Burkholderia
Identification and functional analysis of adhesins expressed by Burkholderia pseudomallei
David Low, University of California, Santa Barbara
Abstract:
B. pseudomallei is an NIAID Category B Priority Pathogen and the threat it presents as a potential agent of bioterrorism is highlighted by 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. This proposal focused on adherence of B. pseudomallei to eukaryotic cell surfaces, which is often an early and often crucial step in bacterial pathogenesis. Proteins that mediate adherence (adhesins), function to target pathogens to specific host tissues and can facilitate invasion of host cells. The overall goal of this proposal is to identify the repertoire of adhesin-encoding genes present in the B. pseudomallei genome and to determine their roles in adherence to oral and respiratory epithelial tissues. B. pseudomallei mutants, containing single and multiple mutations in each of the adhesin operons, will be analyzed in collaboration with Project 2 (Peggy Cotter) using mouse models to assess the roles of adhesins in pathogenesis. This work will have direct application to the development of an effective vaccine against melioidosis. The first aim is to identify the repertoire of adhesins expressed by B. pseudomallei. B. pseudomallei grown under a variety of different conditions will be tested for its ability to adhere to various biologically relevant eukaryotic cells. Targeted and random mutagenesis approaches will be used to identify genes encoding adhesins and their roles in adherence to eukaryotic cells will be assessed by constructing non-polar deletion mutations in each adhesin gene cluster. B. pseudomallei strains containing multiple adhesin gene mutations will also be constructed and evaluated since adhesins may have overlapping target specificities. The second aim is to assess the expression profiles and role(s) of each of the adhesins identified in Aim 1 in pathogenesis. Antisera will be raised against each pilus/adhesin protein and FACS analysis used to determine the profiles of adhesin expression under different growth conditions. Expression of adhesins in human melioidosis will be determined using patient antisera. The role(s) of B. pseudomallei adhesins in pathogenesis will be assessed using adhesin mutants. The third aim is to identify locally- and globally-acting factors that control the expression of B. pseudomallei adhesins. It is likely that B. pseudomallei adhesin genes are regulated by both local and global regulatory factors, which will be identified in this aim. This information will allow us to determine if multiple adhesins are regulated by common pathways and if regulatory cross-talk occurs between adhesin operons, providing the molecular basis for understanding how adhesin expression/pathogenesis is regulated by environmental conditions.
Investigate the role(s) of the Burkholderia pseudomallei filamentous hemagglutinin-like protein and other putative virulence factors in infection and immunity
Peggy Cotter, University of California, Santa Barbara
Abstract:
Burkholderia pseudomallei, the causative agent of melioidosis, is an NIAID 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. This project has two main goals. One is to develop animal models for investigating how specific B. pseudomallei virulence factors contribute to the establishment and course of respiratory infection, the induction of inflammation and innate immunity, and the development of adaptive and potentially protective immunity. These models will be used to evaluate the in vivo functions of B. pseudomallei virulence factors and regulatory systems identified in projects 4.1, 4.2 and 4.3. These models will also be used to test potential component vaccine candidates identified in these various projects. The second goal of Project 4.2 is to determine if a specific B. pseudomallei gene with the potential to encode a filamentous hemagglutinin (FHA)-like protein does in fact represent a bona fide Burkholderia virulence gene. FHA is a multifunctional virulence factor expressed by Bordetella pertussis that represents a major adhesin, an immunomodulatory factor, and an important immunoprotective component of newly formulated acellular pertussis vaccines. We will determine if the B. pseudomallei FHA-like protein functions analogously to FHA and test its potential as a vaccine component.
