidosis. As a flagellated bacterium encoding type III secretion systems, B. pseudomallei may trigger numerous host pathogen recognition receptors. TLR5 is a flagellin sensor located on the plasma membrane. NLRC4, along with NAIP proteins, assembles a canonical caspase-1-dependent inflammasome in the cytoplasm that responds to flagellin (in mice) and type III secretion system components (in mice and humans). In a murine model of respiratory melioidosis, Tlr5 and Nlrc4 each contributed to survival. Mice deficient in both Tlr5 and Nlrc4 were not more susceptible than single knockout animals. Deficiency of Casp1/Casp11 resulted in impaired bacterial control in the lung and spleen; in the lung much of this effect was attributable to Nlrc4, despite relative preservation of pulmonary IL-1β production in Nlrc4^−/− mice. Histologically, deficiency of Casp1/Casp11 imparted more severe pulmonary inflammation than deficiency of Nlrc4. The human NLRC4 region polymorphism rs6757121 was associated with survival in melioidosis patients with pulmonary involvement. Co-inheritance of rs6757121 and a functional TLR5 polymorphism had an additive effect on survival. Our results show that NLRC4 and TLR5, key components of two flagellin sensing pathways, each contribute to host defense in respiratory melioidosis.     

Expression and Function of Macrophage Migration Inhibitory Factor (MIF) in Melioidosis

Background

Macrophage migration inhibitory factor (MIF) has emerged as a pivotal mediator of innate immunity and has been shown to be an important effector molecule in severe sepsis. Melioidosis, caused by Burkholderia pseudomallei, is an important cause of community-acquired sepsis in Southeast-Asia. We aimed to characterize the expression and function of MIF in melioidosis.

Methodology and Principal Findings

MIF expression was determined in leukocytes and plasma from 34 melioidosis patients and 32 controls, and in mice infected with B. pseudomallei. MIF function was investigated in experimental murine melioidosis using anti-MIF antibodies and recombinant MIF. Patients demonstrated markedly increased MIF mRNA leukocyte and MIF plasma concentrations. Elevated MIF concentrations were associated with mortality. Mice inoculated intranasally with B. pseudomallei displayed a robust increase in pulmonary and systemic MIF expression. Anti-MIF treated mice showed lower bacterial loads in their lungs upon infection with a low inoculum. Conversely, mice treated with recombinant MIF displayed a modestly impaired clearance of B. pseudomallei. MIF exerted no direct effects on bacterial outgrowth or phagocytosis of B. pseudomallei.

Conclusions

MIF concentrations are markedly elevated during clinical melioidosis and correlate with patients'' outcomes. In experimental melioidosis MIF impaired antibacterial defense.     

Immunogenic Burkholderia pseudomallei Outer Membrane Proteins as Potential Candidate Vaccine Targets

Background

Burkholderia pseudomallei is the causative agent of melioidosis, a disease of significant morbidity and mortality in both human and animals in endemic areas. There is no vaccine towards the bacterium available in the market, and the efficacy of many of the bacterium''s surface and secreted proteins are currently being evaluated as vaccine candidates.

Methodology/Principal Findings

With the availability of the B. pseudomallei whole genome sequence, we undertook to identify genes encoding the known immunogenic outer membrane protein A (OmpA). Twelve OmpA domains were identified and ORFs containing these domains were fully annotated. Of the 12 ORFs, two of these OmpAs, Omp3 and Omp7, were successfully cloned, expressed as soluble protein and purified. Both proteins were recognised by antibodies in melioidosis patients'' sera by Western blot analysis. Purified soluble fractions of Omp3 and Omp7 were assessed for their ability to protect BALB/c mice against B. pseudomallei infection. Mice were immunised with either Omp3 or Omp7, subsequently challenged with 1×10⁶ colony forming units (cfu) of B. pseudomallei via the intraperitoneal route, and examined daily for 21 days post-challenge. This pilot study has demonstrated that whilst all control unimmunised mice died by day 9 post-challenge, two mice (out of 4) from both immunised groups survived beyond 21 days post-infection.

Conclusions/Significance

We have demonstrated that B. pseudomallei OmpA proteins are immunogenic in mice as well as melioidosis patients and should be further assessed as potential vaccine candidates against B. pseudomallei infection.     

Osteopontin Impairs Host Defense during Established Gram-Negative Sepsis Caused by Burkholderia pseudomallei (Melioidosis)

Background

Melioidosis, caused by infection with Burkholderia (B.) pseudomallei, is a severe illness that is endemic in Southeast Asia. Osteopontin (OPN) is a phosphorylated glycoprotein that is involved in several immune responses including induction of T-helper 1 cytokines and recruitment of inflammatory cells.

