Ganjana Lertmemongkolchai

A Burkholderia pseudomallei protein microarray reveals serodiagnostic and cross-reactive antigens

Understanding the way in which the immune system responds to infection is central to the development of vaccines and many diagnostics. To provide insight into this area, we fabricated a protein microarray containing 1,205 Burkholderia pseudomallei proteins, probed it with 88 melioidosis patient sera, and identified 170 reactive antigens. This subset of antigens was printed on a smaller array and probed with a collection of 747 individual sera derived from 10 patient groups including melioidosis patients from Northeast Thailand and Singapore, patients with different infections, healthy individuals from the USA, and from endemic and nonendemic regions of Thailand. We identified 49 antigens that are significantly more reactive in melioidosis patients than healthy people and patients with other types of bacterial infections. We also identified 59 cross-reactive antigens that are equally reactive among all groups, including healthy controls from the USA. Using these results we were able to devise a test that can classify melioidosis positive and negative individuals with sensitivity and specificity of 95% and 83%, respectively, a significant improvement over currently available diagnostic assays. Half of the reactive antigens contained a predicted signal peptide sequence and were classified as outer membrane, surface structures or secreted molecules, and an additional 20% were associated with pathogenicity, adaptation or chaperones. These results show that microarrays allow a more comprehensive analysis of the immune response on an antigen-specific, patient-specific, and population-specific basis, can identify serodiagnostic antigens, and contribute to a more detailed understanding of immunogenicity to this pathogen.

Genomic transcriptional profiling identifies a candidate blood biomarker signature for the diagnosis of septicemic melioidosis

BackgroundMelioidosis is a severe infectious disease caused by Burkholderia pseudomallei, a Gram-negative bacillus classified by the National Institute of Allergy and Infectious Diseases (NIAID) as a category B priority agent. Septicemia is the most common presentation of the disease with a 40% mortality rate even with appropriate treatments. Better diagnostic tests are therefore needed to improve therapeutic efficacy and survival rates.ResultsWe have used microarray technology to generate genome-wide transcriptional profiles (>48,000 transcripts) from the whole blood of patients with septicemic melioidosis (n = 32), patients with sepsis caused by other pathogens (n = 31), and uninfected controls (n = 29). Unsupervised analyses demonstrated the existence of a whole blood transcriptional signature distinguishing patients with sepsis from control subjects. The majority of changes observed were common to both septicemic melioidosis and sepsis caused by other infections, including genes related to inflammation, interferon-related genes, neutrophils, cytotoxic cells, and T-cells. Finally, class prediction analysis identified a 37 transcript candidate diagnostic signature that distinguished melioidosis from sepsis caused by other organisms with 100% accuracy in a training set. This finding was confirmed in 2 independent validation sets, which gave high prediction accuracies of 78% and 80%, respectively. This signature was significantly enriched in genes coding for products involved in the MHC class II antigen processing and presentation pathway.ConclusionsBlood transcriptional patterns distinguish patients with septicemic melioidosis from patients with sepsis caused by other pathogens. Once confirmed in a large scale trial this diagnostic signature might constitute the basis of a differential diagnostic assay.

Human Polymorphonuclear Neutrophil Responses to Burkholderia pseudomallei in Healthy and Diabetic Subjects

ABSTRACT The major predisposing factor for melioidosis is diabetes mellitus, but no immunological mechanisms have been investigated to explain this. In this study, polymorphonuclear neutrophil (PMN) responses to Burkholderia pseudomallei, the causative agent of melioidosis, in healthy and diabetic Thai subjects were determined by flow cytometry. The results showed that B. pseudomallei displayed reduced uptake by PMNs compared to Salmonella enterica serovar Typhimurium and Escherichia coli. Additionally, intracellular survival of B. pseudomallei was detected throughout a 24-h period, indicating the intrinsic resistance of B. pseudomallei to killing by PMNs. Moreover, PMNs from diabetic subjects displayed impaired phagocytosis of B. pseudomallei, reduced migration in response to interleukin-8, and an inability to delay apoptosis. These data show that B. pseudomallei is intrinsically resistant to phagocytosis and killing by PMNs. These observations, together with the impaired migration and apoptosis in diabetes mellitus, may explain host susceptibility in melioidosis.

Multinucleated Giant Cell Formation and Apoptosis in Infected Host Cells Is Mediated by Burkholderia pseudomallei Type III Secretion Protein BipB

