Paul J Brett

University of South Alabama, Mobile, Alabama, United States

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Publications (37)128.74 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Burkholderia pseudomallei, the etiologic agent of melioidosis, is an opportunistic pathogen that harbors a wide array of secretion systems, including a type II secretion system (T2SS), three type III secretion systems (T3SS), and six type VI secretion systems (T6SS). The proteins exported by these systems provide B. pseudomallei with a growth advantage in vitro and in vivo, but relatively little is known about the full repertoire of exoproducts associated with each system. In this study we constructed deletion mutations in gspD and gspE, T2SS genes encoding an outer membrane secretin and a cytoplasmic ATPase, respectively. The secretion profiles of B. pseudomallei MSHR668 and its T2SS mutants were noticeably different when analyzed by SDS-PAGE. We utilized liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify proteins present in the supernatants of B. pseudomallei MSHR668 and ΔgspD grown in rich and minimal media. The MSHR668 supernatants contained 48 proteins that were either absent, or substantially reduced, in the ΔgspD supernatants. Many of these proteins were putative hydrolytic enzymes, including twelve proteases, two phospholipases, and a chitinase. Biochemical assays validated the LC-MS/MS results and demonstrated that the export of protease, phospholipase C, and chitinase activities were T2SS-dependent. Previous studies have failed to identify the mechanism of secretion of TssM, a deubiquitinase that plays an integral role in regulating the innate immune response. Here we present evidence that TssM harbors an atypical signal sequence and that its secretion is mediated by the T2SS. This study provides the first in-depth characterization of the B. pseudomallei T2SS secretome.
    Infection and immunity. 05/2014;
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    ABSTRACT: Burkholderia pseudomallei, the etiologic agent of melioidosis, is a CDC Tier 1 Select Agent that causes severe disease in both humans and animals. Diagnosis and treatment of melioidosis can be challenging, and in the absence of optimal chemotherapeutic intervention acute disease is frequently fatal. Melioidosis is an emerging infectious disease for which no licensed vaccines currently exist. Due to the potential malicious use of B. pseudomallei, as well as its impact on public health in endemic regions, there is significant interest in developing vaccines for immunization against this disease. In the present study, type-A O-polysaccharide (OPS) and manno-heptose capsular polysaccharide (CPS) antigens were isolated from non-pathogenic, Select Agent-excluded strains of B. pseudomallei and covalently linked to carrier proteins. Using these conjugates (OPS2B1 and CPS2B1) it was demonstrated that, although high titer IgG responses against the OPS or CPS components of the glycoconjugates could be raised in BALB/c mice, only those animals immunized with CPS2B1 were protected against an intraperitoneal challenge of B. pseudomallei. Extending upon these studies, it was also demonstrated that when the mice were immunized with a combination of CPS2B1 and recombinant B. pseudomallei LolC, rather than with CPS2B1 or LolC individually, they exhibited higher survival rates when challenged with a lethal dose of B. pseudomallei. Collectively, these results suggest that CPS-based glycoconjugates are promising candidates for the development of subunit vaccines for immunization against melioidosis.
    Infection and immunity. 05/2014;
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    ABSTRACT: 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.
    PLoS Neglected Tropical Diseases 03/2014; 8(3):e2727. · 4.57 Impact Factor
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    ABSTRACT: O-Polysaccharides (OPS) were isolated from purified Burkholderia pseudomallei and Burkholderia mallei lipopolysaccharides by mild-acid hydrolysis and gel-permeation chromatography. 1-D and 2-D (1)H and (13)C NMR spectroscopy experiments revealed that the OPS antigens were unbranched heteropolymers with the following structures: Collectively, our results demonstrate that the predominant OPS antigens expressed by B. pseudomallei and B. mallei isolates are structurally more complex than previously described and provide evidence that different capping residues are used by these closely related pathogens to terminate chain elongation. Additionally, they confirm that Burkholderia thailandensis and B. pseudomallei express OPS antigens that are essentially identical to one another.