Methodology and Principal Findings

OPN levels were determined in plasma from 33 melioidosis patients and 31 healthy controls, and in wild-type (WT) mice intranasally infected with B. pseudomallei. OPN function was studied in experimental murine melioidosis using WT and OPN knockout (KO) mice. Plasma OPN levels were elevated in patients with severe melioidosis, even more so in patients who went on to die. In patients who recovered plasma OPN concentrations had decreased after treatment. In experimental melioidosis in mice plasma and pulmonary OPN levels were also increased. Whereas WT and OPN KO mice were indistinguishable during the first 24 hours after infection, after 72 hours OPN KO mice demonstrated reduced bacterial numbers in their lungs, diminished pulmonary tissue injury, especially due to less necrosis, and decreased neutrophil infiltration. Moreover, OPN KO mice displayed a delayed mortality as compared to WT mice. OPN deficiency did not influence the induction of proinflammatory cytokines.

Conclusions

These data suggest that sustained production of OPN impairs host defense during established septic melioidosis.     

The role of short-chain dehydrogenase/oxidoreductase, induced by salt stress, on host interaction of B. pseudomallei

Background

Burkholderia pseudomallei is the causative agent of melioidosis, a frequently occurring disease in northeastern Thailand, where soil and water high in salt content are common. Using microarray analysis, we previously showed that B. pseudomallei up-regulated a short-chain dehydrogenase/oxidoreductase (SDO) under salt stress. However, the importance of SDO in B. pseudomallei infection is unknown. This study aimed to explore the function of B. pseudomallei SDO, and to investigate its role in interactions between B. pseudomallei and host cells.

Results

Bioinformatics analysis of B. pseudomallei SDO structure, based on homology modeling, revealed a NAD⁺ cofactor domain and a catalytic triad containing Ser149, Tyr162, and Lys166. This is similar to Bacillus megaterium glucose 1-dehydrogenase. To investigate the role of this protein, we constructed a B. pseudomallei SDO defective mutant, measured glucose dehydrogenase (GDH) activity, and tested the interactions with host cells. The B. pseudomallei K96243 wild type exhibited potent GDH activity under condition containing 300 mM NaCl, while the mutant showed activity levels 15 times lower. Both invasion into the A549 cell line and early intracellular survival within the J774A.1 macrophage cell were impaired in the mutant. Complementation of SDO was able to restore the mutant ability to produce GDH activity, invade epithelial cells, and survive in macrophages.

Conclusions

Our data suggest that induced SDO activity during salt stress may facilitate B. pseudomallei invasion and affect initiation of successful intracellular infection. Identifying the role of B. pseudomallei SDO provides a better understanding of the association between bacterial adaptation and pathogenesis in melioidosis.     

First Genome Sequence of a Burkholderia pseudomallei Isolate in China,Strain BPC006, Obtained from a Melioidosis Patient in Hainan

Melioidosis, caused by Burkholderia pseudomallei, is considered to be endemic to Northern Australia and Southeast Asia, with high mortality and relapse rates, regardless of powerful antibiotic therapy. Here we report the first genome sequence of Burkholderia pseudomallei strain BPC006, obtained from a melioidosis patient in Hainan, China. The genome sizes of the 2 chromosomes were determined to be 4,001,777 bp and 3,153,284 bp.     

Backbone and side chain 1H, 13C, and 15N NMR assignments for the organic hydroperoxide resistance protein (Ohr) from Burkholderia pseudomallei

Burkholderia pseudomallei is a NIAID Category B microorganism responsible for melioidosis. Here we report backbone and side chain NMR assignments for the 139-residue, homodimeric, organic hydroperoxide resistance protein (Ohr) from this organism.     

Laboratory diagnosis of melioidosis: Past,present and future

Melioidosis is an emerging, potentially fatal disease caused by Burkholderia pseudomallei, which requires prolonged antibiotic treatment to prevent disease relapse. However, difficulties in laboratory diagnosis of melioidosis may delay treatment and affect disease outcomes. Isolation of B. pseudomallei from clinical specimens has been improved with the use of selective media. However, even with positive cultures, identification of B. pseudomallei can be difficult in clinical microbiology laboratories, especially in non-endemic areas where clinical suspicion is low. Commercial identification systems may fail to distinguish between B. pseudomallei and closely related species such as Burkholderia thailandensis. Genotypic identification of suspected isolates can be achieved by sequencing of gene targets such as groEL which offer higher discriminative power than 16S rRNA. Specific PCR-based identification of B. pseudomallei has also been developed using B. pseudomallei-specific gene targets such as Type III secretion system and Tat-domain protein. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry, a revolutionary technique for pathogen identification, has been shown to be potentially useful for rapid identification of B. pseudomallei, although existing databases require optimization by adding reference spectra for B. pseudomallei. Despite these advances in bacterial identification, diagnostic problems encountered in culture-negative cases remain largely unresolved. Although various serological tests have been developed, they are generally unstandardized “in house” assays and have low sensitivities and specificities. Although specific PCR assays have been applied to direct clinical and environmental specimens, the sensitivities for diagnosis remain to be evaluated. Metabolomics is an uprising tool for studying infectious diseases and may offer a novel approach for exploring potential diagnostic biomarkers. The metabolomics profiles of B. pseudomallei culture supernatants can be potentially distinguished from those of related bacterial species including B. thailandensis. Further studies using bacterial cultures and direct patient samples are required to evaluate the potential of metabolomics for improving diagnosis of melioidosis.     

