Molecular mechanisms of F. tularensis pathogenesis and immunity

Denise Monack, Stanford University
Project period: May 2007 – April 2009

Abstract:
Francisella tularensis is a highly infectious gram-negative coccobacillus that causes the zoonosis tularemia and is a potential bioterror Category A agent. F. tularensis survives and replicates within macrophages, a phenotype that correlates with virulence, yet little is understood about the molecular basis of its pathogenesis. The long-term goal of this project is to understand how F. tularensis subverts host defenses. We hypothesize that F. tularensis produces gene products that alter the trafficking of the initial bacterial phagosome, as well as additional molecules that lyse the phagosome, thereby allowing bacteria to escape into the cytoplasm where they can survive and replicate. The objective of this proposal is to use molecular genetic approaches to identify F. tularensis molecules that are involved in lysis of the bacterial phagosome and to identify molecules that are required for intracellular growth/survival. In Aim 1, additional mglA-regulated genes will be assessed for their contribution to the escape from the phagosome. In Aim 2, targeted mutations in the genes identified in Aim 1 and random transposon insertion mutants will be constructed and characterized for their ability to replicate in macrophages and to alter phagosomal trafficking.

Restoration of SNARE-dependent exocytosis to Botulinum neurotoxin intoxicated neurons

Mauricio Montal, University of California, San Diego
Project period: May 2006 - April 2008

Abstract:
Botulinum toxin (BoNT) is a bioweapon without an antidote. It is imperative, urgent, and ethical to uncover reliable and selective antidotes for BoNTs. Our program is aimed towards antidote design focusing on the preservation of the SNARE (soluble NSF attachment protein receptor) complex – the synaptic vesicle docking/fusion complex and the substrate of the BoNT light chain (LC) protease. The immediate goal is to demonstrate that restoration of SNARE-mediated exocytosis to BoNT intoxicated neurons may be achieved by increasing the propensity to assemble BoNT–resistant SNARE complexes. The project involves making and delivering peptide decoys to BoNT/A intoxicated neurons, which form complexes with SNAP-25 (synaptosome-associated protein of 25 kDa) coil-forming domains before the assembly of the SNARE complex. The consequence of this intervention is to accrue two populations of SNAP-25 within neurons: one population competent for SNARE complex formation and ensued exocytosis; the other, bound to decoys rendering it accessible to BoNT/A cleavage. The net effect would be restoration of exocytosis. We already have proof-of-principle for this approach. Furthermore, there is now a crystal structure of the BoNT/A LC in complex with the C-terminal core domain of SNAP-25, which will accelerate the structure-based design of additional decoys. This strategy is not limited to peptides: peptidomimetics are a valid option, as demonstrated with our discovery of a blocker of the NMDA receptor channel, using combinatorial libraries and a modest high-throughput screen. And the approach is readily extendable to synaptobrevin and syntaxin, the other two components of the SNARE complex and substrates of other BoNT isoforms. Mixtures of decoys targeted to the three SNARE components may provide a more effective path to preserve SNAREs, which are competent in regulated exocytosis. The assays involve BoNT exposed chromaffin and PC12 cells as well as Aplysia neurons, with the concomitant measurements of regulated exocytosis in secretory cells, and of synaptic transmission in Aplysia neurons. Realistic deployment of antidotes requires selective delivery. The molecular decoys targeted to SNAP-25 act in the neutral cytosolic pH; accordingly, decoys will be conjugated with membrane-permeabilizing oligopeptides composed exclusively of D-arginine thereby increasing both their resistance to proteolysis and bioavailability. 
This program is of prime significance to biodefense. Experimental validation of the activity of the postulated “molecular decoys” should facilitate the design of structure-based SNARE peptide segments or peptidomimetics, which may restore synaptic exocytosis to botulinum neurotoxin, intoxicated neurons. Major outcomes of this focused and innovative program would be lead discovery and structural blueprints for antidote design.

Implementation of alternating laser excitation (ALEX) fluorescence spectroscopy for detection of BOTOX

