Mutations of francisella novicida that alter the mechanism of its phagocytosis by murine macrophages

Xin He Lai, Renee L. Shirley, Lidia Crosa, Duangjit Kanistanon, Rebecca Tempel, Robert K. Ernst, Larry A. Gallagher, Colin Manoil, Fred Heffron

    Research output: Contribution to journalArticle

    27 Citations (Scopus)

    Abstract

    Infection with the bacterial pathogen Francisella tularensis tularensis (F. tularensis) causes tularemia, a serious and debilitating disease. Francisella tularensis novicida strain U112 (abbreviated F. novicida), which is closely related to F.tularensis, is pathogenic for mice but not for man, making it an ideal model system for tularemia. Intracellular pathogens like Francisella inhibit the innate immune response, thereby avoiding immune recognition and death of the infected cell.Because activation of inflammatory pathways may lead to cell death, we reasoned that we could identify bacterial genes involved in inhibiting inflammation by isolating mutants that killed infected cells faster than the wild-type parent. We screened a comprehensive transposon library of F. novicida for mutant strains that increased the rate of cell death following infection in J774 macrophage-like cells, as compared to wild-type F. novicida. Mutations in 28 genes were identified as being hypercytotoxic to both J774 and primary macrophages of which 12 were less virulent in a mouse infection model. Surprisingly, we found that F. novicida with mutations in four genes (lpcC, manB, manC and kdtA) were taken up by and killed macrophages at a much higher rate than the parent strain, even upon treatment with cytochalasin D (cytD), a classic inhibitor of macrophage phagocytosis. At least 10-fold more mutant bacteria were internalized by macrophages as compared to the parent strain if the bacteria were first fixed with formaldehyde, suggesting a surface structure is required for the high phagocytosis rate. However, bacteria were required to be viable for macrophage toxicity. The four mutant strains do not make a complete LPS but instead have an exposed lipid A. Interestingly, other mutations that result in an exposed LPS core were not taken up at increased frequency nor did they kill host cells more than the parent. These results suggest an alternative, more efficient macrophage uptake mechanism for Francisella that requires exposure of a specific bacterial surface structure(s) but results in increased cell death following internalization of live bacteria.

    Original languageEnglish (US)
    Article numbere11857
    JournalPLoS One
    Volume5
    Issue number7
    DOIs
    StatePublished - 2010

    Fingerprint

    Francisella tularensis subsp. novicida
    Francisella
    Macrophages
    phagocytosis
    Phagocytosis
    macrophages
    mutation
    Mutation
    mice
    cell death
    Bacteria
    Cell Death
    Cell death
    tularemia
    Francisella tularensis
    Tularemia
    mutants
    Genes
    bacteria
    Pathogens

    ASJC Scopus subject areas

    • Agricultural and Biological Sciences(all)
    • Biochemistry, Genetics and Molecular Biology(all)
    • Medicine(all)

    Cite this

    Lai, X. H., Shirley, R. L., Crosa, L., Kanistanon, D., Tempel, R., Ernst, R. K., ... Heffron, F. (2010). Mutations of francisella novicida that alter the mechanism of its phagocytosis by murine macrophages. PLoS One, 5(7), [e11857]. https://doi.org/10.1371/journal.pone.0011857

    Mutations of francisella novicida that alter the mechanism of its phagocytosis by murine macrophages. / Lai, Xin He; Shirley, Renee L.; Crosa, Lidia; Kanistanon, Duangjit; Tempel, Rebecca; Ernst, Robert K.; Gallagher, Larry A.; Manoil, Colin; Heffron, Fred.

    In: PLoS One, Vol. 5, No. 7, e11857, 2010.

    Research output: Contribution to journalArticle

    Lai, XH, Shirley, RL, Crosa, L, Kanistanon, D, Tempel, R, Ernst, RK, Gallagher, LA, Manoil, C & Heffron, F 2010, 'Mutations of francisella novicida that alter the mechanism of its phagocytosis by murine macrophages', PLoS One, vol. 5, no. 7, e11857. https://doi.org/10.1371/journal.pone.0011857
    Lai, Xin He ; Shirley, Renee L. ; Crosa, Lidia ; Kanistanon, Duangjit ; Tempel, Rebecca ; Ernst, Robert K. ; Gallagher, Larry A. ; Manoil, Colin ; Heffron, Fred. / Mutations of francisella novicida that alter the mechanism of its phagocytosis by murine macrophages. In: PLoS One. 2010 ; Vol. 5, No. 7.
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