    Carbohydrate research 08/2013; 381C:6-11. · 2.03 Impact Factor
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    Mary N Burtnick, Paul J Brett
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    ABSTRACT: Burkholderia mallei is a facultative intracellular pathogen that causes glanders in humans and animals. Previous studies have demonstrated that the cluster 1 type VI secretion system (T6SS-1) expressed by this organism is essential for virulence in hamsters and is positively regulated by the VirAG two-component system. Recently, we have shown that T6SS-1 gene expression is up-regulated following internalization of this pathogen into phagocytic cells and that this system promotes multinucleated giant cell formation in infected tissue culture monolayers. In the present study, we further investigated the complex regulation of this important virulence factor. To assess T6SS-1 expression, B. mallei strains were cultured in various media conditions and Hcp1 production was analyzed by Western immunoblotting. Transcript levels of several VirAG-regulated genes (bimA, tssA, hcp1 and tssM) were also determined using quantitative real time PCR. Consistent with previous observations, T6SS-1 was not expressed during growth of B. mallei in rich media. Curiously, growth of the organism in minimal media (M9G) or minimal media plus casamino acids (M9CG) facilitated robust expression of T6SS-1 genes whereas growth in minimal media plus tryptone (M9TG) did not. Investigation of this phenomenon confirmed a regulatory role for VirAG in this process. Additionally, T6SS-1 gene expression was significantly down-regulated by the addition of iron and zinc to M9CG. Other genes under the control of VirAG did not appear to be as tightly regulated by these divalent metals. Similar results were observed for B. pseudomallei, but not for B. thailandensis. Collectively, our findings indicate that in addition to being positively regulated by VirAG, B. mallei and B. pseudomallei T6SS-1 gene expression is negatively regulated by iron and zinc.
    PLoS ONE 01/2013; 8(10):e76767. · 3.73 Impact Factor
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    ABSTRACT: O-polysaccharide (OPS) was isolated from purified Burkholderia thailandensis E264 lipopolysaccharide by mild-acid hydrolysis and gel-permeation chromatography. Glycosyl composition and methylation analyses along with 1D and 2D (1)H and (13)C NMR spectroscopy experiments revealed that the OPS antigen was an unbranched heteropolymer with the following structure: Collectively, these results suggest that B. thailandensis OPS is structurally more complex than B. pseudomallei OPS and provide evidence of the signal used by B. thailandensis to terminate chain elongation.
    Carbohydrate research 10/2012; 363C:23-28. · 2.03 Impact Factor
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    ABSTRACT: Capsular polysaccharides (CPSs) were isolated from O-polysaccharide deficient strains of Burkholderia mallei and Burkholderia pseudomallei using a modified hot phenol/water extraction procedure. Glycosyl composition, methylation, MALDI-TOF MS analyses as well as (1)H NMR spectroscopy including COSY, TOCSY, NOESY, HMBC and HSQC experiments identified the presence of two distinct CPS antigens in the samples exhibiting the following structures: This study confirms the ability of B. mallei to express a 6-deoxy-heptan CPS and represents the first report of a mannan CPS being expressed by these bacterial pathogens.
