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ABSTRACT: Septicemia is the most severe form of melioidosis caused by the Gram-negative bacterium, Burkholderia pseudomallei. Here we show that levels of IL-27p28 transcript and protein were both significantly elevated in patients with sepsis, particularly melioidosis and in patients with unfavorable disease outcome. Moreover, human monocytes/macrophages and neutrophils were the major source of IL-27 during infection. The addition of exogenous IL-27 in vitro resulted in significantly increased bacterial survival, reduced B. pseudomallei-induced oxidative burst and enhanced IL-1β and TNF-α production by purified neutrophils from healthy subjects. Finally, blockade of endogenous IL-27 in neutrophils using soluble IL-27 receptor antagonist prior to infection led to significantly reduced survival of bacteria and decreased IL-1β, but not TNF-α production. These results indicate a potential role for IL-27 in the suppression of anti-bacterial defense mechanisms that might contribute to disease severity in sepsis. The targeting of this cytokine may be beneficial in the management of human sepsis.
European Journal of Immunology 09/2012; · 5.10 Impact Factor
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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.
Infection and immunity 08/2012; 80(11):3921-9. · 4.21 Impact Factor
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ABSTRACT: Burkholderia pseudomallei induces the formation of multinucleated giant cells in cell monolayers. After infection of human macrophage-like U937 cells with B. pseudomallei, addition of monoclonal antibodies against integrin-associated protein (CD47), E-selectin (CD62E), a fusion regulatory protein (CD98), and E-cadherin (CD324) suppressed multinucleated giant cells in a concentration-dependent manner while monoclonal antibodies against other surface molecules did not inhibit fusion despite binding to the cell surface. Flow cytometric analysis showed increased expression of CD47 and CD98, but not CD62E and CD324, upon B. pseudomallei infection. Our data suggest the involvement of specific cellular factors in the process of B. pseudomallei-induced fusion.
Microbes and Infection 07/2011; 13(12-13):1006-11. · 3.10 Impact Factor
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ABSTRACT: Burkholderia pseudomallei is an intracellular pathogen and the causative agent of melioidosis, a life-threatening disease of humans. Within host cells, superoxide is an important mediator of pathogen killing. In this study, we have identified the B. pseudomallei K96243 sodC gene, shown that it has superoxide dismutase activity, and constructed an allelic deletion mutant of this gene. Compared with the wild-type, the mutant was more sensitive to killing by extracellular superoxide, but not to superoxide generated intracellularly. The sodC mutant showed a markedly decreased survival in J774A.1 mouse macrophages, and reduced numbers of bacteria were recovered from human polymorphonuclear neutrophils (PMNs) when compared with the wild-type. The numbers of wild-type or mutant bacteria recovered from human diabetic neutrophils were significantly lower than from normal human neutrophils. The sodC mutant was attenuated in BALB/c mice. Our results indicate that SodC plays a key role in the virulence of B. pseudomallei, but that diabetics are not more susceptible to infection because of a reduced ability of PMNs to kill by superoxide.
Microbiology 06/2011; 157(Pt 8):2392-400. · 3.06 Impact Factor
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ABSTRACT: We aimed to determine the antibody and T cell responses to Burkholderia pseudomallei of humans to select candidate vaccine antigens.
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.
Twenty-seven proteins distinctively reacted with human plasma following environmental exposure or clinical melioidosis. We compared the responses according to the patient's 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.
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.
The Journal of Infectious Diseases 02/2011; 203(7):1002-11. · 6.41 Impact Factor
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ABSTRACT: Burkholderia pseudomallei causes melioidosis, a disease with a wide range of possible outcomes, from seroconversion and dormancy to sepsis and death. This spectrum of host-pathogen interactions poses challenging questions about the heterogeneity in immunity to B. pseudomallei. Models show protection to be dependent on CD4(+) cells and IFN-γ, but little is known about specific target antigens. Having previously implicated the ABC transporter, LolC, in protective immunity, we here use epitope prediction, HLA-binding studies, HLA-transgenic models and studies of T cells from seropositive individuals to characterize HLA-restricted LolC responses. Immunized mice showed long-lasting memory to the protein, whereas predictive algorithms identified epitopes within LolC that subsequently demonstrated strong HLA class II binding. Immunization of HLA-DR transgenics with LolC stimulated T-cell responses to four of these epitopes. Furthermore, the responsiveness of HLA transgenics to LolC revealed a hierarchy supportive of HLA polymorphism-determined differential susceptibility. Seropositive human donors of diverse HLA class II types showed T-cell responses to LolC epitopes, which are conserved among Burkholderia species including Burkholderia cenocepacia, associated with life-threatening cepacia complex in cystic fibrosis patients and Burkholderia mallei, which causes glanders. These findings suggest a role for LolC epitopes in multiepitope vaccine design for melioidosis and related diseases.
