Flagellin-modulated inflammasome pathways characterize the human alveolar macrophage response to Burkholderia pseudomallei, a lung-tropic pathogen.

Melioidosis is an emerging tropical infection caused by inhalation, inoculation, or ingestion of the flagellated, facultatively intracellular pathogen Burkholderia pseudomallei. The melioidosis case fatality rate is often high, and pneumonia, the most common presentation, doubles the risk of death. The alveolar macrophage is a sentinel pulmonary host defense cell, but the human alveolar macrophage in B. pseudomallei infection has never been studied. The objective of this study was to investigate the host-pathogen interaction of B. pseudomallei infection with the human alveolar macrophage and to determine the role of flagellin in modulating inflammasome-mediated pathways. We found that B. pseudomallei infects primary human alveolar macrophages but is gradually restricted in the setting of concurrent cell death. Electron microscopy revealed cytosolic bacteria undergoing division, indicating that B. pseudomallei likely escapes the alveolar macrophage phagosome and may replicate in the cytosol, where it triggers immune responses. In paired human blood monocytes, uptake and intracellular restriction of B. pseudomallei are similar to those observed in alveolar macrophages, but cell death is reduced. The alveolar macrophage cytokine response to B. pseudomallei is characterized by marked interleukin (IL)-18 secretion compared to monocytes. Both cytotoxicity and IL-18 secretion in alveolar macrophages are partially flagellin dependent. However, the proportion of IL-18 release that is driven by flagellin is greater in alveolar macrophages than in monocytes. These findings suggest differential flagellin-mediated inflammasome pathway activation in the human alveolar macrophage response to B. pseudomallei infection and expand our understanding of intracellular pathogen recognition by this unique innate immune lung cell.

Synthesis of BLF1-containing trimethyl chitosan nanoparticles and evaluation of its immunogenicity and protection in syrian mice by oral and subcutaneous injections.

The bacterium Burkholderia pseudomallei is the cause of melioidosis infectious disease. In this bacterium, the BLF1 protein wide inhibits the synthesis of proteins in human cells. This disease is reported to cause a death rate of 40% in some parts of the world. Currently, no effective vaccine is available against this bacterial infection. In this study, therefore, a Nano vaccine was synthesized based on the trimethyl chitosan (TMC) polymer containing the BLF1 recombinant protein, and its immunogenicity and protection in Syrian mice were evaluated by oral and subcutaneous injections. The BLF1 recombinant protein expression was induced in Escherichia coli Bl21 (DE3) and purified by the affinity chromatography technique. Recombinant protein-containing nanoparticles (NPs) were then synthesized by the ionotropic gelation method. After oral and subcutaneous injections, antibody titration was assessed by the indirect ELISA assay. Finally, murine groups were challenged using the BLF1 toxin. The results indicated that the immune system showed more antibody titration in subcutaneous injection than in the oral form. However, the results were reversed in the challenge results, and the survival rate was more significant in the oral injection.

Septicemic Melioidosis Detection Using Support Vector Machine with Five Immune Cell Types.

Melioidosis, caused by Burkholderia pseudomallei (B. pseudomallei), predominantly occurs in the tropical regions. Of various types of melioidosis, septicemic melioidosis is the most lethal one with a mortality rate of 40%. Early detection of the disease is paramount for the better chances of cure. In this study, we developed a novel approach for septicemic melioidosis detection, using a machine learning technique-support vector machine (SVM). Several SVM models were built, and 19 features characterized by the corresponding immune cell types were generated by Cell type Identification Estimating Relative Subsets Of RNA Transcripts (CIBERSORT). Using these features, we trained a binomial SVM model on the training set and evaluated it on the independent testing set. Our findings indicated that this model performed well with means of sensitivity and specificity up to 0.962 and 0.979, respectively. Meanwhile, the receiver operating characteristic (ROC) curve analysis gave area under curves (AUCs) ranging from 0.952 to 1.000. Furthermore, we found that a concise SVM model, built upon a combination of CD8+ T cells, resting CD4+ memory T cells, monocytes, M2 macrophages, and activated mast cells, worked perfectly on the detection of septicemic melioidosis. Our data showed that its mean of sensitivity was up to 0.976 while that of specificity up to 0.993. In addition, the ROC curve analysis gave AUC close to 1.000. Taken together, this SVM model is a robust classification tool and may serve as a complementary diagnostic technique to septicemic melioidosis.