    Carbohydrate research 02/2012; 349:90-4. · 2.03 Impact Factor
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    ABSTRACT: Burkholderia mallei the etiologic agent of glanders, causes severe disease in humans and animals and is a potential agent of biological warfare and terrorism. Diagnosis and treatment of glanders can be challenging, and in the absence of chemotherapeutic intervention, acute human disease is invariably fatal. At present, there are no human or veterinary vaccines available for immunization against disease. One of the goals of our research, therefore, is to identify and characterize protective antigens expressed by B. mallei and use them to develop efficacious glanders vaccine candidates. Previous studies have demonstrated that the O-polysaccharide (OPS) expressed by B. mallei is both a virulence factor and a protective antigen. Recently, we demonstrated that Burkholderia thailandensis, a closely related but non-pathogenic species, can be genetically manipulated to express OPS antigens that are recognized by B. mallei OPS-specific monoclonal antibodies (mAbs). As a result, these antigens have become important components of the various OPS-based subunit vaccines that we are currently developing in our laboratory. In this study, we describe a method for isolating B. mallei-like OPS antigens from B. thailandensis oacA mutants. Utilizing these purified OPS antigens, we also describe a simple procedure for coupling the polysaccharides to protein carriers such as cationized bovine serum albumin, diphtheria toxin mutant CRM197 and cholera toxin B subunit. Additionally, we demonstrate that high titer IgG responses against purified B. mallei LPS can be generated by immunizing mice with the resulting constructs. Collectively, these approaches provide a rational starting point for the development of novel OPS-based glycoconjugates for immunization against glanders.
    Frontiers in Cellular and Infection Microbiology 01/2012; 2:148.
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    ABSTRACT: Burkholderia pseudomallei and Burkholderia mallei, the etiologic agents of melioidosis and glanders, respectively, cause severe disease in humans and animals and are considered potential agents of biological warfare and terrorism. Diagnosis and treatment of infections caused by these pathogens can be challenging and, in the absence of chemotherapeutic intervention, acute disease is frequently fatal. At present, there are no human or veterinary vaccines available for immunization against these emerging/re-emerging infectious diseases. One of the long term objectives of our research, therefore, is to identify and characterize protective antigens expressed by B. pseudomallei and B. mallei and use them to develop efficacious vaccine candidates. Previous studies have demonstrated that the 6-deoxy-heptan capsular polysaccharide (CPS) expressed by these bacterial pathogens is both a virulence determinant and a protective antigen. Consequently, this carbohydrate moiety has become an important component of the various subunit vaccines that we are currently developing in our laboratory. In the present study, we describe a reliable method for isolating CPS antigens from O-polysaccharide (OPS) deficient strains of B. pseudomallei; including a derivative of the select agent excluded strain Bp82. Utilizing these purified CPS samples, we also describe a simple procedure for covalently linking these T-cell independent antigens to carrier proteins. In addition, we demonstrate that high titer IgG responses can be raised against the CPS component of such constructs. Collectively, these approaches provide a tangible starting point for the development of novel CPS-based glycoconjugates for immunization against melioidosis and glanders.
    Frontiers in Cellular and Infection Microbiology 01/2012; 2:108.
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    ABSTRACT: The Burkholderia pseudomallei K96243 genome encodes six type VI secretion systems (T6SSs), but little is known about the role of these systems in the biology of B. pseudomallei. In this study, we purified recombinant Hcp proteins from each T6SS and tested them as vaccine candidates in the BALB/c mouse model of melioidosis. Recombinant Hcp2 protected 80% of mice against a lethal challenge with K96243, while recombinant Hcp1, Hcp3, and Hcp6 protected 50% of mice against challenge. Hcp6 was the only Hcp constitutively produced by B. pseudomallei in vitro; however, it was not exported to the extracellular milieu. Hcp1, on the other hand, was produced and exported in vitro when the VirAG two-component regulatory system was overexpressed in trans. We also constructed six hcp deletion mutants (Δhcp1 through Δhcp6) and tested them for virulence in the Syrian hamster model of infection. The 50% lethal doses (LD(50)s) for the Δhcp2 through Δhcp6 mutants were indistinguishable from K96243 (<10 bacteria), but the LD(50) for the Δhcp1 mutant was >10(3) bacteria. The hcp1 deletion mutant also exhibited a growth defect in RAW 264.7 macrophages and was unable to form multinucleated giant cells in this cell line. Unlike K96243, the Δhcp1 mutant was only weakly cytotoxic to RAW 264.7 macrophages 18 h after infection. The results suggest that the cluster 1 T6SS is essential for virulence and plays an important role in the intracellular lifestyle of B. pseudomallei.