European Journal of Immunology 01/2011; 41(1):107-15. · 5.10 Impact Factor
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Patcharaporn Tippayawat,
Maneerat Pinsiri,
Darawan Rinchai,
Donporn Riyapa,
Amornrat Romphruk,
Yunn-Hwen Gan,
Raymond L Houghton,
Philip L Felgner,
Richard W Titball,
Mark P Stevens,
Edouard E Galyov,
Gregory J Bancroft, Ganjana Lertmemongkolchai
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ABSTRACT: Melioidosis is a severe infectious disease caused by the saprophytic facultative intracellular pathogen Burkholderia pseudomallei. The disease is endemic in Southeast Asia and Northern Australia, and no effective vaccine exists. To describe human cell-mediated immune responses to B. pseudomallei and to identify candidate antigens for vaccine development, the ability of antigen-pulsed monocyte-derived dendritic cells (moDCs) to trigger autologous T-cell responses to B. pseudomallei and its products was tested. moDCs were prepared from healthy individuals exposed or not exposed to B. pseudomallei, based on serological evidence. These were pulsed with heat-killed B. pseudomallei or purified antigens, including ABC transporters (LolC, OppA, and PotF), Bsa type III secreted proteins (BipD and BopE), tandem repeat sequence-containing proteins (Rp1 and Rp2), flagellin, and heat shock proteins (Hsp60 and Hsp70), prior to being mixed with autologous T-cell populations. After pulsing of cells with either heat-killed B. pseudomallei, LolC, or Rp2, coculturing the antigen-pulsed moDCs with T cells elicited gamma interferon production from CD4(+) T cells from seropositive donors at levels greater than those for seronegative donors. These antigens also induced granzyme B (cytotoxic) responses from CD8(+) T cells. Activation of antigen-specific CD4(+) T cells required direct contact with moDCs and was therefore not dependent on soluble mediators. Rp peptide epitopes recognized by T cells in healthy individuals were identified. Our study provides valuable novel data on the induction of human cell-mediated immune responses to B. pseudomallei and its protein antigens that may be exploited in the rational development of vaccines to combat melioidosis.
Infection and immunity 11/2010; 79(1):305-13. · 4.21 Impact Factor
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ABSTRACT: Burkholderia thailandensis is a close relative of Burkholderia pseudomallei. These organisms are very similar, but B. thailandensis is far less virulent than B. pseudomallei. Nucleotide sequencing and analysis of 14 B. thailandensis isolates revealed variation in the regions coding for the type III secreted BipD protein. The degree of B. thailandensis BipD sequence variation was greater than that found in B. pseudomallei. Western blot analysis indicated that, unlike B. pseudomallei, B. thailandensis type III secreted proteins including BipD and BopE could not be detected in the supernatant of culture medium unless induced by acidic conditions. In addition, culturing B. thailandensis under acidic growth conditions (pH 4.5) can induce the ability of this bacterium to invade human respiratory epithelial cells A549. The identification of an environmental stimulus that increases the invasion capability of B. thailandensis invasion is of value for those who would like to use this bacterium as a model to study B. pseudomallei virulence.
The Journal of Microbiology 08/2010; 48(4):526-32. · 1.10 Impact Factor
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ABSTRACT: Previous studies of T cell activation in dengue infection have focused on restriction of specific T cell receptors (TCRs) and classical MHC molecules. However, bystander T cell activation, which is TCR independent, occurs via cytokines in other viral infections, both in vitro and in vivo, and enables T cells to bypass certain control checkpoints. Moreover, clinical and pathological evidence has pointed to cytokines as the mediators of dengue disease severity. Therefore, we investigated bystander T cell induction by dengue viral antigen.
Whole blood samples from 55 Thai schoolchildren aged 13-14 years were assayed for in vitro interferon-gamma (IFN-γ) induction in response to inactivated dengue serotype 2 antigen (Den2). The contribution of TCR-dependent and independent pathways was tested by treatment with cyclosporin A (CsA), which inhibits TCR-dependent activation of T cells. ELISA results revealed that approximately 72% of IFN-γ production occurred via the TCR-dependent pathway. The major IFN-γ sources were natural killer (NK) (mean ± SE = 55.2 ± 3.3), CD4+T (24.5 ± 3.3) and CD8+T cells (17.9 ± 1.5), respectively, as demonstrated by four-color flow cytometry. Interestingly, in addition to these cells, we found CsA-resistant IFN-γ producing T cells (CD4+T = 26.9 ± 3.6% and CD8+T = 20.3 ± 2.1%) implying the existence of activated bystander T cells in response to dengue antigen in vitro. These bystander CD4+ and CD8+T cells had similar kinetics to NK cells, appeared after 12 h and were inhibited by anti-IL-12 neutralization indicating cytokine involvement.
This study described immune cell profiles and highlighted bystander T cell activation in response to dengue viral antigens of healthy people in an endemic area. Further studies on bystander T cell activation in dengue viral infection may reveal the immune mechanisms that protect or enhance pathogenesis of secondary dengue infection.
BMC Immunology 01/2010; 11:47. · 2.53 Impact Factor
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Philip L Felgner,
Matthew A Kayala,
Adam Vigil,
Chad Burk,
Rie Nakajima-Sasaki,
Jozelyn Pablo,
Douglas M Molina,
Siddiqua Hirst,
Janet S W Chew,
Dongling Wang, [......],
Melanie Duffield,
Ron Yang,
Julien Neel,
Narisara Chantratita,
Greg Bancroft, Ganjana Lertmemongkolchai,
D Huw Davies,
Pierre Baldi,
Sharon Peacock,
Richard W Titball
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ABSTRACT: 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.
Proceedings of the National Academy of Sciences 08/2009; 106(32):13499-504. · 9.68 Impact Factor
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ABSTRACT: MOTIVATION: Finding diagnostic patterns for fighting diseases like Burkholderia pseudomallei using biomarkers involves two key issues. First, exhausting all subsets of testable biomarkers (antigens in this context) to find a best one is computationally infeasible. Therefore, a proper optimization approach like evolutionary computation should be investigated. Second, a properly selected function of the antigens as the diagnostic pattern which is commonly unknown is a key to the diagnostic accuracy and the diagnostic effectiveness in clinical use. RESULTS: A conversion function is proposed to convert serum tests of antigens on patients to binary values based on which Boolean functions as the diagnostic patterns are developed. A genetic programming approach is designed for optimizing the diagnostic patterns in terms of their accuracy and effectiveness. During optimization, it is aimed to maximize the coverage (the rate of positive response to antigens) in the infected patients and minimize the coverage in the non-infected patients while maintaining the fewest number of testable antigens used in the Boolean functions as possible. The final coverage in the infected patients is 96.55% using 17 of 215 (7.4%) antigens with zero coverage in the non-infected patients. Among these 17 antigens, BPSL2697 is the most frequently selected one for the diagnosis of Burkholderia Pseudomallei. The approach has been evaluated using both the cross-validation and the Jack-knife simulation methods with the prediction accuracy as 93% and 92%, respectively. A novel approach is also proposed in this study to evaluate a model with binary data using ROC analysis.
Bioinformatics 07/2009; 25(17):2256-62. · 5.47 Impact Factor
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Patcharaporn Tippayawat,
Wipawee Saenwongsa,
Jirawan Mahawantung,
Duangchan Suwannasaen,
Ploenchan Chetchotisakd,
Direk Limmathurotsakul,
Sharon J Peacock,
Philip L Felgner,
Helen S Atkins,
Richard W Titball,
Gregory J Bancroft, Ganjana Lertmemongkolchai
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ABSTRACT: 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-gamma) is critical for resistance, but in humans the characteristics of IFN-gamma production and the bacterial antigens that are recognized by the cell-mediated immune response have not been defined.
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-gamma responses were analyzed by ELISPOT and flow cytometry.
B. pseudomallei was a potent activator of human peripheral blood NK cells for innate production of IFN-gamma. 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 (T(EMRA)) phenotype and correlated with the titer of anti-B. pseudomallei antibodies in the serum.
Individuals living in a melioidosis-endemic region show clear evidence of T cell priming for the ability to make IFN-gamma 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.
PLoS Neglected Tropical Diseases 02/2009; 3(4):e407. · 4.69 Impact Factor
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Bioinformatics. 01/2009; 25:2256-2262.
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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.
Infection and immunity 11/2008; 77(1):456-63. · 4.21 Impact Factor
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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.
Journal of Clinical Microbiology 10/2007; 45(9):2894-901. · 4.15 Impact Factor
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Caroline A Rowland, Ganjana Lertmemongkolchai,
Alison Bancroft,
Ashraful Haque,
M Stephen Lever,
Kate F Griffin,
Matthew C Jackson,
Michelle Nelson,
Anne O'Garra,
Richard Grencis,
Gregory J Bancroft,
Roman A Lukaszewski
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ABSTRACT: Burkholderia mallei is a gram-negative bacterium which causes the potentially fatal disease glanders in humans; however, there is little information concerning cell-mediated immunity to this pathogen. The role of gamma interferon (IFN-gamma) during B. mallei infection was investigated using a disease model in which infected BALB/c mice normally die between 40 and 60 days postinfection. IFN-gamma knockout mice infected with B. mallei died within 2 to 3 days after infection, and there was uncontrolled bacterial replication in several organs, demonstrating the essential role of IFN-gamma in the innate immune response to this pathogen. Increased levels of IFN-gamma, interleukin-6 (IL-6), and monocyte chemoattractant protein 1 were detected in the sera of immunocompetent mice in response to infection, and splenic mRNA expression of IFN-gamma, IL-6, IL-12p35, and IL-27 was elevated 24 h postinfection. The effects of IL-18, IL-27, and IL-12 on stimulation of the rapid IFN-gamma production were investigated in vitro by analyzing IFN-gamma production in the presence of heat-killed B. mallei. IL-12 was essential for IFN-gamma production in vitro; IL-18 was also involved in induction of IFN-gamma, but IL-27 was not required for IFN-gamma production in response to heat-killed B. mallei. The main cellular sources of IFN-gamma were identified in vitro as NK cells, CD8+ T cells, and TCRgammadelta T cells. Our data show that B. mallei is susceptible to cell-mediated immune responses which promote expression of type 1 cytokines. This suggests that development of effective vaccines against glanders should target the production of IFN-gamma.
Infection and Immunity 10/2006; 74(9):5333-40. · 4.16 Impact Factor
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ABSTRACT: Antigen-specific T cells are important sources of interferon (IFN)-gamma for acquired immunity to intracellular pathogens, but they can also produce IFN- gamma directly via a "bystander" activation pathway in response to proinflammatory cytokines. We investigated the in vivo role of cytokine- versus antigen-mediated T cell activation in resistance to the pathogenic bacterium Burkholderia pseudomallei. IFN-gamma, interleukin (IL)-12, and IL-18 were essential for initial bacterial control in infected mice. B. pseudomallei infection rapidly generated a potent IFN-gamma response from natural killer (NK) cells, NK T cells, conventional T cells, and other cell types within 16 h after infection, in an IL-12- and IL-18-dependent manner. However, early T cell- and NK cell-derived IFN-gamma responses were functionally redundant in cell depletion studies, with IFN-gamma produced by other cell types, such as major histocompatibility complex class II(int) F4/80(+) macrophages being sufficient for initial resistance. In contrast, B. pseudomallei-specific CD4(+) T cells played an important role during the later stage of infection. Thus, the T cell response to primary B. pseudomallei infection is biphasic, an early cytokine-induced phase in which T cells appear to be functionally redundant for initial bacterial clearance, followed by a later antigen-induced phase in which B. pseudomallei-specific T cells, in particular CD4(+) T cells, are important for host resistance.
The Journal of Infectious Diseases 03/2006; 193(3):370-9. · 6.41 Impact Factor
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ABSTRACT: Burkholderia pseudomallei, the causative agent of melioidosis, can be isolated from soil and water. To persist, adapt and survive within and outside their human host, bacteria rely on regulatory mechanisms that allow them to respond rapidly to stressful situations. We have examined the possible role of B. pseudomallei alternative sigma factor sigma(E) (RpoE) in the stress response and found that rpoE and its putative regulators (bprE-rseB-mucD) are transcribed in a single transcriptional unit. Inactivation of the rpoE operon changed the B. pseudomallei phenotype. Changes included increased susceptibility to killing by menadione and H(2)O(2), susceptibility to high osmolarity, reduced ability to form biofilms, and reduced survival in macrophage J774A.1. Therefore, we conclude that rpoE controls gene expression that contributes, at least in part, to B. pseudomallei adaptation to adverse environmental conditions.
FEMS Microbiology Letters 12/2005; 252(2):243-9. · 2.04 Impact Factor
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Journal of Bacteriology 10/2005; 187(18):6556-60. · 3.83 Impact Factor
