Bacterial Zoonoses

Intracellular biology of Francisella tularensis

Marcus A. Horwitz, UC Los Angeles

Abstract:
Francisella tularensis is a facultative intracellular bacterial pathogen that causes a serious and potentially life threatening illness by surviving and multiplying within host cells, primarily macrophages. Since the bacterium has extraordinarily high infectivity, causes serious morbidity and mortality, and is easily dispersed, it is also considered a potential agent of bioterrorism. While currently available antibiotics are effective in treating tularemia, F. tularensis can be engineered to carry antibiotic resistance genes. For these reasons, new approaches to treatment and prevention of tularemia are needed. However, devising such strategies requires an improved understanding of the interaction between F. tularensis and its host cells. We have demonstrated that fully virulent F. tularensis invades macrophages and then enters a phagosomal compartment that exhibits arrested maturation before the bacterium escapes into the cytoplasm. We propose to further define the intracellular biology of F. tularensis and to identify host and bacterial proteins that are important to the bacterial intracellular survival by using wild type, mutant and killed strains. We will also determine the role of specific host and bacterial proteins in F. tularensis’ intracellular trafficking by using siRNA techniques and by the direct examination of the phenotype of targeted bacterial mutants. We will also determine whether F. tularensis mutants that are defective in phagosome-lysosome fusion or phagosome escape can be restored to wild-type phenotype by delivering the protein corresponding to the mutated gene into the host cell cytoplasm or into the F. tularensis phagosome.

Molecular mechanisms of F. tularensis pathogenesis and immunity

Denise Monack, Stanford University

Abstract:
Francisella tularensis is a highly infectious gram-negative coccobacillus that causes the zoonosis tularemia and is a Category A agent. A hallmark of tularemia is the ability of the bacterium to grow in mammalian hosts before the onset of a protective cell-mediated immune response. Mammalian hosts are endowed with numerous antimicrobial effector functions but F. tularensis has evolved mechanisms to subvert host defenses. It is very striking that this small bacterium can infect its host via a variety of different infection routes, each of which involves a different host tissue site with a vastly different microenvironment. Given that F. tularensis is so successful at infecting its host via multiple tissue sites, our hypothesis is that, in addition to a core set of genes that are needed for general survival and growth in vivo, F. tularensis possess additional genes that are required in specific tissues or microniches. Thus, our overarching goal is to identify novel core and tissue-specific virulence factors in F. tularensis. In the first aim, we will identify tissue-specific (e.g. lung-, spleen-, and skin-specific) F. tularensis virulence factors using our well-established microarray-based negative selection methodology following intranasal, intraperitoneal and intradermal routes of inoculation. In the second and third aims, we will validate the tissue-specificity of novel virulence factors and characterize the molecular mechanisms in our mouse models of infection and in in vitro tissue culture assays.

Infection and immunity in reservoir hosts

Alan Barbour, UC Irvine

Abstract:
Our long-term goal is the sustainable reduction of human infections of Lyme borreliosis and other vector-borne zoonoses, such as plague and West Nile encephalitis, by targeting vaccines to the reservoir hosts of Borrelia burgdorferi and other agents. Two important considerations for this prevention strategy are the following: (1) Of the possible reservoir hosts for the agent under study, which one or ones are key for maintenance of the pathogen in nature? (2) To what extent do these reservoir species, such as Peromyscus leucopus, differ qualitatively and quantitatively from usual laboratory animal models, such as Mus musculus, in their protective immune responses to infection and immunization? These laboratory-based studies will initially focus on B. burgdorferi infection and will provide means to answer question (1) through the development of inexpensive assays to identify the source of the last blood meal for the arthropod, as well as to test hypotheses for question (2) by comparing immune responses of candidate reservoirs to those of M. musculus for infection and vaccination. Building upon proof-of-concepts, the project’s aims are the following: (1) to further develop and evaluate sensitive proteomics-based assays to identify the vertebrate species that last provided the blood meal (and the source of infection) for unengorged ticks of Ixodes scapularis; (2) to improve PCR-based assays to identify and characterize residual vertebrate mitochondrial DNA in flat ticks by high throughput PCR-MS technology; (3) to identify protective immunogens of B. burgdorferi for key reservoir species by using genome-wide protein arrays developed to study the immune responses of infected humans; and (4) to assess protection against tick-transmitted infection after immunization of key reservoir species in the laboratory with OspA and/or other vaccine candidates identified in this project.

