title: Evasion of IFN-γ signaling by Francisella novicida is dependent upon Francisella outer membrane protein C.
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.
  
  
  RESULTS
  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.
authors.affiliation: South Texas Center for Emerging Infectious Diseases and Department of Biology, University of Texas at San Antonio, San Antonio, Texas, United States of America.
authors.forename: Kalyan C
authors.initials: KC
authors.lastname: Nallaparaju
authors.forename: Jieh-Juen
authors.initials: JJ
authors.lastname: Yu
authors.forename: Stephen A
authors.initials: SA
authors.lastname: Rodriguez
authors.forename: Xhavit
authors.initials: X
authors.lastname: Zogaj
authors.forename: Srikanth
authors.initials: S
authors.lastname: Manam
authors.forename: M Neal
authors.initials: MN
authors.lastname: Guentzel
authors.forename: Janakiram
authors.initials: J
authors.lastname: Seshu
authors.forename: Ashlesh K
authors.initials: AK
authors.lastname: Murthy
authors.forename: James P
authors.initials: JP
authors.lastname: Chambers
authors.forename: Karl E
authors.initials: KE
authors.lastname: Klose
authors.forename: Bernard P
authors.initials: BP
authors.lastname: Arulanandam
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D005260:Female; D005603:Francisella / Q000187:drug effects / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity*; D005816:Genetic Complementation Test; D007371:Interferon-gamma / Q000494:pharmacology*; D008264:Macrophages / Q000382:microbiology; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D009573:Nitrites / Q000378:metabolism; D011112:Polymyxin B / Q000494:pharmacology; D014406:Tularemia / Q000382:microbiology; D014774:Virulence / Q000235:genetics
chemical_list: D000900:Anti-Bacterial Agents; D001425:Bacterial Outer Membrane Proteins; D009573:Nitrites; D007371:Interferon-gamma; D011112:Polymyxin B