Francisella tularensis is a facultative intracellular bacterial pathogen that causes serious and potentially life threatening illness by surviving and multiplying within host cells, primarily macrophages. Because 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 enter macrophages via spacious pseudopod loops and that the bacterium then enters a phagosomal compartment that exhibits arrested maturation in that it acquires limited amounts of lysosome associated membrane glycoproteins, does not acquire cathepsin D, and is only minimally acidified prior to escape of the bacterium into the macrophage cytoplasm. We propose to define further the intracellular biology of F. tularensis and to identify the virulence mechanisms that allow it to evade phagosome-lysososome fusion, phagosome acidification, and to escape into the host cell cytoplasm. We shall identify host and bacterial proteins that are important to resistance to phagosome-lysosome fusion and phagosome escape by a mass spectrometry based proteomic comparison of purified phagosomes containing live wild type F. tularensis, killed F. tularensis, and selected F. tularensis mutants that fail to escape and that traffic differently within the host cell. We shall identify bacterial genes required for growth in macrophages that are required for altering the intracellular trafficking and phagosome escape by screening a transposon mutant library. We shall determine whether specific host and bacterial proteins that we identify by these methods are required for uptake of the bacteria by looping phagocytosis, resistance to phagosomelysosome fusion, or phagosome escape by using siRNA techniques and by examining the phenotype of targeted bacterial mutants by immunofluorescence microscopy and transmission electron microscopy. We shall 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. This will provide information regarding the possible site of action of these bacterial proteins.
These studies will increase our understanding of how F. tularensis subverts host cell membrane traffic and escapes into the cytoplasm and will identify specific bacterial molecules and pathways that are involved in bacterial virulence. Identification of these molecules and pathways will guide the development of new strategies for prevention and treatment of tularemia.
|Hertz, Tomer; Beatty, P Robert; MacMillen, Zachary et al. (2017) Antibody Epitopes Identified in Critical Regions of Dengue Virus Nonstructural 1 Protein in Mouse Vaccination and Natural Human Infections. J Immunol 198:4025-4035|
|Marques, Adriana R; Yang, Xiuli; Smith, Alexis A et al. (2017) Citrate Anticoagulant Improves the Sensitivity of Borreliella (Borrelia) burgdorferi Plasma Culture. J Clin Microbiol 55:3297-3299|
|Parameswaran, Poornima; Wang, Chunling; Trivedi, Surbhi Bharat et al. (2017) Intrahost Selection Pressures Drive Rapid Dengue Virus Microevolution in Acute Human Infections. Cell Host Microbe 22:400-410.e5|
|Bortell, Nikki; Flynn, Claudia; Conti, Bruno et al. (2017) Osteopontin Impacts West Nile virus Pathogenesis and Resistance by Regulating Inflammasome Components and Cell Death in the Central Nervous System at Early Time Points. Mediators Inflamm 2017:7582437|
|Barbour, Alan G (2017) Infection resistance and tolerance in Peromyscus spp., natural reservoirs of microbes that are virulent for humans. Semin Cell Dev Biol 61:115-122|
|Nualnoi, Teerapat; Norris, Michael H; Tuanyok, Apichai et al. (2017) Development of Immunoassays for Burkholderia pseudomallei Typical and Atypical Lipopolysaccharide Strain Typing. Am J Trop Med Hyg 96:358-367|
|Nakajima, Rie; Escudero, Raquel; Molina, Douglas M et al. (2016) Towards Development of Improved Serodiagnostics for Tularemia by Use of Francisella tularensis Proteome Microarrays. J Clin Microbiol 54:1755-65|
|Zeltina, Antra; Bowden, Thomas A; Lee, Benhur (2016) Emerging Paramyxoviruses: Receptor Tropism and Zoonotic Potential. PLoS Pathog 12:e1005390|
|Ziegler, Christopher M; Eisenhauer, Philip; Bruce, Emily A et al. (2016) A novel phosphoserine motif in the LCMV matrix protein Z regulates the release of infectious virus and defective interfering particles. J Gen Virol 97:2084-9|
|Park, Arnold; Yun, Tatyana; Hill, Terence E et al. (2016) Optimized P2A for reporter gene insertion into Nipah virus results in efficient ribosomal skipping and wild-type lethality. J Gen Virol 97:839-43|
Showing the most recent 10 out of 457 publications