BpOmpW Antigen Stimulates the Necessary Protective T-Cell Responses Against Melioidosis.

Melioidosis is a potentially fatal bacterial disease caused by Burkholderia pseudomallei and is estimated to cause 89,000 deaths per year in endemic areas of Southeast Asia and Northern Australia. People with diabetes mellitus are most at risk of melioidosis, with a 12-fold increased susceptibility for severe disease. Interferon gamma (IFN-γ) responses from CD4 and CD8 T cells, but also from natural killer (NK) and natural killer T (NKT) cells, are necessary to eliminate the pathogen. We previously reported that immunization with B. pseudomallei OmpW (BpOmpW antigen) protected mice from lethal B. pseudomallei challenge for up to 81 days. Elucidating the immune correlates of protection of the protective BpOmpW vaccine is an essential step prior to clinical trials. Thus, we immunized either non-insulin-resistant C57BL/6J mice or an insulin-resistant C57BL/6J mouse model of type 2 diabetes (T2D) with a single dose of BpOmpW. BpOmpW induced strong antibody responses, stimulated effector CD4+ and CD8+ T cells and CD4+ CD25+ Foxp3+ regulatory T cells, and produced higher IFN-γ responses in CD4+, CD8+, NK, and NKT cells in non-insulin-resistant mice. The T-cell responses of insulin-resistant mice to BpOmpW were comparable to those of non-insulin-resistant mice. In addition, as a precursor to its evaluation in human studies, humanized HLA-DR and HLA-DQ (human leukocyte antigen DR and DQ isotypes, respectively) transgenic mice elicited IFN-γ recall responses in an enzyme-linked immune absorbent spot (ELISpot)-based study. Moreover, human donor peripheral blood mononuclear cells (PBMCs) exposed to BpOmpW for 7 days showed T-cell proliferation. Finally, plasma from melioidosis survivors with diabetes recognized our BpOmpW vaccine antigen. Overall, the range of approaches used strongly indicated that BpOmpW elicits the necessary immune responses to combat melioidosis and bring this vaccine closer to clinical trials.

Interactions Between Pathogenic Burkholderia and the Complement System: A Review of Potential Immune Evasion Mechanisms.

The genus Burkholderia contains over 80 different Gram-negative species including both plant and human pathogens, the latter of which can be classified into one of two groups: the Burkholderia pseudomallei complex (Bpc) or the Burkholderia cepacia complex (Bcc). Bpc pathogens Burkholderia pseudomallei and Burkholderia mallei are highly virulent, and both have considerable potential for use as Tier 1 bioterrorism agents; thus there is great interest in the development of novel vaccines and therapeutics for the prevention and treatment of these infections. While Bcc pathogens Burkholderia cenocepacia, Burkholderia multivorans, and Burkholderia cepacia are not considered bioterror threats, the incredible impact these infections have on the cystic fibrosis community inspires a similar demand for vaccines and therapeutics for the prevention and treatment of these infections as well. Understanding how these pathogens interact with and evade the host immune system will help uncover novel therapeutic targets within these organisms. Given the important role of the complement system in the clearance of bacterial pathogens, this arm of the immune response must be efficiently evaded for successful infection to occur. In this review, we will introduce the Burkholderia species to be discussed, followed by a summary of the complement system and known mechanisms by which pathogens interact with this critical system to evade clearance within the host. We will conclude with a review of literature relating to the interactions between the herein discussed Burkholderia species and the host complement system, with the goal of highlighting areas in this field that warrant further investigation.

Whole Blood Transcriptome Analysis Reveals the Correlation between Specific Immune Cells and Septicemic Melioidosis.

