Identification of a novel Francisella tularensis factor required for intramacrophage survival and subversion of innate immune response

Abstract

Francisella tularensis, the causative agent of tularemia, is one of the deadliest agents of biological warfare and bioterrorism. Extremely high virulence of this bacterium is associated with its ability to dampen or subvert host innate immune response. The objectives of this study were to identify factors and understand the mechanisms of host innate immune evasion by F. tularensis. We identified and explored the pathogenic role of a mutant interrupted at gene locus FTL_0325, which encodes an OmpA-like protein. Our results establish a pathogenic role of FTL_0325 and its ortholog FTT0831c in the virulent F. tularensis SchuS4 strain in intramacrophage survival and suppression of proinflammatory cytokine responses. This study provides mechanistic evidence that the suppressive effects on innate immune responses are due specifically to these proteins and that FTL_0325 and FTT0831c mediate immune subversion by interfering with NF-κB signaling. Furthermore, FTT0831c inhibits NF-κB activity primarily by preventing the nuclear translocation of p65 subunit. Collectively, this study reports a novel F. tularensis factor that is required for innate immune subversion caused by this deadly bacterium.

FIGURE 1.

FTL_0325 mutant induces significantly increased proinflammatory cytokines in macrophages. Macrophage cell culture invasion assay was performed in MH-S cells (A) and BMDMs (B) derived from C57BL/6 mice. The macrophages were infected with 100 m.o.i. of FTL_0325 mutant or WT F. tularensis LVS. The intracellular replication was quantitated at the indicated times and expressed as log₁₀ cfu. TNF-α levels were measured in culture supernatants from infected MH-S cells (C), BMDMs (D), and untreated or phorbol 12-myristate 13-acetate differentiated THP1 cells (E). F, the macrophages were infected with 100 m.o.i. of indicated mutants or WT F. tularensis LVS. The intracellular replication was quantitated 24 h PI and expressed as log₁₀ cfu. G, TNF-α levels were measured in culture supernatants from infected MHS cells 24 h PI. The values represent the mean ± S.D. (A and C) of quadruplicate samples from one of the three experiments conducted or mean ± S.E. (B, D, E, F, and G) and cumulative of three independent experiments conducted. The p values were determined using one-way ANOVA.

FIGURE 2.

Localization of OmpA-like protein of F. tularensis. A, surface localization of FTL_0325 by immunofluorescent staining with polyclonal anti-FTT0831c antibodies and Alexa Fluor 488-conjugated goat anti-rat IgG secondary antibodies. Anti-F. tularensis LPS monoclonal and polyclonal anti-KatG antibodies were used as positive and negative controls, respectively (left panel). Bacteria were treated with proteinase-K, washed, and stained (right panel) (magnification ×63). B, shown is subcellular localization of FTT0831c in F. tularensis SchuS4 by immunoblotting of sucrose density gradient fractions. Sequential fractions were collected from gradients, and immunoblotting was performed using anti-FTT0831c antibodies. Known outer membrane proteins FopA and Mip and inner membrane protein SecY were used as controls for localization.

FIGURE 3.

Elevated proinflammatory cytokines by FTL_0325 mutant is not due to increased release of TLR ligands. A macrophage cell culture invasion assay was performed in BMDMs derived from WT (A) and TLR2^−/− C57BL/6 (B) mice. The macrophages were infected with 100 m.o.i. of FTL_0325 mutant or WT F. tularensis LVS. The intracellular replication was quantitated at the indicated times and expressed as log₁₀ cfu. TNF-α levels were measured in culture supernatants from WT BMDMs (C) and TLR2^−/− (D) macrophages. E, the macrophages were infected with 100 m.o.i. of UV-killed FTL_0325 mutant or UV-killed WT F. tularensis LVS, and TNF-α levels were measured in culture supernatants 24 h later. The results are expressed as the mean ± S.E. and are representative of at least two-three independent experiments. The p values were determined using one-way ANOVA.

FIGURE 4.

FTL_0325 mutant escapes from the phagosomes. A, shown is detection of IL-1β activation. Lysates from BMDMs infected with 100 m.o.i. of LVS or FTL_0325 mutant at 24 h PI were run on a SDS-gel, transferred, and blotted with anti-IL-1β antibodies. Blotting with anti-β actin antibodies was used as the loading control. B, levels of secreted IL-1β were measured in culture supernatants from infected BMDMs at the indicated times. C, quantification of intramacrophage bacterial lysis. Macrophages were infected with an m.o.i. of 100 with the indicated strains carrying pFNLTP6:luciferase construct. The macrophages were lysed at the indicated times, and luminescence was measured in a luminometer (PerkinElmer Life Sciences). F. novicida was used as a positive control. The results are representative of two to three independent experiments, expressed as the mean ± S.D., and the p values were determined using one-way ANOVA. In C, comparisons are shown between F. novicida and LVS or FTL_0325 mutant strain. ND, not determined.

FIGURE 5.

Hyper-induced proinflammatory cytokine response in FTL_0325 mutant-infected macrophages is due to enhanced activation of NF-κB. MH-S cells were transfected with NF-κB-luciferase construct and infected with FTL_0325 mutant and F. tularensis LVS at 100 m.o.i. for 4 h. The cells were lysed, and luciferase activity was monitored using the Dual Luciferase Assay kit (Promega). The data are expressed as percent luciferase activity and represent the mean ± S.D. from one of the three independent experiments. The statistical significance of the results was examined with Student's t test, and p values were recorded.

FIGURE 6.

FTT0831c protein inhibits NF-κB activity. A, a macrophage cell culture invasion assay was performed in MH-S cells. The macrophages were infected with 100 m.o.i. of ΔFTT0831c mutant, WT F. tularensis SchuS4, and the transcomplemented ΔFTT0831c+pFTT0831c strain. The intracellular replication was quantitated and is expressed as Log₁₀ cfu. B, the TNF-α levels were measured in culture supernatants from infected MH-S cells 24 h PI. The values represent the mean ± S.D. of six simples each and are representative of two experiments conducted. The p values were determined using one-way ANOVA. C, shown is confirmation of FTT0831c expression by Western blot analysis of HEK293T cells transfected with 1000 ng of pcDNA:FTT0831c vector. D, shown is a dose-dependent reduction in NF-κB activity in HEK293T cells transfected with indicated concentration of pcDNA expressing FTT0831c. The luciferase activity was determined as a measure of NF-κB activity in HEK293T cells treated with TNF-α. E, IL-8 mRNA expression in HEK293T cells transfected with pcDNA:FTT0831c (1000 ng) by quantitative real-time PCR is shown. F, shown is determination of ISRE-dependent luciferase activity in HEK293T cells transfected with Cignal ISRE-Luciferase reporter and empty or FTT0831c expressing pcDNA (1000 ng) 18 h post-treatment with IFNβ. Data presented in D, E, and F are derived from quadruplicate samples, are cumulative of two independent experiments, and are represented as the mean ± S.E. The p values were determined by one-way ANOVA.

FIGURE 7.

FTT0831c protein inhibits NF-κB activity by preventing nuclear translocation of p65 subunit. A, HEK293T cells expressing FTT0831c were co-transfected with vector expressing p65 subunit. p65-induced NF-κB luciferase reporter activity was measured. B, HEK293T cells were transfected as in A and treated with TNF-α for 30 min. Immunofluorescence staining was performed to detect the cellular localization of p65subunit of NF-κB (magnification 20× and 100×; red, p65; blue, nucleus). C, quantification of nuclear localization of p65 subunit of NF-κB is shown. At least 200 cells were counted in randomly chosen fields for each condition and expressed as percent cells with p65 staining. