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ABSTRACT: Cycle inhibiting factor (Cif) is produced by pathogenic intracellular bacteria and injected into the host cells via a type III secretion system. Cif is known to interfere with the eukaryotic cell cycle by inhibiting the function of cullin RING E3 ubiquitin ligases (CRLs). Cullin proteins form the scaffold protein of CRLs and are modified with the ubiquitin-like protein Nedd8, which exerts important conformational control required for CRL activity. Cif has recently been shown to catalyze the deamidation of Gln40 in Nedd8 to Glu. Here, we addressed how Nedd8 deamidation inhibits CRL activity. Our results indicate that Burkholderia pseudomallei Cif (also known as CHBP) inhibits the deconjugation of Nedd8 in vivo by inhibiting binding of the deneddylating COP9 signalosome (CSN) complex. We provide evidence that the reduced binding of CSN and the inhibition of CRL activity by Cif are due to interference with Nedd8-induced conformational control, which is dependent on the interaction between the Nedd8 hydrophobic patch and the cullin winged-helix B subdomain. Of note, mutation of Gln40 to Glu in ubiquitin, an additional target of Cif, inhibits the interaction between the hydrophobic surface of ubiquitin and the ubiquitin-binding protein p62/SQSTM1, showing conceptually that Cif activity can impair ubiquitin/ubiquitin-like protein non-covalent interactions. Our results also suggest that Cif may exert additional cellular effects by interfering with the association between ubiquitin and ubiquitin-binding proteins.
Journal of Molecular Biology 08/2011; 413(2):430-7. · 4.00 Impact Factor
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ABSTRACT: Type III and type VI secretion systems (T3SSs and T6SSs, respectively) are critical virulence determinants in several Gram-negative pathogens. In Burkholderia pseudomallei, the T3SS-3 and T6SS-1 clusters have been implicated in bacterial virulence in mammalian hosts. We recently discovered a regulatory cascade that coordinately controls the expression of T3SS-3 and T6SS-1. BsaN is a central regulator located within T3SS-3 for the expression of T3SS-3 effectors and regulators for T6SS-1 such as VirA-VirG (VirAG) and BprC. Whereas T6SS-1 gene expression was completely dependent on BprC when bacteria were grown in medium, the expression inside host cells was dependent on the two-component sensor-regulator VirAG, with the exception of the tssAB operon, which was dependent primarily on BprC. VirAG and BprC initiate different transcriptional start sites within T6SS-1, and VirAG is able to activate the hcp1 promoter directly. We also provided novel evidence that virAG, bprC, and tssAB are critical for T6SS-1 function in macrophages. Furthermore, virAG and bprC regulator mutants were avirulent in mice, demonstrating the absolute dependence of T6SS-1 expression on these regulators in vivo.
Infection and immunity 06/2011; 79(8):3064-73. · 4.21 Impact Factor
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ABSTRACT: Burkholderia pseudomallei is a highly versatile pathogen capable of infecting many species of animals and plants. It is the causative agent of melioidosis, a medically important infectious disease in humans with a wide spectrum of disease manifestations. Its versatility as a pathogen is reflected in its huge 7.2Mb genome and the many virulence mechanisms it possesses, including three different type III secretion systems (T3SSs). Recent elucidation of the regulatory network of T3SS3 and the characterization of several T3SS proteins have enabled us to construct a model of the B. pseudomallei T3SS3 apparatus and evaluate the role it plays in disease pathogenesis.
Trends in Microbiology 10/2010; 18(12):561-8. · 7.91 Impact Factor
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ABSTRACT: A major and critical virulence determinant of many Gram-negative bacterial pathogens is the Type III Secretion Systems (T3SS). T3SS3 in Burkholderia pseudomallei is critical for bacterial virulence in mammalian infection models but its regulation is unknown. B. pseudomallei is the causative agent of melioidosis, a potentially fatal disease endemic in Southeast Asia and northern Australia. While screening for bacterial transposon mutants with a defective T3SS function, we discovered a TetR family regulator (bspR) responsible for the control of T3SS3 gene expression. The bspR mutant exhibited significant virulence attenuation in mice. BspR acts through BprP, a novel transmembrane regulator located adjacent to the currently delineated T3SS3 region. BprP in turn regulates the expression of structural and secretion components of T3SS3 and the AraC family regulator bsaN. BsaN and BicA likely form a complex to regulate the expression of T3SS3 effectors and other regulators which in turn affect the expression of Type VI Secretion Systems (T6SS). The complete delineation of the bspR initiated T3SS regulatory cascade not only contributes to the understanding of B. pseudomallei pathogenesis but also provides an important example of how bacterial pathogens could co-opt and integrate various regulatory motifs to form a new regulatory network adapted for its own purposes.
Molecular Microbiology 03/2010; 76(3):677-89. · 5.01 Impact Factor
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ABSTRACT: The gram-negative rod Burkholderia pseudomallei is the causative agent of melioidosis, a potentially fatal disease which is endemic in tropical and subtropical areas. The bacterium multiplies intracellularly within the cytosol, induces the formation of actin tails, and can spread directly from cell to cell. Recently, it has been shown that B. pseudomallei can induce caspase-1-dependent cell death in macrophages. The aim of the present study was to further elucidate the role of caspase-1 during B. pseudomallei infection. In vivo experiments with caspase-1(-/-) mice revealed a high susceptibility to B. pseudomallei challenge. This phenotype was associated with a significantly higher bacterial burden 2 days after infection and decreased gamma interferon (IFN-gamma) and interleukin-18 cytokine levels 24 h after infection compared to control animals. caspase-1(-/-) bone marrow-derived macrophages (BMM) exhibited strong caspase-3 expression and reduced cell damage compared to wild-type (WT) cells during early B. pseudomallei infection, indicating "classical" apoptosis, whereas WT BMM showed signs of rapid caspase-1-dependent cell death. Moreover, we found that caspase-1(-/-) BMM had a strongly increased bacterial burden compared to WT cells 3 h after infection under conditions where no difference in cell death could be observed between both cell populations at this time point. We therefore suggest that caspase-1-dependent rapid cell death might contribute to resistance by reducing the intracellular niche for B. pseudomallei, but, in addition, caspase-1 might also have a role in controlling intracellular replication of B. pseudomallei in macrophages. Moreover, caspase-1-dependent IFN-gamma production is likely to contribute to resistance in murine melioidosis.
Infection and immunity 02/2009; 77(4):1589-95. · 4.21 Impact Factor
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ABSTRACT: Burkholderia pseudomallei is the causative agent of melioidosis. While adaptive immunity has been shown to be important for host resistance to B. pseudomallei, the direct interaction of the bacteria with adaptive immune cells such as T and B cells is not well known. To address this question, we infected Jurkat T cells, as well as human primary CD4(+) and CD8(+) T cells, with live B. pseudomallei. We found that live bacterial infection could costimulate T cells to produce interleukin-2 (IL-2) and gamma interferon (IFN-gamma) in the presence of anti-CD3 cross-linking antibodies. Bacterial supernatant could also costimulate T cells, and this was due to the presence of flagellin in the supernatant. However, T cells infected with bacterial mutants lacking flagellin showed strong impairment in IL-2 but only a slight impairment in IFN-gamma production. When cross-linking of CD3 is replaced by IL-2, live bacterial infection was still able to costimulate human primary T cells to produce IFN-gamma and flagellin is only a minor ligand contributing to this costimulation. Thus, live B. pseudomallei could potentially costimulate T cells not only in an antigen-specific manner but also in a nonspecific manner through bystander activation via IL-2.
Infection and immunity 07/2008; 76(6):2541-50. · 4.21 Impact Factor
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ABSTRACT: Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease that can result in asymptomatic, chronic, or acute illness. In acute melioidosis, high levels of proinflammatory cytokines and chemokines are found in organs and blood, and neutrophils play a key role in controlling the infection. We showed that B. pseudomallei activates NF-kappaB via Toll-like receptor (TLR) 2, TLR4, and TLR5 but can also activate NF-kappaB and induce interleukin (IL)-8 without involving TLRs. TLR-independent activation depends on a functional Bsa type III secretion system (T3SS) and requires internalization. The mitogen-activated protein kinase (MAPK) inhibitors for p38 and c-Jun N-terminal kinase (JNK) severely impaired IL-8 induction by B. pseudomallei and reduced bacterial internalization. Furthermore, the T3SS mutant induced less JNK phosphorylation than did wild-type bacteria. Thus, in cells with no or low expression of TLRs, such as mucosal epithelial cells, B. pseudomallei can induce IL-8 via NF-kappaB and MAPK pathways, aided by Bsa T3SS.
The Journal of Infectious Diseases 05/2008; 197(11):1537-47. · 6.41 Impact Factor
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ABSTRACT: Burkholderia pseudomallei is the causative agent for melioidosis, an infectious disease endemic in South-east Asia and northern Australia. Infection can result in a wide spectrum of clinical outcomes, including asymtomatic, acute or chronic conditions. The ability of the bacteria to survive intracellularly within phagocytes and non-phagocytes is postulated to help this pathogen persist in the body during latent chronic conditions. In some Gram-negative bacteria, such as Shigella and Salmonella, the ability to evade macrophage killing involves inducing rapid macrophage cell death. In several of these instances, these bacteria activate cellular caspase-1 to induce cell death, which is increasingly described to exhibit features more characteristic of oncosis than classical apoptosis. We found that B. pseudomallei is also capable of inducing caspase-1 dependent death in macrophages and this process requires a functional bsa Type III Secretion System (TTSS). Bacterial internalization and pore formation in the cell membrane is necessary for death. Furthermore, cell death is accompanied by the release of IL-1beta and IL-18. We believe that this novel description of macrophage death induced by B. pseudomallei could shed light on the pathogenesis of the bacteria in disease.
Cellular Microbiology 11/2005; 7(10):1447-58. · 5.46 Impact Factor
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ABSTRACT: Melioidosis is a life-threatening bacterial infection caused by Burkholderia pseudomallei. Some antibiotics used to treat melioidosis can induce filamentation in B. pseudomallei. Despite studies on the mechanism of virulence of the bacteria, the properties of B. pseudomallei filaments and their impact on virulence have not been investigated before. To understand the characteristics of antibiotic-induced filaments, we performed in vitro assays to compare several aspects of virulence between normal, nonfilamentous and filamentous B. pseudomallei. Normal, nonfilamentous B. pseudomallei could cause the lysis of monocytic cells, while filaments induced by sublethal concentrations of ceftazidime, ofloxacin, or trimethoprim show decreased lysis of monocytic cells, especially after prolonged antibiotic exposure. The motility of the filamentous bacteria was reduced compared to that of nonfilamentous bacteria. However, the filamentation was reversible when the antibiotics were removed, and the revertant bacteria recovered their motility and ability to lyse monocytic cells. Meanwhile, antibiotic resistance developed in revertant bacteria exposed to ceftazidime at the MIC. Our study highlights the danger of letting antibiotic concentration drop to the MIC or sub-MICs during antibiotic treatment of melioidosis. This could potentially give rise to a temporary reduction of bacterial virulence, only to result in bacteria that are equally virulent but more resistant to antibiotics, should the antibiotics be reduced or removed.
Antimicrobial Agents and Chemotherapy 04/2005; 49(3):1002-9. · 4.84 Impact Factor