Melioidosis is a serious infectious disease caused by the environmental Gram-negative bacillus Burkholderia pseudomallei. It has been shown that the host immune system, mainly comprising various types of immune cells, fights against the disease. The present study was to specify correlation between septicemic melioidosis and the levels of multiple immune cells. First, the genes with differential expression patterns between patients with septicemic melioidosis (B. pseudomallei) and health donors (control/healthy) were identified. These genes being related to cytokine binding, cell adhesion molecule binding, and MHC relevant proteins may influence immune response. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed 23 enriched immune response pathways. We further leveraged the microarray data to investigate the relationship between immune response and septicemic melioidosis, using the CIBERSORT analysis. Comparison of the percentages of 22 immune cell types in B. pseudomallei vs. control/healthy revealed that those of CD4 memory resting cells, CD8+ T cells, B memory cells, and CD4 memory activated cells were low, whereas those of M0 macrophages, neutrophils, and gamma delta T cells were high. The multivariate logistic regression analysis further revealed that CD8+ T cells, M0 macrophages, neutrophils, and naive CD4+ cells were strongly associated with the onset of septicemic melioidosis, and M2 macrophages and neutrophils were associated with the survival in septicemic melioidosis. Taken together, these data point to a complex role of immune cells on the development and progression of melioidosis.

Melioidosis Patient Survival Correlates With Strong IFN-γ Secreting T Cell Responses Against Hcp1 and TssM.

Melioidosis, caused by the Gram-negative bacterium Burkholderia pseudomallei, is a serious infectious disease with diverse clinical manifestations. The morbidity and mortality of melioidosis is high in Southeast Asia and no licensed vaccines currently exist. This study was aimed at evaluating human cellular and humoral immune responses in Thai adults against four melioidosis vaccine candidate antigens. Blood samples from 91 melioidosis patients and 100 healthy donors from northeast Thailand were examined for immune responses against B. pseudomallei Hcp1, AhpC, TssM and LolC using a variety of cellular and humoral immune assays including IFN-γ ELISpot assays, flow cytometry and ELISA. PHA and a CPI peptide pool were also used as control stimuli in the ELISpot assays. Hcp1 and TssM stimulated strong IFN-γ secreting T cell responses in acute melioidosis patients which correlated with survival. High IFN-γ secreting CD4+ T cell responses were observed during acute melioidosis. Interestingly, while T cell responses of melioidosis patients against the CPI peptide pool were low at the time of enrollment, the levels increased to the same as in healthy donors by day 28. Although high IgG levels against Hcp1 and AhpC were detected in acute melioidosis patients, no significant differences between survivors and non-survivors were observed. Collectively, these studies help to further our understanding of immunity against disease following natural exposure of humans to B. pseudomallei as well as provide important insights for the selection of candidate antigens for use in the development of safe and effective melioidosis subunit vaccines.

Hijacking of the Host's Immune Surveillance Radars by Burkholderia pseudomallei.

Burkholderia pseudomallei (B. pseudomallei) causes melioidosis, a potentially fatal disease for which no licensed vaccine is available thus far. The host-pathogen interactions in B. pseudomallei infection largely remain the tip of the iceberg. The pathological manifestations are protean ranging from acute to chronic involving one or more visceral organs leading to septic shock, especially in individuals with underlying conditions similar to COVID-19. Pathogenesis is attributed to the intracellular ability of the bacterium to 'step into' the host cell's cytoplasm from the endocytotic vacuole, where it appears to polymerize actin filaments to spread across cells in the closer vicinity. B. pseudomallei effectively evades the host's surveillance armory to remain latent for prolonged duration also causing relapses despite antimicrobial therapy. Therefore, eradication of intracellular B. pseudomallei is highly dependent on robust cellular immune responses. However, it remains ambiguous why certain individuals in endemic areas experience asymptomatic seroconversion, whereas others succumb to sepsis-associated sequelae. Here, we propose key insights on how the host's surveillance radars get commandeered by B. pseudomallei.

Multicomponent Gold-Linked Glycoconjugate Vaccine Elicits Antigen-Specific Humoral and Mixed TH1-TH17 Immunity, Correlated with Increased Protection against Burkholderia pseudomallei.

