Comparative Genomics and an Insect Model Rapidly Identify Novel Virulence Genes of

Department of Microbiology, University of Georgia, Athens, GA 30602, USA.
Journal of bacteriology (Impact Factor: 2.81). 05/2008; 190(7):2306-13. DOI: 10.1128/JB.01735-07
Source: PubMed


Burkholderia pseudomallei and its host-adapted deletion clone Burkholderia mallei cause the potentially fatal human diseases melioidosis and glanders, respectively. The antibiotic resistance profile and
ability to infect via aerosol of these organisms and the absence of protective vaccines have led to their classification as
major biothreats and select agents. Although documented infections by these bacteria date back over 100 years, relatively
little is known about their virulence and pathogenicity mechanisms. We used in silico genomic subtraction to generate their
virulome, a set of 650 putative virulence-related genes shared by B. pseudomallei and B. mallei but not present in five closely related nonpathogenic Burkholderia species. Although most of these genes are clustered in putative operons, the number of targets for mutant construction and
verification of reduced virulence in animal models is formidable. Therefore, Galleria mellonella (wax moth) larvae were evaluated as a surrogate host; we found that B. pseudomallei and B. mallei, but not other phylogenetically related bacteria, were highly pathogenic for this insect. More importantly, four previously
characterized B. mallei mutants with reduced virulence in hamsters or mice had similarly reduced virulence in G. mellonella larvae. Site-specific inactivation of selected genes in the computationally derived virulome identified three new potential
virulence genes, each of which was required for rapid and efficient killing of larvae. Thus, this approach may provide a means
to quickly identify high-probability virulence genes in B. pseudomallei, B. mallei, and other pathogens.

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    • "Type strain; isolated in 1944 from a human case of glanders Yabuuchi et al., 1992 GRS 23344 ATCC 23344 derivative; sucrose-resistant, sacB: Pm r , Zeo s Schell et al., 2008 BM210 GRS 23344 derivative; wcbB: Pm r , Zeo s This study "
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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 11/2012; 2:148. DOI:10.3389/fcimb.2012.00148 · 3.72 Impact Factor
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    • "The experiments reported here were undertaken to correlate disease phenotypes in the murine model with the genetic composition of C. jejuni isolates tested and to extend our understanding of the ability of the murine model to replicate the spectrum of C. jejuniassociated disease seen in humans. Similar approaches using combinations of in vivo and in silico methods have been used to define the " virulome, " or the set of genes necessary for pathogenicity , of Brucella suis and Burkholderia mallei [18] [19]. "
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    ABSTRACT: Human Campylobacter jejuni infection can result in an asymptomatic carrier state, watery or bloody diarrhea, bacteremia, meningitis, or autoimmune neurological sequelae. Infection outcomes of C57BL/6 IL-10(-/-) mice orally infected with twenty-two phylogenetically diverse C. jejuni strains were evaluated to correlate colonization and disease phenotypes with genetic composition of the strains. Variation between strains was observed in colonization, timing of development of clinical signs, and occurrence of enteric lesions. Five pathotypes of C. jejuni in C57BL/6 IL-10(-/-) mice were delineated: little or no colonization, colonization without disease, colonization with enteritis, colonization with hemorrhagic enteritis, and colonization with neurological signs with or without enteritis. Virulence gene content of ten sequenced strains was compared in silico; virulence gene content of twelve additional strains was compared using a C. jejuni pan-genome microarray. Neither total nor virulence gene content predicted pathotype; nor was pathotype correlated with multilocus sequence type. Each strain was unique with regard to absences of known virulence-related loci and/or possession of point mutations and indels, including phase variation, in virulence-related genes. An experiment in C. jejuni 11168-infected germ-free mice showed that expression levels of ninety open reading frames (ORFs) were significantly up- or down-regulated in the mouse cecum at least two-fold compared to in vitro growth. Genomic content of these ninety C. jejuni 11168 ORFs was significantly correlated with the capacity to colonize and cause enteritis in C57BL/6 IL-10(-/-) mice. Differences in gene expression levels and patterns are thus an important determinant of pathotype in C. jejuni strains in this mouse model.
    Microbial Pathogenesis 08/2012; 54(1). DOI:10.1016/j.micpath.2012.08.001 · 1.79 Impact Factor
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    • "Several surrogate hosts have been used as alternatives to mammals to study virulence factors and host-pathogen interactions with B. pseudomalleiB. mallei, and B. thailandensis, including Galleria mellonella larvae (wax worms) [15,16], Dictyostelium discoideum (phagocytic amoeba) [17], Caenorhabditis elegans (soil nematode) [18-20], and Solanum lycopersicum (tomato plantlets) [21]. These alternative hosts have allowed the identification of new Burkholderia virulence determinants and have confirmed the importance of virulence factors previously characterized using rodent models of infection. "
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    ABSTRACT: Burkholderia pseudomallei and Burkholderia mallei are gram-negative pathogens responsible for the diseases melioidosis and glanders, respectively. Both species cause disease in humans and animals and have been designated as category B select agents by the Centers for Disease Control and Prevention (CDC). Burkholderia thailandensis is a closely related bacterium that is generally considered avirulent for humans. While it can cause disease in rodents, the B. thailandensis 50% lethal dose (LD50) is typically ≥ 104-fold higher than the B. pseudomallei and B. mallei LD50 in mammalian models of infection. Here we describe an alternative to mammalian hosts in the study of virulence and host-pathogen interactions of these Burkholderia species. Madagascar hissing cockroaches (MH cockroaches) possess a number of qualities that make them desirable for use as a surrogate host, including ease of breeding, ease of handling, a competent innate immune system, and the ability to survive at 37°C. MH cockroaches were highly susceptible to infection with B. pseudomallei, B. mallei and B. thailandensis and the LD50 was <10 colony-forming units (cfu) for all three species. In comparison, the LD50 for Escherichia coli in MH cockroaches was >105 cfu. B. pseudomallei, B. mallei, and B. thailandensis cluster 1 type VI secretion system (T6SS-1) mutants were all attenuated in MH cockroaches, which is consistent with previous virulence studies conducted in rodents. B. pseudomallei mutants deficient in the other five T6SS gene clusters, T6SS-2 through T6SS-6, were virulent in both MH cockroaches and hamsters. Hemocytes obtained from MH cockroaches infected with B. pseudomallei harbored numerous intracellular bacteria, suggesting that this facultative intracellular pathogen can survive and replicate inside of MH cockroach phagocytic cells. The hemolymph extracted from these MH cockroaches also contained multinuclear giant cells (MNGCs) with intracellular B. pseudomallei, which indicates that infected hemocytes can fuse while flowing through the insect's open circulatory system in vivo. The results demonstrate that MH cockroaches are an attractive alternative to mammals to study host-pathogen interactions and may allow the identification of new Burkholderia virulence determinants. The importance of T6SS-1 as a virulence factor in MH cockroaches and rodents suggests that the primary role of this secretion system is to target evasion of the innate immune system.
    BMC Microbiology 06/2012; 12(1):117. DOI:10.1186/1471-2180-12-117 · 2.73 Impact Factor
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