Type III Secretion in Burkholderia pseudomallei
Jeff F. Miller, University of California, Los Angeles
Abstract:
Type III secretion systems allow Gram-negative bacteria to inject protein effector molecules into the cytoplasm or plasma membrane of eukaryotic cells. Secreted effectors are capable of modulating a wide range of host cell functions and they play a major role in the virulence strategies of bacteria that encode them. Since type III secretion control systems are almost always coupled to global regulators that control virulence, understanding how type III secretion is regulated provides an expedient means to identify previously unknown determinants of pathogenesis. Burkholderia pseudomallei contains three gene clusters that are predicted to encode ?injection-type? type III secretion systems. Relatively little is know about their individual and combinatorial functions during infection. The goals of this project are to conduct a comprehensive assessment of the roles in pathogenesis of the B. pseudomallei type III secretion systems and to use an understanding of the regulation of type III secretion to identify co-regulated virulence factors. In addition to highlighting mechanisms of pathogenesis, we expect these studies will identify potential immunogens for the development of vaccines.
Targeting the B. pseudomallei capsule for immunodiagnosis
Thomas Kozel, University of Nevada Reno
Abstract:
Type III secretion systems allow Gram-negative bacteria to inject protein effector molecules into the cytoplasm or plasma membrane of eukaryotic cells. Secreted effectors are capable of modulating a wide range of host cell functions and they play a major role in the virulence strategies of bacteria that encode them. Since type III secretion control systems are almost always coupled to global regulators that control virulence, understanding how type III secretion is regulated provides an expedient means to identify previously unknown determinants of pathogenesis. Burkholderia pseudomallei contains three gene clusters that are predicted to encode ?injection-type? type III secretion systems. Relatively little is know about their individual and combinatorial functions during infection. The goals of this project are to conduct a comprehensive assessment of the roles in pathogenesis of the B. pseudomallei type III secretion systems and to use an understanding of the regulation of type III secretion to identify co-regulated virulence factors. In addition to highlighting mechanisms of pathogenesis, we expect these studies will identify potential immunogens for the development of vaccines.
Role of Rho GTPases in B. pseudomallei infection
Ulla Knaus, The Scripps Research Institute
Project period: May 2006 - April 2008
Abstract:
Melioidosis is a highly infectious disease caused by exposure to B. pseudomallei. B. pseudomallei interaction with host cells and evasion of host immune response is mediated by a type III secretion apparatus, endosomal escape, intracellular replication, and actin tail-based motility and cell fusion. Many of these features are connected to actin changes and are more than likely triggered by Rho GTPases, critical regulators of actin rearrangements. We propose to use Rho GTPase-deficient innate immune cells to connect specific GTPase functions to B. pseudomallei infection. Elucidating how B. pseudomallei hijack regulators of cellular actin dynamics is essential to combating this pathogen.
Occurrence and ecologic associations of virulence genes in Burkholderia pseudomallei
David Wagner, Northern Arizona University
Project period: May 2006 - April 2008
Abstract:
We will use bioinformatic analyses, high-throughput genetic assays, and new and existing DNA/isolate collections to examine how the presence/absence of specific virulence genes varies across populations of Burkholderia pseudomallei and to determine if this variation may be due to growth in different ecologic settings.
For our first aim, we will examine how the presence/absence of known and putative virulence genes varies among isolates of B. pseudomallei. To date, there has been very little research on how the presence/absence of virulence genes may vary among populations of B. pseudomallei. We will work to use bioinformatic analyses to identify known or putative virulence genes that vary in presence/absence among the available genome sequences of B. pseudomallei, and to use high-throughput genetic assays to examine how the presence/absence of these genes varies across large isolate collections that include strains from diverse settings (e.g., human vs. environmental) and locations (i.e., Australia vs. Thailand). Our specific research questions for this aim include: Do clinical and environmental isolates differ in the presence/absence of virulence genes? Do Thai and Australian isolates differ in the presence/absence of virulence genes?
For the second aim of our research, we will use in vitro experiments to examine how growth under different ecologic conditions influences the virulence of B. pseudomallei. Although B. pseudomallei occurs in a variety of ecologic settings, including soil and water, very little is known about how growth in these different settings may influence virulence. We will grow B. pseudomallei strains under several different ecologic settings and examine the influence of these conditions on strain virulence. Our specific research questions for this aim include: Does growth under different ecologic conditions influence the presence/absence of virulence genes? Does growth under different ecologic conditions influence the expression of virulence genes? Do clinical and environmental isolates differ in their ability to survive in different ecologic settings?