Burkholderia pseudomallei is the causative agent of melioidosis, a fatal disease with a high mortality rate. The intrinsic resistance to commonly used antibiotics combined with the complex bacterial life cycle has hampered the development of preventive and therapeutic interventions and vaccines. Furthermore, the need of humoral and cell-mediated immunity in protection against B. pseudomallei has complicated the development of effective vaccines. Antigen delivery vaccine platforms that promote humoral and cellular responses while maintaining a safe profile are a roadblock to developing subunit vaccines against intracellular pathogens. Gold nanoparticles (AuNPs) were used for the delivery of multicomponent antigens with the goal of inducing vaccine-mediated immunity, promoting protection against melioidosis disease. Different nanoglycoconjugates using predicted immunogenic protein candidates, Hcp1, FlgL, OpcP, OpcP1, OmpW, and hemagglutinin, were covalently coupled to AuNPs, together with the lipopolysaccharide (LPS) from Burkholderia thailandensis, which acted as an additional antigen. Animals immunized with individually coupled (AuNP-protein-LPS) formulations containing OpcP or OpcP1, together with CpG as an adjuvant, showed a significant increase in protection, whereas a nanovaccine combination (AuNP-Combo2-LPS) showed significant and complete protection against a lethal intranasal B. pseudomallei challenge. Animals immunized with AuNP-Combo2-LPS showed robust humoral antigen-specific (IgG and IgA) responses with higher IgG2c titer, indicating a TH1-skewed response and promotion of macrophage uptake. In addition, immunization with the nanovaccine combination resulted in a mixed antigen-specific TH1-TH17 cytokine profile after immunization. This study provides the basis for an elegant and refined multicomponent glycoconjugate vaccine formulation capable of eliciting both humoral and cell-mediated responses against lethal B. pseudomallei challenge. IMPORTANCE Melioidosis is a complex human disease associated with a wide range of complications caused by the Gram-negative bacillus Burkholderia pseudomallei. The global burden of melioidosis is estimated to have 165,000 cases per year and 89,000 fatal outcomes. The endemicity of B. pseudomallei includes a wide range of tropical regions in Asia, Africa, Latin America, and Australia. Therefore, a viable alternative to prevent human infections is the development of an effective vaccine; however, no approved vaccine for human use is available. This study provides a vaccine strategy against B. pseudomallei and an immune-stimulatory platform to induce strong humoral and T-cell-mediated immunity.

Release of pro-inflammatory cytokines TNF-α, IFN-γ and IL-6 by Burkholderia pseudomallei- stimulated peripheral blood mononucleocytes of acute myeloid leukemia patients.

Acute myeloid leukemia (AML) is a malignant disease progressed from abnormal production of immature myeloid cells, which is often associated with concurrent infections after diagnosis. It was widely established that infections are the major contributors to mortality in this group due to the prevalency of neutropenia. Gram-negative Burkholderia pseudomallei is the causative agent of melioidosis. This disease had been reported in several neutropenic cancer patients undergoing chemotherapy resulting in severe clinical presentations and high mortalities which is in need of critical attention. Studies show that cytokines are important mediators of melioidosis progression and low neutrophil counts are associated with progression of its severity. However, to date, there are no reports on cytokine production in neutropenic cancer patients who are prone to melioidosis. Hence, here we assessed the cytokine production in neutropenic AML patients by introducing B. pseudomallei to their peripheral blood mononuclear cell (PBMC) culture in vitro. We observed that inflammatory response related cytokines namely TNF-α, IFN-γ IL-6 and IL-10 were highly circulated in infected PBMCs suggesting that these cytokines may play important roles in the progression of severity in melioidosis infected neutropenic patients.

Identification of defective early immune responses to Burkholderia pseudomallei infection in a diet-induced murine model of type 2 diabetes.

Co-occurrence of bacterial infections with type 2 diabetes (T2D) is a global problem. Melioidosis caused by Burkholderia pseudomallei is 10 times more likely to occur in patients with T2D, than in normoglycemic individuals. Using an experimental model of T2D, we observed that greater susceptibility in T2D was due to differences in proportions of infiltrating leucocytes and reduced levels of MCP-1, IFN-γ and IL-12 at sites of infection within 24 h post-infection. However, by 72 h the levels of inflammatory cytokines and bacteria were markedly higher in visceral tissue and blood in T2D mice. In T2D, dysregulated early immune responses are responsible for the greater predisposition to B. pseudomallei infection.

