Burkholderia

Burkholderia pseudomallei autotransporter and TPS proteins

Peggy Cotter, UC Santa Barbara

Abstract:
Burkholderia pseudomallei, the causative agent of melioidosis, is a Category B priority pathogen. It causes serious and often lethal human disease and represents both a worldwide emerging infectious disease problem and a bioterrorism threat due to its extremely low infectious dose, the ability to initiate infection by an aerosol route, an intrinsic resistance to commonly used antibiotics, and the lack of a currently available vaccine. Little is known about the molecular bases of B. pseudomallei pathogenesis. Autotransporter (AT) and Two Partner Secretion (TPS) pathway proteins are large, surface-localized and secreted proteins that function as virulence factors in many Gram-negative bacterial pathogens. Available genome sequence information indicates that B. pseudomallei has the potential to produce at least ten AT proteins and three TPS pathway proteins. This project is focused on determining the roles of the B. pseudomallei AT and TPS pathway proteins in pathogenesis. Specifically, we will: 1) characterize the expression and localization of each predicted AT and TPS protein in B. pseudomallei; 2) investigate the roles of the predicted AT and TPS proteins in vivo using mouse, nematode and amoeba models; and 3) investigate the mechanism of action of predicted AT and TPS proteins at the molecular level using a variety of in vitro assays. In addition to contributing to our knowledge of how B. pseudomallei causes disease at the molecular level, our results are expected to be directly applicable to the development of point-of-care diagnostics, effective therapeutics and efficacious vaccines.

Type III Secretion in Burkholderia pseudomallei

Jeff F. Miller, UC Los Angeles

Abstract:
Type III secretion systems (T3SSs) allow Gram-negative bacteria to inject protein effector molecules into eukaryotic cells. T3SS effectors perform an impressive array of functions to manipulate the eukaryotic hosts and represent one of the most complex mechanisms of protein translocation in biology. B. pseudomallei contains three “injection-type” T3SS gene clusters. Of these, only T3SS-3 has been addressed in the literature and the analysis is rudimentary. We have devised in silico screens that allow the identification of T3SS effector genes. Out of 18 candidate effectors, we have partially characterized four. Our goal is to systematically characterize the Type III “secretome” of B. pseudomallei, taking into account the broad host specificity and enormous phylogenetic diversity of the species. Our specific aims are to: 1) complete a comprehensive and systematic identification of T3SS substrates; 2) evaluate the roles of T3SS effectors in mammalian, nematode and amoeba models of infection; and 3) identify the intracellular changes, targets and mechanisms of action of selected T3SS effectors.

Genomic correlates with differential virulence in melioidosis animal models

Paul Keim, Northern Arizona University

Abstract:
This project will examine the effects of genomic diversity on B. pseudomallei virulence by surveying a large collection of strains in mouse and rat melioidosis models. We will also examine the infection route-by-strain effect in mouse-inbred lines. Differential virulence will be correlated with high-resolution strain genotyping to identify putative controlling factors, such as genomic islands. Potential virulence factors will be subsequently examined by gene knockout constructs. Proteomic and gene expression patterns of high- and low-virulence strains will be characterized to better understand the virulence phenotypes. Our primary hypothesis is that genomic variations control the differential virulence that exists among B. pseudomallei strains. Our specific aims are to: 1) identify and assemble relevant diverse clinical and environmental strains for testing differential virulence in selected animal models; 2) genomically characterize selected strains to identify genetic correlates of differential virulence; 3) test representative strains to measure their differential virulence in animal models; 4) correlate genomic composition to virulence data; and 5) test virulence determinants by gene knockouts, antigenic response surveys and functional genomics..

Antigenemia immunoassay for point-of-care diagnosis of inhalational melioidosis

Thomas Kozel, University of Nevada, Reno

Abstract:
The goal of this study is to develop a point-of-care immunoassay that detects secreted bacterial antigens in serum and urine for early diagnosis of melioidosis. A point-of-care immunoassay for diagnosis of melioidosis could greatly impact patient outcome because a high percentage of patients with acute septicemia die before culture results are available, and the antibiotics used for empirical treatment of septicemia are not effective for B. pseudomallei. A novel approach to target discovery termed In vivo Microbial Antigen Discovery (InMAD) will be used to identify candidate protein and polysaccharide antigens. InMAD will use direct proteomic examination of serum and urine from infected BALB/c mice and humans to identify B. pseudomallei proteins that have been shed into body fluids during infection. In a complementary approach, immunization of syngeneic BALB/c mice with filtered serum or urine from infected BALB/c mice will be used to raise antibodies specific for the shed bacterial proteins. These antibodies will be used to probe a proteome array to identify B. pseudomallei proteins that are recognized by the immune serum. The specific aims are to i) construct immunoassays in ELISA format for detection of potential polysaccharide targets using monoclonal antibodies (mAbs); ii) use InMAD to identify candidate target proteins that are secreted in vivo during B. pseudomallei infection; iii) generate mAbs specific for candidate target proteins; iv) construct immunoassays in point-of-care format for detection of B. pseudomallei polysaccharides and proteins; and v) evaluate prototype immunoassays using murine models of pulmonary melioidosis and archived samples from humans with melioidosis. The ultimate product will be an immunoassay that identifies the presence of two or more distinct B. pseudomallei-specific antigens.

Analysis of regulatory networks controlling virulence in Burkholderia pseudomallei

Imke Schroeder, UC Los Angeles

Abstract:
Burkholderia pseudomallei is a highly versatile bacterium that colonizes vastly different environments including soil, humans and other mammals, and possibly protozoa and plants. The flexibility to adapt to and thrive in such diverse habitats is reflected in its 7.24 Mb genome which harbors a rich assortment of metabolic loci and a large selection of genes that are predicted to modulate pathogenicity and host/cell interactions. The goal of this project is to identify regulatory elements and establish regulatory networks that govern the expression of virulence and colonization determinants. Little is known about the coordinate regulation of virulence factors in B. pseudomallei. Using known virulence determinants as the target, we plan to identify and characterize global transcription factors that regulate the expression of unrelated virulence genes. A combination of in-frame deletion mutagenesis by allelic exchange, ectopic expression using a controllable promoter, and DNA microarray analysis will be employed to characterize control circuits. Our specific aim includes the determination of the role of “local” regulatory factors on type IIII secretion system (T3SS) gene expression. We hypothesize that “local” T3SS regulators are part of a global regulatory network that governs their expression in response to multiple signal input. Using this approach, we plan to predict regulatory networks controlling virulence genes that will be integrated into the experimental analysis of transcriptional regulators to assemble a more complete picture of potentially overlapping regulatory circuits. Understanding regulatory networks is essential for conceptualizing pathogenesis and to identify additional virulence factors that could be useful for developing antimicrobial agents and vaccines, and may be exploited to serve a more effective rapid diagnosis of melioidosis.