Here we have assessed the role of a type III translocator protein, BipB, in the cell biology and virulence of Burkholderia pseudomallei. Genetic inactivation of bipB reduced multinucleated giant cell formation, cell-to-cell spreading of bacteria, and induction of apoptosis of J774A.1 macrophages. The bipB mutant was also significantly attenuated following intranasal challenge of BALB/c mice, whereas virulence was fully restored by complementation with a functional bipB gene. Burkholderia pseudomallei, the etiological agent of melioidosis in humans and animals, is a gram-negative bacterium. Melioidosis is endemic in southeast Asia and tropical Australia and has been reported sporadically elsewhere (6). Currently, there is no vaccine against melioidosis. Uniquely among intracellular bacterial pathogens, B. pseudomallei induces host cell fusion leading to multinucleated giant cell (MNGC) formation in tissue culture models of infection (14). This novel phenotype may be relevant to pathogenesis, since granuloma formation and generation of MNGC are also found in tissues of humans with melioidosis (23). In addition to inducing MNGC formation, B. pseudomallei is able to spread from cell to cell and induce apoptotic death in infected host cells (14). The molecular mechanisms of these pathogenic characteristics have not been elucidated. Analysis of the B. pseudomallei genome and several other studies have demonstrated the presence of a type III secretion system (TTSS) (for reviews, see references 3, 12, 17, 20, and 22). A knockout mutant of B. pseudomallei lacking a functional bipD gene, a homologue of Salmonella enterica serovar Typhimurium sipD, on the TTSS3/bsa cluster of TTSS exhibited reduced replication in murine macrophage-like cells (20), was significantly attenuated in BALB/c mice and gave partial protection against subsequent challenge with wild-type B. pseudomallei (19). These data correlated with the recent report that the TTSS3/bsa cluster is required for the pathogenicity of B. pseudomallei (21). In addition to BipD, B. pseudomallei BipB and BipC (46 and 30% amino acid identity to Salmonella SipB and SipC, respectively) have been identified in the TTSS3/bsa cluster (3). Here, we report on the role of BipB in the pathogenesis of infection with B. pseudomallei. With Salmonella organisms, purified SipB integrates into artificial membranes and induces liposome fusion (10), and it is required for inducing apoptosis in murine macrophages (11). By analogy with SipB, therefore, we investigated the role of BipB for MNGC formation, cell-to-cell spreading, and induction of apoptosis in infected host cells. We also examined the virulence of a B. pseudomallei bipB mutant in a murine model of melioidosis.

Neutrophil Extracellular Traps Exhibit Antibacterial Activity against Burkholderia pseudomallei and Are Influenced by Bacterial and Host Factors

ABSTRACT Burkholderia pseudomallei is the causative pathogen of melioidosis, of which a major predisposing factor is diabetes mellitus. Polymorphonuclear neutrophils (PMNs) kill microbes extracellularly by the release of neutrophil extracellular traps (NETs). PMNs play a key role in the control of melioidosis, but the involvement of NETs in killing of B. pseudomallei remains obscure. Here, we showed that bactericidal NETs were released from human PMNs in response to B. pseudomallei in a dose- and time-dependent manner. B. pseudomallei-induced NET formation required NADPH oxidase activation but not phosphatidylinositol-3 kinase, mitogen-activated protein kinases, or Src family kinase signaling pathways. B. pseudomallei mutants defective in the virulence-associated Bsa type III protein secretion system (T3SS) or capsular polysaccharide I (CPS-I) induced elevated levels of NETs. NET induction by such mutants was associated with increased bacterial killing, phagocytosis, and oxidative burst by PMNs. Taken together the data imply that T3SS and the capsule may play a role in evading the induction of NETs. Importantly, PMNs from diabetic subjects released NETs at a lower level than PMNs from healthy subjects. Modulation of NET formation may therefore be associated with the pathogenesis and control of melioidosis.

Human Immune Responses to Burkholderia pseudomallei Characterized by Protein Microarray Analysis

BACKGROUND We aimed to determine the antibody and T cell responses to Burkholderia pseudomallei of humans to select candidate vaccine antigens. METHODS For antibody profiling, a protein microarray of 154 B. pseudomallei proteins was probed with plasma from 108 healthy individuals and 72 recovered patients. Blood from 20 of the healthy and 30 of the recovered individuals was also obtained for T cell assays. RESULTS Twenty-seven proteins distinctively reacted with human plasma following environmental exposure or clinical melioidosis. We compared the responses according to the patients history of subsequent relapse, and antibody response to BPSL2765 was higher in plasma from individuals who had only 1 episode of disease than in those with recurrent melioidosis. A comparison of antibody and T cell responses to 5 B. pseudomallei proteins revealed that BimA and flagellin-induced responses were similar but that BPSS0530 could induce T cell responses in healthy controls more than in recovered patients. CONCLUSIONS By combining large-scale antibody microarrays and assays of T cell-mediated immunity, we identified a panel of novel B. pseudomallei proteins that show distinct patterns of reactivity in different stages of human melioidosis. These proteins may be useful candidates for development of subunit-based vaccines and in monitoring the risks of treatment failure and relapse.