Role of Burkholderia pseudomallei-Specific IgG2 in Adults with Acute Melioidosis, Thailand.

Melioidosis is a life-threatening infectious disease caused by the gram-negative bacillus Burkholderia pseudomallei. An effective vaccine is needed, but data on protective immune responses in human melioidosis are lacking. We used ELISA and an antibody-dependent cellular phagocytosis assay to identify the major features of protective antibodies in patients with acute melioidosis in Thailand. We found that high levels of B. pseudomallei-specific IgG2 are associated with protection against death in a multivariable logistic regression analysis adjusting for age, diabetes, renal disease, and neutrophil count. Serum from melioidosis survivors enhanced bacteria uptake into human monocytes expressing FcγRIIa-H/R131, an intermediate-affinity IgG2-receptor, compared with serum from nonsurvivors. We did not find this enhancement when using monocytes carrying the low IgG2-affinity FcγRIIa-R131 allele. The findings indicate the importance of IgG2 in protection against death in human melioidosis, a crucial finding for antibody-based therapeutics and vaccine development.

Burkholderia pseudomallei sepsis with osteoarticular melioidosis of the hip in a patient with diabetes mellitus.

Melioidosis is caused by the tropical soil pathogen Burkholderia pseudomallei Infection, usually in the form of pneumonia, disproportionately affects people with a risk factor for immune dysregulation and mortality remains high even with treatment. Climate change and increasing rates of diabetes render the populations of endemic areas increasingly vulnerable to the disease, which is emerging as a serious global health threat. We present here a case of a 68-year-old man from northern Australia with sepsis and osteoarticular melioidosis of the hip, and explore the links between diabetes mellitus and melioidosis, particularly with respect to musculoskeletal infection.

Burkholderia pseudomallei triggers canonical inflammasome activation in a human primary macrophage-based infection model.

Most of the current knowledge on Burkholderia pseudomallei-induced inflammasome activation and cell death in macrophages is derived from murine systems. Little is known about the involved bacterial structures and mechanisms in primary human macrophages. This is of particular relevance since murine and human macrophages as well as primary cells and cell lines differ in many aspects of inflammasome activation, including the proteins involved in the recognition of bacterial patterns. In this study, we therefore aimed (i) to establish an in vitro B. pseudomallei infection model with human monocyte-derived primary macrophages from single donors as these cells more closely resemble macrophages in the human host and (ii) to analyze B. pseudomallei-triggered cell death and bacterial elimination in those cells. Our results show that B. pseudomallei-infected primary human macrophages not only release the inflammasome-independent pro-inflammatory cytokines IL-8 and TNF-α, but are also engaged in canonical inflammasome activation as evidenced by caspase-1 and gasdermin D processing. Absence of the B. pseudomallei T3SS-3 needle protein BsaL, a potent activator of the canonical inflammasome, abolished lytic cell death, reduced IL-1ß release, and caspase-1 and gasdermin D processing. IFN-γ, known to promote non-canonical inflammasome activation, did not influence pyroptosis induction or IL-1ß release from infected primary human macrophages. Nevertheless, it reduced intracellular B. pseudomallei loads, an effect which was partially antagonist by the inhibition of NADPH oxidase. Overall, our data implicate T3SS-3 dependent inflammasome activation and IFN-γ induced immune mechanisms as critical defense mechanisms of human macrophages against B. pseudomallei. In addition, our infection model provides a versatile tool to study human host-pathogen interactions and has the potential to elucidate the role of human individual genetic variations in B. pseudomallei infections.

The Peptidoglycan-associated lipoprotein Pal contributes to the virulence of Burkholderia mallei and provides protection against lethal aerosol challenge.