    Infection and immunity 02/2011; 79(4):1512-25. · 4.21 Impact Factor
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    ABSTRACT: Previous studies have shown that the O polysaccharides (OPS) expressed by Burkholderia mallei are similar to those produced by Burkholderia thailandensis except that they lack the 4-O-acetyl modifications on their 6-deoxy-α-l-talopyranosyl residues. In the present study, we describe the identification and characterization of an open reading frame, designated oacA, expressed by B. thailandensis that accounts for this phenomenon. Utilizing the B. thailandensis and B. mallei lipopolysaccharide (LPS)-specific monoclonal antibodies Pp-PS-W and 3D11, Western immunoblot analyses demonstrated that the LPS antigens expressed by the oacA mutant, B. thailandensis ZT0715, were antigenically similar to those produced by B. mallei ATCC 23344. In addition, immunoblot analyses demonstrated that when B. mallei ATCC 23344 was complemented in trans with oacA, it synthesized B. thailandensis-like LPS antigens. To elucidate the structure of the OPS moieties expressed by ZT0715, purified samples were analyzed via nuclear magnetic resonance spectroscopy. As predicted, these studies demonstrated that the loss of OacA activity influenced the O acetylation phenotype of the OPS moieties. Unexpectedly, however, the results indicated that the O methylation status of the OPS antigens was also affected by the loss of OacA activity. Nonetheless, it was revealed that the LPS moieties expressed by the oacA mutant reacted strongly with the B. mallei LPS-specific protective monoclonal antibody 9C1-2. Based on these findings, it appears that OacA is required for the 4-O acetylation and 2-O methylation of B. thailandensis OPS antigens and that ZT0715 may provide a safe and cost-effective source of B. mallei-like OPS to facilitate the synthesis of glanders subunit vaccine candidates.
    Infection and immunity 02/2011; 79(2):961-9. · 4.21 Impact Factor
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    ABSTRACT: Detection of microbial antigens in clinical samples can lead to rapid diagnosis of an infection and administration of appropriate therapeutics. A major barrier in diagnostics development is determining which of the potentially hundreds or thousands of antigens produced by a microbe are actually present in patient samples in detectable amounts against a background of innumerable host proteins. In this report, we describe a strategy, termed in vivo microbial antigen discovery (InMAD), that we used to identify circulating bacterial antigens. This technique starts with "InMAD serum," which is filtered serum that has been harvested from BALB/c mice infected with a bacterial pathogen. The InMAD serum, which is free of whole bacterial cells, is used to immunize syngeneic BALB/c mice. The resulting "InMAD immune serum" contains antibodies specific for the soluble microbial antigens present in sera from the infected mice. The InMAD immune serum is then used to probe blots of bacterial lysates or bacterial proteome arrays. Bacterial antigens that are reactive with the InMAD immune serum are precisely the antigens to target in an antigen immunoassay. By employing InMAD, we identified multiple circulating antigens that are secreted or shed during infection using Burkholderia pseudomallei and Francisella tularensis as model organisms. Potential diagnostic targets identified by the InMAD approach included bacterial proteins, capsular polysaccharide, and lipopolysaccharide. The InMAD technique makes no assumptions other than immunogenicity and has the potential to be a broad discovery platform to identify diagnostic targets from microbial pathogens. IMPORTANCE: Effective treatment of microbial infection is critically dependent on early diagnosis and identification of the etiological agent. One means for rapid diagnosis is immunoassay for antigens that are shed into body fluids during infection. Immunoassays can be inexpensive, rapid, and adaptable to a point-of-care format. A major impediment to immunoassay for diagnosis of infectious disease is identification of appropriate antigen targets. This report describes a strategy that can be used for identification of microbial antigens that are shed into serum during infection by the biothreats Burkholderia pseudomallei and Francisella tularensis. Termed InMAD (in vivo microbial antigen discovery), the strategy has the potential for application to a broad spectrum of microbial pathogens.
    mBio 01/2011; 2(4). · 5.62 Impact Factor
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    ABSTRACT: Burkholderia pseudomallei and Burkholderia mallei are closely related gram-negative bacteria that can cause serious diseases in humans and animals. This review summarizes the current and rapidly expanding knowledge on the specific virulence factors employed by these pathogens and their roles in the pathogenesis of melioidosis and glanders. In particular, the contributions of recently identified virulence factors are described in the context of the intracellular lifestyle of these pathogens. Throughout this review, unique and shared virulence features of B. pseudomallei and B. mallei are discussed.
