Evasion of IFN-γ Signaling by Francisella novicida Is Dependent upon Francisella Outer Membrane Protein C

Date
2011-03-31
Authors
Nallaparaju, Kalyan C.
Yu, Jieh-Juen
Rodriguez, Stephen A.
Zogaj, Xhavit
Manam, Srikanth
Guentzel, M. Neal
Seshu, Janakiram
Murthy, Ashlesh K.
Chambers, James P.
Klose, Karl E.
Journal Title
Journal ISSN
Volume Title
Publisher
Public Library of Science
Abstract

Background: Francisella tularensis is a Gram-negative facultative intracellular bacterium and the causative agent of the lethal disease tularemia. An outer membrane protein (FTT0918) of F. tularensis subsp. tularensis has been identified as a virulence factor. We generated a F. novicida (F. tularensis subsp. novicida) FTN_0444 (homolog of FTT0918) fopC mutant to study the virulence-associated mechanism(s) of FTT0918.

Methods and Findings: The ΔfopC strain phenotype was characterized using immunological and biochemical assays. Attenuated virulence via the pulmonary route in wildtype C57BL/6 and BALB/c mice, as well as in knockout (KO) mice, including MHC I, MHC II, and µmT (B cell deficient), but not in IFN-γ or IFN-γR KO mice was observed. Primary bone marrow derived macrophages (BMDM) prepared from C57BL/6 mice treated with rIFN-γ exhibited greater inhibition of intracellular ΔfopC than wildtype U112 strain replication; whereas, IFN-γR KO macrophages showed no IFN-γ-dependent inhibition of ΔfopC replication. Moreover, phosphorylation of STAT1 was downregulated by the wildtype strain, but not the fopC mutant, in rIFN-γ treated macrophages. Addition of NG-monomethyl-L-arginine, an NOS inhibitor, led to an increase of ΔfopC replication to that seen in the BMDM unstimulated with rIFN-γ. Enzymatic screening of ΔfopC revealed aberrant acid phosphatase activity and localization. Furthermore, a greater abundance of different proteins in the culture supernatants of ΔfopC than that in the wildtype U112 strain was observed.

Conclusions: F. novicida FopC protein facilitates evasion of IFN-γ-mediated immune defense(s) by down-regulation of STAT1 phosphorylation and nitric oxide production, thereby promoting virulence. Additionally, the FopC protein also may play a role in maintaining outer membrane stability (integrity) facilitating the activity and localization of acid phosphatases and other F. novicida cell components.

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Citation
Nallaparaju, K. C., Yu, J.-J., Rodriguez, S. A., Zogaj, X., Manam, S., Guentzel, M. N., . . . Arulanandam, B. P. (2011). Evasion of IFN-γ Signaling by Francisella novicida Is Dependent upon Francisella Outer Membrane Protein C. PLOS ONE, 6(3), e18201. doi:10.1371/journal.pone.0018201
Department
Molecular Microbiology and Immunology
Neuroscience, Developmental and Regenerative Biology