BURKHOLDERIA MALLEI: is a highly pathogenic bacterium that causes the fatal zoonosis glanders. The organism specifies multiple membrane proteins, which represent prime targets for the development of countermeasures given their location at the host-pathogen interface. We investigated one of these proteins, Pal, and discovered that it is involved in the ability of B. mallei to resist complement-mediated killing and replicate inside host cells in vitro, is expressed in vivo and induces antibodies during the course of infection, and contributes to virulence in a mouse model of aerosol infection. A mutant in the pal gene of the B. mallei wild-type strain ATCC 23344 was found to be especially attenuated, as BALB/c mice challenged with the equivalent of 5,350 LD50 completely cleared infection. Based on these findings, we tested the hypothesis that a vaccine containing the Pal protein elicits protective immunity against aerosol challenge. To achieve this, the pal gene was cloned in the vaccine vector Parainfluenza Virus 5 (PIV5) and mice immunized with the virus were infected with a lethal dose of B. mallei. These experiments revealed that a single dose of PIV5 expressing Pal provided 80% survival over a period of 40 days post-challenge. In contrast, only 10% of mice vaccinated with a PIV5 control virus construct survived infection. Taken together, our data establish that the Peptidoglycan-associated lipoprotein Pal is a critical virulence determinant of B. mallei and effective target for developing a glanders vaccine.

Utility of endobronchial ultrasound guided trans-bronchial needle aspiration in diagnosis of melioidosis - case series and review of literature.

Endobronchial ultrasound guided transbronchial needle aspiration (EBUS-TBNA) is routinely performed for diagnostic evaluation of mediastinal lymphadenopathy due to various etiologies with excellent sensitivity and specificity. Melioidosis can have atypical features like isolated mediastinal lymphadenopathy mimicking as tuberculosis or lymphoma. Differentiation of such atypical melioidosis presentation become difficult due to similar clinical, radiological and even similar EBUS lymph node characteristics. Role of EBUS TBNA in diagnosing melioidosis is under investigated and sparsely reported. We describe two cases of melioidosis diagnosed by point of care rapid lateral flow immunoassay antigen testing and culture of EBUS-TBNA samples from necrotic mediastinal lymph nodes.

Immune responses in beta-thalassaemia: heme oxygenase 1 reduces cytokine production and bactericidal activity of human leucocytes.

Patients with beta-thalassaemia increase the risk of bacterial infections, particularly Burkholderia pseudomallei (Bp), the causative agent of melioidosis in Thailand. Impaired immune cell functions may be the cause of this susceptibility, but detailed mechanisms have not been defined. In this study, we observed impaired production of IFN-gamma and IL-10 by whole blood from beta-thalassaemia patients upon stimulation with a range of bacteria-derived stimuli. In contrast, IFN-gamma response via TCR and plasma IgG specific for Bp were still intact. Importantly, mRNA expression of heme oxygenase 1 (HO-1), a potential modulator of immune function, was increased in whole blood from beta-thalassaemia patients, either with or without stimulation with Bp in vitro. Induction of HO-1 by hemin or CoPP in vitro reduced production of IFN-gamma and IL-10 from healthy human PBMCs and decreased bacterial clearance activity of whole blood from healthy controls and beta-thalassaemia, while inhibition of HO-1 by SnPP enhanced both functions in healthy controls. These results were confirmed to some extent in purified human monocytes of healthy controls. Our results suggest a mechanism that excess hemin of beta-thalassaemia patients is a significant cause of immune suppression via HO-1 induction and may underlie the susceptibility of these individuals to severe bacterial infection.

Dysregulation of TNF-α and IFN-γ expression is a common host immune response in a chronically infected mouse model of melioidosis when comparing multiple human strains of Burkholderia pseudomallei.