Armin Reimair, Nesher Technologies, Inc.
Project period: May 2006 - April 2008

Abstract:
Nesher Technologies, Inc. (NTI) licensed from UCLA the IP for a revolutionary suite of ultrasensitive biodetection technologies - developed at the Single Molecule Biophysics Laboratory (headed by Prof. Shimon Weiss) - involving alternating laser excitation (ALEX) fluorescence spectroscopy.  NTI proposes to apply ALEX for development of an ultrasensitive, highly specific, rapid cost-efficient, homogeneous assay system to accurately detect and quantify botulinum toxins in human serum, overcoming limitations of current diagnostic methodologies.  Outstanding sensitivity, specificity, and accuracy are achieved as two very high-affinity antibodies; labeled with two different fluorescent dyes, need to be bound at the same time to mutually exclusive epitopes on the target.  By counting the number of coincident fluorescent bursts in a femto-liter confocal detection volume, the actual number (or concentration) of the target can be derived in a real time fashion, eliminating the need for signal amplification.  The dual-color coincidence detection by laser alternation allows virtual exclusion of the majority of background noises, eliminating the need for washing steps.  Addition of more laser excitation sources allows a high level of multiplexing (with an ultimate capacity of  > 10,000 targets per standard patient sample).  Over the initial funding period, we will develop and ALEX-based assay using two high-affinity fluorescently labeled antibodies - developed by Prof. Jim Marks via molecular evolution using yeast display – to detect Botulinum Neurotoxin type A (BoNT/A), and demonstrate the quantitative nature of our methodology in serum background. Our specific aims are:

• Expression and purification of an engineered high-affinity antibody pair specific for BoNT/A
• Conjugation of two different fluorescent dyes to these antibodies
• Evaluation of BoNT/A detection with ALEX- based single molecule fluorescence spectroscopy

In the event of an intentional exposure of a large number of people to bioterror agents such as botulinum toxins, the broad availability of a rapid, reliable, ultra-sensitive and highly specific presymptomatic diagnostic assay that allows discrimination between many different agents in a single patient sample will be critical for correct diagnosis and appropriate emergency responses.  This in turn will maximize the effectiveness of anti-toxin treatment and limit the need for extended intensive care, mitigating a catastrophic outcome.

The interface between the host immune response and dengue virus (DENV) pathogenesis

Sujan Shresta, La Jolla Institute for Allergy and Immunology
Project period: May 2006 - April 2008

Abstract:
Dengue virus (DENV) causes dengue fever (DF) and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), the most prevalent arthropod-borne viral illnesses in humans worldwide. Studies suggest that DHF/DSS is an immunopathogenic disease, in which the interferon (IFN) system may be dysregulated. However, the precise role of the IFN-dependent immunity in response to DENV infection in vivo is not fully defined, due to the lack of an adequate animal model. Our long-term goals are to determine how the immune system interacts with DENV in vivo and to develop a better small animal model for DHF/DSS. We will test the hypothesis that the IFN-dependent, signal transducer and activator of transcription 1 (STAT1)-independent pathway signals via IFN-? receptors to control primary DENV infection in mice. Using genetically-deficient mouse strains and our recently generated DENV strains that cause a more relevant disease in mice, the research will examine the role of various IFN-dependent, STAT1-independent pathways in controlling primary DENV infection in vivo (Aim I) and at the cellular level in bone marrow-derived dendritic cells (Aim II). Furthermore, it will identify viral components that modulate the various IFN-dependent pathways in vivo (Aim III). Overall, this research will elucidate the immune response to DENV infection in vivo and provide novel insights into STAT1-dependent versus STAT1-independent responses. By identifying the molecular basis of the primary immune response to DENV infection in vivo, this project will set the foundation for investigating sequential DENV infection in vivo, and it will provide new opportunities for optimizing a mouse model of DENV disease through immunologic manipulation.

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?

Mapping targets of T-cell responses in Coxiella burnetii

Bjoern Peters, La Jolla Institute for Allergy and Immunology - Samuel James, Texas A & M
Project period: May 2006 - April 2008

Abstract:
Coxiella burnetii is considered a potential bioterror threat due to its extremely high infectivity and ability to survive in adverse conditions. We propose to identify the molecular targets of T-cell responses in the course of C. burnetii infections in mice, using a bioinformatics guided minimal epitope mapping approach. The identification of epitopes will improve our knowledge of disease pathogenesis, and will demonstrate for the first time the feasibility of directly mapping epitopes in a pathogen with a proteome of this size. We will establish the presence and magnitude of CD8* vs. CD4* T-cell responses in infected mice. This will prioritize the focus of the rest of this study on eater MHC-I or MHC-II restricted epitopes. We will identify a set of candidate peptides by applying state of the art computational T-cell epitope prediction algorithms to scan the entire C. burnetii proteome. The peptides will be ranked by their probability of being antigenic. From this set we will test the top 400 peptides for recognition by T-cells from C. burnetii infected mice. We will then determine the ability of recognized peptides to convey protective immunity against challenge. With demonstrated success, this approach can be applied to identify epitopes recognized in acute and chronically infected human patients. This has clinical importance for the development of specific diagnostics and the rational design of prophylactic vaccines.