    Annual review of microbiology 01/2010; 64:495-517. · 12.80 Impact Factor
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    ABSTRACT: Burkholderia mallei is a facultative intracellular pathogen that causes severe disease in animals and humans. Recent studies have shown that the cluster 1 type VI secretion system (T6SS-1) expressed by this organism is essential for survival in a hamster model of glanders. To better understand the role of T6SS-1 in the pathogenesis of disease, studies were initiated to examine the interactions of B. mallei tssE mutants with RAW 264.7 murine macrophages. Results obtained by utilizing modified gentamicin protection assays indicated that although the tssE mutants were able to survive within RAW 264.7 cells, significant growth defects were observed in comparison to controls. In addition, analysis of infected monolayers by differential interference contrast and fluorescence microscopy demonstrated that the tssE mutants lacked the ability to induce multinucleated giant cell formation. Via the use of fluorescence microscopy, tssE mutants were shown to undergo escape from lysosome-associated membrane protein 1-positive vacuoles. Curiously, however, following entry into the cytosol, the mutants exhibited actin polymerization defects resulting in inefficient intra- and intercellular spread characteristics. Importantly, all mutant phenotypes observed in this study could be restored by complementation. Based upon these findings, it appears that T6SS-1 plays a critical role in growth and actin-based motility following uptake of B. mallei by RAW 264.7 cells.
    Infection and immunity 11/2009; 78(1):88-99. · 4.21 Impact Factor
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    ABSTRACT: Burkholderia mallei, a category B biothreat agent, is a facultative intracellular pathogen that causes the zoonotic disease glanders. The B. mallei VirAG two-component regulatory system activates the transcription of approximately 60 genes, including a large virulence gene cluster encoding a type VI secretion system (T6SS). The B. mallei tssM gene encodes a putative ubiquitin-specific protease that is physically linked to, and transcriptionally coregulated with, the T6SS gene cluster. Mass spectrometry and immunoblot analysis demonstrated that TssM was secreted in a virAG-dependent manner in vitro. Surprisingly, the T6SS was found to be dispensable for the secretion of TssM. The C-terminal half of TssM, which contains Cys and His box motifs conserved in eukaryotic deubiquitinases, was purified and biochemically characterized. Recombinant TssM hydrolyzed multiple ubiquitinated substrates and the cysteine at position 102 was critical for enzymatic activity. The tssM gene was expressed within 1 h after uptake of B. mallei into RAW 264.7 murine macrophages, suggesting that the TssM deubiquitinase is produced in this intracellular niche. Although the physiological substrate(s) is currently unknown, the TssM deubiquitinase may provide B. mallei a selective advantage in the intracellular environment during infection.