Airborne Transmission of Melioidosis to Humans from Environmental Aerosols Contaminated with B. pseudomallei

Melioidosis results from an infection with the soil-borne pathogen Burkholderia pseudomallei, and cases of melioidosis usually cluster after rains or a typhoon. In an endemic area of Taiwan, B. pseudomallei is primarily geographically distributed in cropped fields in the northwest of this area, whereas melioidosis cases are distributed in a densely populated district in the southeast. We hypothesized that contaminated cropped fields generated aerosols contaminated with B. pseudomallei, which were carried by a northwesterly wind to the densely populated southeastern district. We collected soil and aerosol samples from a 72 km² area of land, including the melioidosis-clustered area and its surroundings. Aerosols that contained B. pseudomallei-specific TTSS (type III secretion system) ORF2 DNA were well distributed in the endemic area but were rare in the surrounding areas during the rainy season. The concentration of this specific DNA in aerosols was positively correlated with the incidence of melioidosis and the appearance of a northwesterly wind. Moreover, the isolation rate in the superficial layers of the contaminated cropped field in the northwest was correlated with PCR positivity for aerosols collected from the southeast over a 2-year period. According to pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) analyses, PFGE Type Ia (ST58) was the predominant pattern linking the molecular association among soil, aerosol and human isolates. Thus, the airborne transmission of melioidosis moves from the contaminated soil to aerosols and/or to humans in this endemic area.     

Type 3 Secretion System Cluster 3 Is a Critical Virulence Determinant for Lung-Specific Melioidosis

Burkholderia pseudomallei, the bacterial agent of melioidosis, causes disease through inhalation of infectious particles, and is classified as a Tier 1 Select Agent. Optical diagnostic imaging has demonstrated that murine respiratory disease models are subject to significant upper respiratory tract (URT) colonization. Because human melioidosis is not associated with URT colonization as a prominent presentation, we hypothesized that lung-specific delivery of B. pseudomallei may enhance our ability to study respiratory melioidosis in mice. We compared intranasal and intubation-mediated intratracheal (IMIT) instillation of bacteria and found that the absence of URT colonization correlates with an increased bacterial pneumonia and systemic disease progression. Comparison of the LD₅₀ of luminescent B. pseudomallei strain, JW280, in intranasal and IMIT challenges of albino C57BL/6J mice identified a significant decrease in the LD₅₀ using IMIT. We subsequently examined the LD₅₀ of both capsular polysaccharide and Type 3 Secretion System cluster 3 (T3SS3) mutants by IMIT challenge of mice and found that the capsule mutant was attenuated 6.8 fold, while the T3SS3 mutant was attenuated 290 fold, demonstrating that T3SS3 is critical to respiratory melioidosis. Our previously reported intranasal challenge studies, which involve significant URT colonization, did not identify a dissemination defect for capsule mutants; however, we now report that capsule mutants exhibit significantly reduced dissemination from the lung following lung-specific instillation, suggesting that capsule mutants are competent to spread from the URT, but not the lung. We also report that a T3SS3 mutant is defective for dissemination following lung-specific delivery, and also exhibits in vivo growth defects in the lung. These findings highlight the T3SS3 as a critical virulence system for respiratory melioidosis, not only in the lung, but also for subsequent spread beyond the lung using a model system uniquely capable to characterize the fate of lung-delivered pathogen.     

Characterization of the Burkholderia mallei tonB Mutant and Its Potential as a Backbone Strain for Vaccine Development

BackgroundIn this study, a Burkholderia mallei tonB mutant (TMM001) deficient in iron acquisition was constructed, characterized, and evaluated for its protective properties in acute inhalational infection models of murine glanders and melioidosis.Conclusions/SignificanceAlthough further work is needed to prevent chronic infection by TMM001 while maintaining immunogenicity, our attenuated strain demonstrates great potential as a backbone strain for future vaccine development against both glanders and melioidosis.     

