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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Specialized Center--Cooperative Agreements (U54)
Project #
5U54AI065359-09
Application #
8462553
Study Section
Special Emphasis Panel (ZAI1-DDS-M)
Project Start
Project End
Budget Start
2013-05-01
Budget End
2014-04-30
Support Year
9
Fiscal Year
2013
Total Cost
$329,663
Indirect Cost
$31,497
Name
University of California Irvine
Department
Type
DUNS #
046705849
City
Irvine
State
CA
Country
United States
Zip Code
92697
Torres, Rodrigo; Lan, Benson; Latif, Yama et al. (2014) Structural snapshots along the reaction pathway of Yersinia pestis RipA, a putative butyryl-CoA transferase. Acta Crystallogr D Biol Crystallogr 70:1074-85
Houghton, Raymond L; Reed, Dana E; Hubbard, Mark A et al. (2014) Development of a prototype lateral flow immunoassay (LFI) for the rapid diagnosis of melioidosis. PLoS Negl Trop Dis 8:e2727
Strotmeier, Jasmin; Mahrhold, Stefan; Krez, Nadja et al. (2014) Identification of the synaptic vesicle glycoprotein 2 receptor binding site in botulinum neurotoxin A. FEBS Lett 588:1087-93
Koellhoffer, Jayne F; Dai, Zhou; Malashkevich, Vladimir N et al. (2014) Structural characterization of the glycoprotein GP2 core domain from the CAS virus, a novel arenavirus-like species. J Mol Biol 426:1452-68
Bennett, Shannon N; Gu, Se Hun; Kang, Hae Ji et al. (2014) Reconstructing the evolutionary origins and phylogeography of hantaviruses. Trends Microbiol 22:473-82
Burtnick, Mary N; Brett, Paul J; DeShazer, David (2014) Proteomic analysis of the Burkholderia pseudomallei type II secretome reveals hydrolytic enzymes, novel proteins, and the deubiquitinase TssM. Infect Immun 82:3214-26
Koskiniemi, Sanna; Garza-Sánchez, Fernando; Sandegren, Linus et al. (2014) Selection of orphan Rhs toxin expression in evolved Salmonella enterica serovar Typhimurium. PLoS Genet 10:e1004255
Sabouri, Amir H; Marcondes, Maria Cecilia Garibaldi; Flynn, Claudia et al. (2014) TLR signaling controls lethal encephalitis in WNV-infected brain. Brain Res 1574:84-95
Vigant, Frederic; Hollmann, Axel; Lee, Jihye et al. (2014) The rigid amphipathic fusion inhibitor dUY11 acts through photosensitization of viruses. J Virol 88:1849-53
Relman, David A (2014) "Inconvenient truths" in the pursuit of scientific knowledge and public health. J Infect Dis 209:170-2

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