    Infection and immunity 02/2009; 77(4):1636-48. · 4.21 Impact Factor
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    ABSTRACT: Treponema denticola harbours a genetic locus with significant homology to most of the previously characterized Treponema pallidum tro operon. Within this locus are five genes (troABCDR) encoding for the components of an ATP-binding cassette cation-transport system (troABCD) and a DtxR-like transcriptional regulator (troR). In addition, a sigma(70)-like promoter and an 18 bp region of dyad symmetry were identified upstream of the troA start codon. This putative operator sequence demonstrated similarity to the T. pallidum TroR (TroR(Tp)) binding sequence; however, the position of this motif with respect to the predicted tro promoters differed. Interestingly, unlike the T. pallidum orthologue, T. denticola TroR (TroR(Td)) possesses a C-terminal Src homology 3-like domain commonly associated with DtxR family members. In the present study, we show that TroR(Td) is a manganese- and iron-dependent transcriptional repressor using Escherichia coli reporter constructs and in T. denticola. In addition, we demonstrate that although TroR(Td) possessing various C-terminal deletions maintain metal-sensing capacities, these truncated proteins exhibit reduced repressor activities in comparison with full-length TroR(Td). Based upon these findings, we propose that TroR(Td) represents a novel member of the DtxR family of transcriptional regulators and is likely to play an important role in regulating both manganese and iron homeostases in this spirochaete.
    Molecular Microbiology 09/2008; 70(2):396-409. · 4.96 Impact Factor
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    ABSTRACT: Burkholderia pseudomallei is a facultative intracellular pathogen capable of surviving and replicating within eukaryotic cells. Recent studies have shown that B. pseudomallei Bsa type III secretion system 3 (T3SS-3) mutants exhibit vacuolar escape and replication defects in J774.2 murine macrophages. In the present study, we characterized the interactions of a B. pseudomallei bsaZ mutant with RAW 264.7 murine macrophages. Following uptake, the mutant was found to survive and replicate within infected RAW 264.7 cells over an 18-h period. In addition, high levels of tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), granulocyte-macrophage colony-stimulating factor (GM-CSF), and RANTES, but not IL-1alpha and IL-1beta, were detected in culture supernatants harvested from infected monolayers. The subcellular location of B. pseudomallei within infected RAW 264.7 cells was determined, and as expected, the bsaZ mutant demonstrated early-vacuolar-escape defects. Interestingly, however, experiments also indicated that this mutant was capable of delayed vacuolar escape. Consistent with this finding, evidence of actin-based motility and multinucleated giant cell formation were observed between 12 and 18 h postinfection. Further studies demonstrated that a triple mutant defective in all three B. pseudomallei T3SSs exhibited the same phenotype as the bsaZ mutant, indicating that functional T3SS-1 and T3SS-2 did not appear to be responsible for the delayed escape phenotype in RAW 264.7 cells. Based upon these findings, it appears that B. pseudomallei may not require T3SS-1, -2, and -3 to facilitate survival, delayed vacuolar escape, and actin-based motility in activated RAW 264.7 macrophages.
    Infection and immunity 08/2008; 76(7):2991-3000. · 4.21 Impact Factor
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    ABSTRACT: Burkholderia mallei is a facultative intracellular pathogen that can cause fatal disease in animals and humans. To better understand the role of phagocytic cells in the control of infections caused by this organism, studies were initiated to examine the interactions of B. mallei with RAW 264.7 murine macrophages. Utilizing modified kanamycin-protection assays, B. mallei was shown to survive and replicate in RAW 264.7 cells infected at multiplicities of infection (moi) of < or = 1. In contrast, the organism was efficiently cleared by the macrophages when infected at an moi of 10. Interestingly, studies demonstrated that the monolayers only produced high levels of TNF-alpha, IL-6, IL-10, GM-CSF, RANTES and IFN-beta when infected at an moi of 10. In addition, nitric oxide assays and inducible nitric oxide synthase (iNOS) immunoblot analyses revealed a strong correlation between iNOS activity and clearance of B. mallei from RAW 264.7 cells. Furthermore, treatment of activated macrophages with the iNOS inhibitor, aminoguanidine, inhibited clearance of B. mallei from infected monolayers. Based upon these results, it appears that moi significantly influence the outcome of interactions between B. mallei and murine macrophages and that iNOS activity is critical for the clearance of B. mallei from activated RAW 264.7 cells.