Development of poxvirus-based vectors as vaccines against biodefense threat agents

Bertram Jacobs, Arizona State University

Abstract:
The overall goal of this project is to develop highly attenuated, replication competent strains of poxviruses, with limited potential to spread from animal to animal in the wild, that can be delivered orally in bait to vaccinate wildlife reservoirs against diseases of human importance, including Lyme disease and plague. The specific aims of the proposal are to: 1) construct various recombinant poxviruses that express the Borrelia OspA protein; and 2) compare constructs for expression, immunogenicity, attenuation and spread in lab-reared mice. By the end of the funding cycle, we hope to have vectors ready to evaluate for immunogenicity and safety in trapped or lab-reared wildlife species, in preparation for submission to regulatory agencies for use as oral wildlife vaccines for decreasing the disease burden to B. burgdorferi. It is anticipated that these vectors would have a significant impact on human disease, by limiting the vector-mediated spread of disease from the wildlife reservoir to humans.

Environmental amoeba as potential reservoirs for Francisella tularensis: exploring a possible link between environmental persistence and pathogenesis

Amy Rasley, Lawrence Livermore National Laboratory

Abstract:
Francisella tularensis subspecies are facultative intracellular pathogens that exhibit remarkably broad host ranges having been estimated to infect more than 200 diverse hosts and vectors. Interestingly, it has been reported that F. tularensis enters and survives in environmental amoeba, suggesting that the amoeba-Francisella interactions are important for Francisella’s environmental persistence. This observation is consistent with the isolation of Francisella species from soil and water, which are prime amoeba habitats. Furthermore, recent studies have demonstrated that interactions between F. tularensis and amoeba may enhance the virulence of this organism. Given that F. tularensis preferentially infects and replicates within host macrophages, probing the Francisella-amoeba interaction may provide insight into pathogenesis as well as environmental persistence. We have recently demonstrated that pathogenic strains of F. tularensis induce a rapid encsytment in the environmental amoeba Acanthamoeba castellanii. The aim of this proposal is to further characterize the interactions of pathogenic F. tularensis with A. castellanii and human monocytes. We propose to: 1) sequence key genes encoding proteins involved in encystment across virulent isolates to correlate sequence differences with encystment efficiencies; 2) determine if virulent F. tularensis strains disrupt the endosomal pathway in A. castellanii; and 3) investigate the role of F. tularensis encystment proteins in reducing the bactericidal properties of human monocytes.

Diverse Yersinia pestis in animal models: comparative virulence and host response

David Wagner, Northern Arizona University

Abstract:
Plague, caused by the bacterium Y. pestis, is one of the most important diseases of human history and a Category A select agent. Most current efforts for addressing plague are focused on preventing human deaths via rapid detection and treatment with antibiotics. However, antibiotic-resistant strains can occur naturally and rapid diagnoses are not always possible, especially in developing countries. Vaccines would offer widespread protection but have not been widely successful to date. Animal models are crucial for understanding host response and pathogenicity of Y. pestis, information that may lead to improved vaccines and therapeutics. A variety of animal models are used to examine host response, but it is not clear if results from one host are applicable to results from another host as few studies employ multiple animal models. The overarching goal of this work is to identify the most appropriate Y. pestis strains and animal models to use in future experimental studies that seek to develop improved plague vaccines and therapeutics. This project will include Y. pestis challenge of both laboratory and sylvatic rodents to better understand differential pathogenicity within Y. pestis and differences in host response across animal models. Three main hypotheses will be examined: H1: Diverse Y. pestis strains differ in pathogenicity, H2: Host response to challenge with Y. pestis varies across animal models, H3: Resistance to plague has developed in a sylvatic, North American rodent host. This preliminary work will provide a basis for more detailed studies of pathogenicity in Y. pestis in multiple animal models.