Development of Real-Time PCR Assays and Evaluation of Their Potential Use for Rapid Detection of Burkholderia pseudomallei in Clinical Blood Specimens

ABSTRACT The early initiation of appropriate antimicrobial therapy is critical for improving the prognosis of patients with septicemic melioidosis. Thus, the use of a rapid molecular diagnosis may affect the outcome of this disease, which has a high mortality rate. We report the development of two TaqMan real-time PCR assays (designated 8653 and 9438) that detect the presence of two novel genes unique to Burkolderia pseudomallei. The analytical sensitivity and specificity of the assays were assessed with 91 different B. pseudomallei isolates, along with 96 isolates and strains representing 28 other bacterial species, including the closely related Burkholderia/Ralstonia. The two assays performed equally well with both purified DNA and crude cell lysates, with 100% analytical specificity for the detection of B. pseudomallei. The limit of detection was 50 fg of DNA (equivalent to six bacterial genomes) per PCR for both assay 8563 and 9438. We also evaluated these assays with DNA extracted from blood specimens taken from 45 patients with culture-confirmed septicemic melioidosis or other septicemias. Of the 28 melioidosis blood specimens, assays 8653 and 9438 gave sensitivities of 71% (20/28) and 54% (15/28), respectively. Effectively, all fatal cases of septicemic melioidosis were detected by 8653. For the 17 non-melioidosis blood specimens, specificities of 82% (14/17) and 88% (15/17) were obtained for assays 8653 and 9438, respectively. The real-time PCR assays developed in this study provide alternative, rapid molecular tools for the specific detection of B. pseudomallei, and this may be of particular use in the early diagnosis and treatment of septicemic melioidosis.

Melioidosis Diagnostic Workshop, 20131

Melioidosis is a severe disease that can be difficult to diagnose because of its diverse clinical manifestations and a lack of adequate diagnostic capabilities for suspected cases. There is broad interest in improving detection and diagnosis of this disease not only in melioidosis-endemic regions but also outside these regions because melioidosis may be underreported and poses a potential bioterrorism challenge for public health authorities. Therefore, a workshop of academic, government, and private sector personnel from around the world was convened to discuss the current state of melioidosis diagnostics, diagnostic needs, and future directions.

Phenotypic and Functional Characterization of Human Memory T Cell Responses to Burkholderia pseudomallei

Background Infection with the Gram-negative bacterium Burkholderia pseudomallei is an important cause of community-acquired lethal sepsis in endemic regions in southeast Asia and northern Australia and is increasingly reported in other tropical areas. In animal models, production of interferon-gamma (IFN-γ) is critical for resistance, but in humans the characteristics of IFN-γ production and the bacterial antigens that are recognized by the cell-mediated immune response have not been defined. Methods Peripheral blood from 133 healthy individuals who lived in the endemic area and had no history of melioidosis, 60 patients who had recovered from melioidosis, and 31 other patient control subjects were stimulated by whole bacteria or purified bacterial proteins in vitro, and IFN-γ responses were analyzed by ELISPOT and flow cytometry. Findings B. pseudomallei was a potent activator of human peripheral blood NK cells for innate production of IFN-γ. In addition, healthy individuals with serological evidence of exposure to B. pseudomallei and patients recovered from active melioidosis developed CD4+ (and CD8+) T cells that recognized whole bacteria and purified proteins LolC, OppA, and PotF, members of the B. pseudomallei ABC transporter family. This response was primarily mediated by terminally differentiated T cells of the effector–memory (TEMRA) phenotype and correlated with the titer of anti-B. pseudomallei antibodies in the serum. Conclusions Individuals living in a melioidosis-endemic region show clear evidence of T cell priming for the ability to make IFN-γ that correlates with their serological status. The ability to detect T cell responses to defined B. pseudomallei proteins in large numbers of individuals now provides the opportunity to screen candidate antigens for inclusion in protein or polysaccharide–conjugate subunit vaccines against this important but neglected disease.

1α,25-dihydroxyvitamin D3 in combination with transforming growth factor-β increases the frequency of Foxp3+ regulatory T cells through preferential expansion and usage of interleukin-2

A high prevalence of vitamin D insufficiency and deficiency exists worldwide, which is associated with an increased incidence and severity of a range of immune‐mediated diseases. This has resulted in considerable interest in the immunodulatory functions of vitamin D. The active form of vitamin D, 1α,25‐dihydroxyvitamin D3 [1,25(OH)2D3], has been shown to increase the frequency of Foxp3+ CD4+ T regulatory (Treg) cells when present at high concentrations or under strong T‐cell stimulation in culture. Supporting evidence exists in vivo for a positive association between serum 25(OH)D and Foxp3+ Treg cell numbers in humans. The aim of this work was to identify the cytokine milieu required in vitro to promote Foxp3+ Treg cells in cultures containing 1,25(OH)2D3 at more moderate concentrations (10−7 m). Stimulation of human CD4+ T cells with a combination of 1,25(OH)2D3 and transforming growth factor‐β (TGF‐β) greatly increased the frequency of Foxp3+ Treg cells, which is proposed to result from the preferential expansion of Foxp3+ Treg cells, as compared with the Foxp3− effector T cells, in culture. The differential effect on proliferation may result from enhanced availability and usage of interleukin‐2 by the Foxp3+ Treg cells compared with Foxp3− effector T cells. In summary, modulation of the cytokine environment to one high in TGF‐β in the presence of 1,25(OH)2D3 (10−7 m) significantly increased Foxp3+ Treg cell frequency. These data provide additional evidence for the important immunomodulatory properties of 1,25(OH)2D3 that exist and may help to control inflammatory diseases.