Deficiency of protease-activated receptor-1 limits bacterial dissemination during severe Gram-negative sepsis (melioidosis)

Protease-activated receptor-1 (PAR-1) is a G-coupled transmembrane receptor expressed by multiple cell types present in the lungs that can be activated by various proteases generated during acute inflammation. In this study we aimed to investigate the role of PAR-1 during melioidosis, a common cause of (pneumo)sepsis in Southeast Asia in a murine model of intranasal inoculation of the causative pathogen Burkholderia (B.) pseudomallei. Our results show that endogenous PAR-1 facilitates bacterial growth and dissemination during murine melioidosis, which is associated with increased cell influxes. However, these observations have no impact on survival.     

Comparative virulence of three different strains of Burkholderia pseudomallei in an aerosol non-human primate model

Melioidosis, caused by the Gram-negative bacterium Burkholderia pseudomallei, is a major cause of sepsis and mortality in endemic regions of Southeast Asia and Northern Australia. B. pseudomallei is a potential bioterrorism agent due to its high infectivity, especially via inhalation, and its inherent resistance to antimicrobials. There is currently no vaccine for melioidosis and antibiotic treatment can fail due to innate drug resistance, delayed diagnosis and treatment, or insufficient duration of treatment. A well-characterized animal model that mimics human melioidosis is needed for the development of new medical countermeasures. This study first characterized the disease progression of melioidosis in the African green monkey (AGM) and rhesus macaque (RM) for non-human primate model down-selection. All AGMs developed acute lethal disease similar to that described in human acute infection following exposure to aerosolized B. pseudomallei strain HBPUB10134a. Only 20% of RMs succumbed to acute disease. Disease progression, immune response and pathology of two other strains of B. pseudomallei, K96243 and MSHR5855, were also compared using AGMs. These three B. pseudomallei strains represent a highly virulent strain from Thailand (HBPUB101034a), a highly virulent strains from Australia (MSHR5855), and a commonly used laboratory strains originating from Thailand (K96243). Animals were observed for clinical signs of infection and blood samples were analyzed for cytokine responses, blood chemistry and leukocyte changes in order to characterize bacterial infection. AGMs experienced fever after exposure to aerosolized B. pseudomallei at the onset of acute disease. Inflammation, abscesses and/or pyogranulomas were observed in lung with all three strains of B. pseudomallei. Inflammation, abscesses and/or pyogranulomas were observed in lymph nodes, spleen, liver and/or kidney with B. pseudomallei, HBPUB10134a and K96243. Additionally, the Australian strain MSHR5855 induced brain lesions in one AGM similar to clinical cases of melioidosis seen in Australia. Elevated serum levels of IL-1β, IL-1 receptor antagonist, IL-6, MCP-1, G-CSF, HGF, IFNγ, MIG, I-TAC, and MIP-1β at terminal end points can be significantly correlated with non-survivors with B. pseudomallei infection in AGM. The AGM model represents an acute model of B. pseudomallei infection for all three strains from two geographical locations and will be useful for efficacy testing of vaccines and therapeutics against melioidosis. In summary, a dysregulated immune response leading to excessive persistent inflammation and inflammatory cell death is the key driver of acute melioidosis. Early intervention in these pathways will be necessary to counter B. pseudomallei and mitigate the pathological consequences of melioidosis.     

Environmental Isolates of Burkholderia pseudomallei in Ceará State, Northeastern Brazil

Melioidosis has been considered an emerging disease in Brazil since the first cases were reported to occur in the northeast region. This study investigated two municipalities in Ceará state where melioidosis cases have been confirmed to occur. Burkholderia pseudomallei was isolated in 26 (4.3%) of 600 samples in the dry and rainy seasons.     

Within-Host Evolution of Burkholderia pseudomallei in Four Cases of Acute Melioidosis

Little is currently known about bacterial pathogen evolution and adaptation within the host during acute infection. Previous studies of Burkholderia pseudomallei, the etiologic agent of melioidosis, have shown that this opportunistic pathogen mutates rapidly both in vitro and in vivo at tandemly repeated loci, making this organism a relevant model for studying short-term evolution. In the current study, B. pseudomallei isolates cultured from multiple body sites from four Thai patients with disseminated melioidosis were subjected to fine-scale genotyping using multilocus variable-number tandem repeat analysis (MLVA). In order to understand and model the in vivo variable-number tandem repeat (VNTR) mutational process, we characterized the patterns and rates of mutations in vitro through parallel serial passage experiments of B. pseudomallei. Despite the short period of infection, substantial divergence from the putative founder genotype was observed in all four melioidosis cases. This study presents a paradigm for examining bacterial evolution over the short timescale of an acute infection. Further studies are required to determine whether the mutational process leads to phenotypic alterations that impact upon bacterial fitness in vivo. Our findings have important implications for future sampling strategies, since colonies in a single clinical sample may be genetically heterogeneous, and organisms in a culture taken late in the infective process may have undergone considerable genetic change compared with the founder inoculum.