F. tularensis is a potent pathogen when aerosolized, wherein ~ 10 organisms can produce disease. Despite a robust inflammatory response in the lung, mortality from pneumonic tularemia can reach 60%, and the fundamental principles of pulmonary host defense against F. tularensis are incompletely understood. We reason that the remarkable efficiency of aerosolized F. tularensis to produce disease must reflect unique microbial features that render local innate host defenses in the lung quantitatively or qualitatively ineffective. We hypothesize that specific surface features of F. tularensis, especially its extracellular CLM, subvert pulmonary innate defenses, resulting in blunted host response in the lung and consequently the observed virulence of aerosolized F. tularensis. As a corollary, we hypothesize that epitopes on CLM may elicit protective immunity, a concept supported by our data demonstrating that an anti-CLM murine monoclonal antibody exerts protection after passive immunization in an experimental infection challenge. To test our overall hypothesis, we propose an integrated approach to elucidate the properties of F. tularensis and its surface components endotoxin and CLM, and of innate pulmonary host defense that provide the basis for the virulence of inhaled F. tularensis. Accordingly, we have created a program consisting of the following three complementary projects intimately linked to an analytical core: Project 1 - Studies of the capsular antigen of F. tularensis. Project 2 - Early airway innate immune responses to F. tularensis. Project 3 - Human DC and macrophages in dysregulation of lung innate immunity by F. tularensis. The BioAnalytical Chemistry Core will serve as the analytical anchor for structural studies for each of the projects, at critical junctures providing expert analysis of composition, posttranslational modification, or structure of microbial or host proteins, carbohydrates, or lipids. Application of our analytical approaches and the biological principles they support to study interactions of F. tularensis with innate immune elements in the lung, will provide novel and fundamental insights into pathogenicity of F. tularensis disease in the airway and contribute to a better understanding of elements of innate immunity in general.
Understanding how F. tularensis withstands, subverts, or evades innate host defense in the airway will provide important insights into the fundamental biology of F. tularensis and elements of innate host defense in general, and provide potential targets for novel pharmacologic agents and vaccines. Learning how to engage more fully innate immune responses to F. tularensis may have direct therapeutic implications of clinical import, and elucidating the immunogenicity of CLM may provide critical insights into developing an effective vaccine. PROJECT 1: Title: - Studies of the Capsular-like Antigen of F. tularensis Project Leader: Apicella, M PROJECT 1 DESCRIPTION (provided by applicant): F. tularensis is a class A bacterial select agent due to its extreme pathogenicity and potential use as a bioweapon. Early studies by Carlisle and Hood have indicated that F. tularensis produces a capsule-like material (CLM). Many bacteria produce capsules and the genes required to produce them encode proteins for transport, biosynthesis and regulation. Homologs of genes implicated in capsular biosynthesis are present in the Francisella genome database. Preliminary studies, using electron microscopy, indicate and confirm that a CLM surrounds F. tularensis. We can now isolate this material, free from Francisella LPS, based on chemical, chromatographic and immunochemical analysis. This material is loosely associated with the bacterial cell, is easily removed from the bacterial surface and is composed of a repeating tetrasaccharide repeat. Using transposon mutagenesis in F. tularensis Schu S4, we have identified a number of mutants with no or limited reactivity to our CLM specific antibody XE8. We have currently identified the site of insertion of 7 of these transposon mutants that have altered CLM expression that will serve as acapsular strains for the studies in this proposal. Based on these observations, we would pose the following hypotheses 1) F. tularensis expresses a capsular-like material that is important in pathogenesis and that we believe is a group 1 capsule;2) Alteration of this capsule-like structure by mutations of the genes involved in biosynthesis and expression of CLM will alter pathogenicity of F. tularensis in cell culture models and in an animal model of respiratory infection;3) Induction of an immune response targeted to the CLM will provide host immunity. The following specific aims will be used to resolve these hypotheses: 1) Characterization of the F. tularensis Schu S4 mutants that are defective in production of CLM, 2) Characterization of the biological role of the F. tularensis Schu S4 CLM, and 3) Study the immunogenicity and protective efficacy of passive and active immunization in murine models of Francisella infection.
The proposed work will focus on characterizing the Francisella CLM which remains poorly understood. There is a significant possibility that efforts to understand this potentially critical virulence factor will not only increase our understanding of Francisella pathogenesis but open up new avenues for therapeutic development, using acapsular strains or antibodies to the CLM.
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