BACKGROUND: Melioidosis is endemic in Southeast Asia and Northern Australia and is caused by the Gram-negative, facultative intracellular pathogen Burkholderia pseudomallei. Diagnosis of melioidosis is often difficult because of the protean clinical presentation of the disease, and it may mimic other diseases, such as tuberculosis. There are many different strains of B. pseudomallei that have been isolated from patients with melioidosis, but it was not clear if they could cause a similar disease in a chronic BALB/c murine model of melioidosis. Hence, we wanted to examine chronically infected mice exposed to different strains of B. pseudomallei to determine if there were differences in the host immune response to the pathogen. RESULTS: We identified common host immune responses exhibited in chronically infected BALB/c mice, although there was some heterogeneity in the host response in chronically infected mice after exposure to different strains of B. pseudomallei. They all displayed pyogranulomatous lesions in their spleens with a large influx of monocytes/macrophages, NK cells, and neutrophils identified by flow cytometry. Sera from chronically infected mice by ELISA exhibited elevated IgG titers to the pathogen, and we detected by Luminex micro-bead array technology a significant increase in the expression of inflammatory cytokines/chemokines, such as IFN-γ, IL-1α, IL-1ß, KC, and MIG. By immunohistochemical and in situ RNA hybridization analysis we found that the increased expression of proinflammatory cytokines (IL-1α, IL-1ß, TNF-α, IFN-γ) was confined primarily to the area with the pathogen within pyogranulomatous lesions. We also found that cultured splenocytes from chronically infected mice could express IFN-γ, TNF-α, and MIP-1α ex vivo without the need for additional exogenous stimulation. In addition by flow cytometry, we detected significant amounts of intracellular expression of TNF-α and IFN-γ without a protein transport blocker in monocytes/macrophages, NK cells, and neutrophils but not in CD4+ or CD8+ T cells in splenocytes from chronically infected mice. CONCLUSION: Taken together the common features we have identified in chronically infected mice when 10 different human clinical strains of B. pseudomallei were examined could serve as biomarkers when evaluating potential therapeutic agents in mice for the treatment of chronic melioidosis in humans.

Development of an immunoassay using recombinant outer membrane protein A and flagellin for diagnosis of goats with melioidosis.

Among domestic animals, melioidosis is one of the most common diseases reported in goat, sheep, and swine. To evaluate the specific antibodies in goats with melioidosis, we developed a serology test using recombinant outer membrane protein A (OmpA) and flagellin (FliC) of Burkholderia pseudomallei as antigens. DNA corresponding to each antigen was cloned into a pET32a vector and expressed in Escherichia coli. Essentially, the recombinant OmpA and FliC were expressed in a soluble form that could be isolated with 95% homogeneity. Both recombinants could be recognized by rabbit antibodies prepared against heat-inactivated B. pseudomallei (1:1,000) on a Western blot. Subsequently, we demonstrated that both recombinants could capture the antibodies present in goat with naturally occurring melioidosis (optimized titer 1:40) while not cross-reacting with the serum samples of goats naturally infected by Corynebacterium pseudotuberculosis or Staphylococcus aureus. Finally, an enzyme-linked immunosorbent assay (ELISA) using 20 goat serum samples without melioidosis and 10 goat serum samples with melioidosis demonstrated that the infected group has significantly higher antibody titer levels than the normal group (P<0.001) when using either OmpA or FliC as an antigen. However, the sensitivity (100%) of the assay using OmpA was superior to that (90%) from using FliC. Serological tests that are commonly used often rely on antigens from crude cell extracts, which pose risks for laboratory-acquired infections and inconsistency in their preparation; however, use of recombinant OmpA is safe; it can potentially be used as a reagent in testing for goat melioidosis.

Caspase-4 Mediates Restriction of Burkholderia pseudomallei in Human Alveolar Epithelial Cells.

Melioidosis is an infectious disease with a high mortality rate responsible for community-acquired sepsis in Southeast Asia and Northern Australia. The causative agent of this disease is Burkholderia pseudomallei, a Gram-negative bacterium that resides in soil and contaminated natural water. After entering into host cells, the bacteria escape into the cytoplasm, which has numerous cytosolic sensors, including the noncanonical inflammatory caspases. Although the noncanonical inflammasome (caspase-11) has been investigated in a murine model of B. pseudomallei infection, its role in humans, particularly in lung epithelial cells, remains unknown. We, therefore, investigated the function of caspase-4 (ortholog of murine caspase-11) in intracellular killing of B. pseudomallei The results showed that B. pseudomallei induced caspase-4 activation at 12 h postinfection in human alveolar epithelial A549 cells. The number of intracellular B. pseudomallei bacteria was increased in the absence of caspase-4, suggesting its function in intracellular bacterial restriction. In contrast, a high level of caspase-4 processing was observed when cells were infected with lipopolysaccharide (LPS) mutant B. pseudomallei The enhanced bacterial clearance in LPS-mutant-infected cells is also correlated with a higher degree of caspase-4 activation. These results highlight the susceptibility of the LPS mutant to caspase-4-mediated intracellular bacterial killing.