Integrative genomic and proteomic analyses of Burkholderia pseudpmallei
Apichai Tuanyok, Northern Arizona University
Project period: May 2007 - April 2009
Abstract:
We will investigate molecular processes that contribute to phenotypic diversity in B. pseudomallei using an integrative approach, including comparative genomic and proteomic analyses of distinct strains. Our current genomic studies with two distinct B. pseudomallei strains have identified variability at conserved regions and accessory genetic elements known as genomic islands. This variation includes gene-loss from the ancestral state and gene-gain via horizontal transfer. We have identified the existence of two distinct B. pseudomallei populations with important biological consequences. We believe that these different types lead to differential disease outcomes, including clinical manifestations and pathological severity. We hypothesize that genomic diversity leads to transcriptome and proteome diversity among B. pseudomallei strains, which is the basis for the differential disease pathology. We propose two different, but integrated, sub-projects. Firstly, we will investigate genomic diversity across a number of strains using comparative genomic hybridization (CGH). This microarray-based technique will be designed to contain all genes from 12+ genomes currently sequenced. This will allow us to examine the diversity of many more strains to understand their “status” vis-à-vis the presence and absence of all known B. pseudomallei genes.. Secondly, we will ask if the diversity identified from genomic analyses predicts translational diversity among the tested strains.
Occurrence and ecologic associations of virulence genes in Burkholderia pseudomallei
Minghsun Liu, University of California, Los Angeles
Project period: May 2007 - April 2008
Abstract:
Macrophages are central to the pathogenesis of many infectious diseases, particularly those caused by intracellular pathogens. In melioidosis, macrophages have been shown to be essential for the early control of Burkholderia pseudornallei infection. Survival of B. pseudomallei in professional phagocytes has long been postulated as the mechanism that allows B. pseudomallei to enter latency and reactivate at a later time. The objective of this project is to define the macrophage transcriptional response to B. pseudornallei and in parallel use a genome-wide screen to identify B. pseudomallei genes required for intracellular survival and growth. Our hypothesis is that the macrophage-B. pseudomallei interaction is key to persistence of B. pseudomallei inside a host. A detailed molecular understanding of this interaction will improve our management of B. pseudomallei as a potential bioweapon and melioidosis as an emerging infectious disease worldwide. Two aims are proposed: 1) Define the transcriptional program of macrophages from wild- type and mutant mice in response to Bukholderia thailandensis and B. pseudornallei infections; and 2) Identify B. pseudomallei genes that are expressed inside macrophages and construct mutants defective in intracellular survival and replication.
In vivo microbial antigen discovery (InMAD) for melioidosis
David Au Coin, University of Nevada Reno
Abstract:
In vivo microbial antigen discovery (InMAD) is a novel technique that utilizes serum from infected mice to identify secreted antigens. Filtered serum from B. pseudomallei infected BALB/c mice will be used to immunize syngeneic BALB/c mice. The hypothesis is that sera from mice that have been infected contain secreted antigens that can be used to generate polyclonal antibodies. The polyclonal antibodies can be used to identify relevant antigens by Western blot and proteomic analysis or by probing of microbial proteome arrays. Monoclonal antibodies can then be developed to the best candidate antigens for eventual use in antigen capture immunoassays for melioidosis. The aims of this project are:
1. Generate polyclonal antibodies against in vivo secreted protein antigens of B. pseudomallei and identify candidate proteins for immunoassay.
2. Begin production of monoclonal antibodies to candidate secreted protein antigens.
The candidate antigens that are identified by this technique will be either secreted or shed during infection. Therefore, these antigens are precisely the proteins that should be evaluated for an antigen detection immunoassay. This antigen discovery process has the potential for use with any biothreat for which a BALB/c model exists.
Relevance: This proposal will develop a novel microbial antigen discovery process that can be translated to any pathogen for which a murine model exists. The antigens that are identified can be exploited for diagnostic or therapeutic use.
