title: The Protease Locus of Francisella tularensis LVS Is Required for Stress Tolerance and Infection in the Mammalian Host.
abstract: Francisella tularensis is the causative agent of tularemia and a category A potential agent of bioterrorism, but the pathogenic mechanisms of F. tularensis are largely unknown. Our previous transposon mutagenesis screen identified 95 lung infectivity-associated F. tularensis genes, including those encoding the Lon and ClpP proteases. The present study validates the importance of Lon and ClpP in intramacrophage growth and infection of the mammalian host by using unmarked deletion mutants of the F. tularensis live vaccine strain (LVS). Further experiments revealed that lon and clpP are also required for F. tularensis tolerance to stressful conditions. A quantitative proteomic comparison between heat-stressed LVS and the isogenic Lon-deficient mutant identified 29 putative Lon substrate proteins. The follow-up protein degradation experiments identified five substrates of the F. tularensis Lon protease (FTL578, FTL663, FTL1217, FTL1228, and FTL1957). FTL578 (ornithine cyclodeaminase), FTL663 (heat shock protein), and FTL1228 (iron-sulfur activator complex subunit SufD) have been previously described as virulence-associated factors in F. tularensis Identification of these Lon substrates has thus provided important clues for further understanding of the F. tularensis stress response and pathogenesis. The high-throughput approach developed in this study can be used for systematic identification of the Lon substrates in other prokaryotic and eukaryotic organisms.
authors.affiliation: Center for Infectious Disease Research, School of Medicine, Tsinghua University, Beijing, China.
authors.forename: Lihong
authors.initials: L
authors.lastname: He
authors.affiliation: Center for Immunology and Microbial Disease, Albany Medical College, Albany, New York, USA.
authors.forename: Manoj Kumar Mohan
authors.initials: MKM
authors.lastname: Nair
authors.affiliation: MOE Key Laboratory of Bioinformatics, School of Life Sciences, Tsinghua University, Beijing, China.
authors.forename: Yuling
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Center for Infectious Disease Research, School of Medicine, Tsinghua University, Beijing, China.
authors.forename: Xue
authors.initials: X
authors.lastname: Liu
authors.affiliation: Center for Infectious Disease Research, School of Medicine, Tsinghua University, Beijing, China.
authors.forename: Mengyun
authors.initials: M
authors.lastname: Zhang
authors.affiliation: Center for Immunology and Microbial Disease, Albany Medical College, Albany, New York, USA.
authors.forename: Karsten R O
authors.initials: KRO
authors.lastname: Hazlett
authors.affiliation: MOE Key Laboratory of Bioinformatics, School of Life Sciences, Tsinghua University, Beijing, China dengh@biomed.tsinghua.edu.cn zhanglab@tsinghua.edu.cn.
authors.forename: Haiteng
authors.initials: H
authors.lastname: Deng
authors.affiliation: Center for Infectious Disease Research, School of Medicine, Tsinghua University, Beijing, China dengh@biomed.tsinghua.edu.cn zhanglab@tsinghua.edu.cn.|Collaborative Innovation Center for Biotherapy, State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medical School, Sichuan University, Chengdu, China.
authors.forename: Jing-Ren
authors.initials: JR
authors.lastname: Zhang
mesh_terms: D000818:Animals; D002460:Cell Line; D004195:Disease Models, Animal; D049071:Endopeptidase Clp / Q000235:genetics / Q000378:metabolism*; D005260:Female; D005604:Francisella tularensis / Q000201:enzymology* / Q000235:genetics / Q000502:physiology*; D017353:Gene Deletion; D056426:Genetic Loci; D006801:Humans; D008264:Macrophages / Q000382:microbiology; D008807:Mice, Inbred BALB C; D049070:Protease La / Q000235:genetics / Q000378:metabolism*; D013312:Stress, Physiological; D014406:Tularemia / Q000382:microbiology* / Q000473:pathology; D037521:Virulence Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D037521:Virulence Factors; D049070:Protease La; D049071:Endopeptidase Clp