    Cellular Microbiology 02/2008; 10(2):487-98. · 4.81 Impact Factor
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    ABSTRACT: Co-ordinated regulation of gene expression is required for the transmission and survival of Borrelia burgdorferi in different hosts. The sigma factor RpoS (sigma(S)), as regulated by RpoN (sigma(54)), has been shown to regulate key virulence factors (e.g. OspC) required for these processes. As important, multiple signals (e.g. temperature, pH, cell density, oxygen) have been shown to increase the expression of sigma(S)-dependent genes; however, little is known about the signal transduction mechanisms that modulate the expression of rpoS. In this report we show that: (i) rpoS has a sigma(54)-dependent promoter that requires Rrp2 to activate transcription; (ii) Rrp2Delta123, a constitutively active form of Rrp2, activated sigma(54)-dependent transcription of rpoS/P-lacZ reporter constructs in Escherichia coli; (iii) quantitative reverse transcription polymerase chain reaction (QRT-PCR) experiments with reporter cat constructs in B. burgdorferi indicated that Rrp2 activated transcription of rpoS in an enhancer-independent fashion; and finally, (iv) rpoN is required for cell density- and temperature-dependent expression of rpoS in B. burgdorferi, but histidine kinase Hk2, encoded by the gene immediately upstream of rrp2, is not essential. Based on these findings, a model for regulation of rpoS has been proposed which provides mechanisms for multiple signalling pathways to modulate the expression of the sigma(S) regulon in B. burgdorferi.
    Molecular Microbiology 08/2007; 65(2):277-93. · 4.96 Impact Factor
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    ABSTRACT: Burkholderia mallei, the aetiologic agent of glanders, causes a variety of illnesses in animals and humans ranging from occult infections to acute fulminating septicaemias. To better understand the role of lipopolysaccharide (LPS) in the pathogenesis of these diseases, studies were initiated to characterize the structural and biological properties of lipid A moieties expressed by this organism. Using a combination of chemical analyses and MALDI-TOF mass spectrometry, B. mallei was shown to express a heterogeneous mixture of tetra- and penta-acylated lipid A species that were non-stoichiometrically substituted with 4-amino-4-deoxy-arabinose residues. The major penta-acylated species consisted of bisphosphorylated d-glucosamine disaccharide backbones possessing two amide linked 3-hydroxyhexadecanoic acids, two ester linked 3-hydroxytetradecanoic acids [C14:0(3-OH)] and an acyloxyacyl linked tetradecanoic acid, whereas, the major tetra-acylated species possessed all but the 3'-linked C14:0(3-OH) residues. In addition, although devoid of hexa-acylated species, B. mallei LPS was shown to be a potent activator of human Toll-like receptor 4 complexes and stimulated human macrophage-like cells (THP-1 and U-937), monocyte-derived macrophages and dendritic cells to produce high levels of TNF-alpha, IL-6 and RANTES. Based upon these results, it appears that B. mallei LPS is likely to play a significant role in the pathogenesis of human disease.
    Molecular Microbiology 02/2007; 63(2):379-90. · 4.96 Impact Factor

Publication Stats

1k Citations
128.74 Total Impact Points

Institutions

  • 2009–2014
    • University of South Alabama
      • Department of Microbiology and Immunology
      Mobile, Alabama, United States
    • Emory University
      • Department of Biochemistry
      Atlanta, GA, United States
  • 2012–2013
    • University of Georgia
      • Complex Carbohydrate Research Center
      Athens, GA, United States
  • 2010
    • University of Leicester
      • Department of Infection, Immunity and Inflammation
      Leicester, ENG, United Kingdom
  • 2007
    • National Institute of Allergy and Infectious Diseases
      Maryland, United States
  • 2003
    • Genomics Institute of the Novartis Research Foundation
      San Diego, California, United States
  • 2002–2003
    • The University of Calgary
      • Department of Microbiology, Immunology and Infectious Diseases
      Calgary, Alberta, Canada
  • 1999
    • U.S. Army Medical Research Institute of Infectious Diseases
      Maryland, United States
    • Mahidol University
      Krung Thep, Bangkok, Thailand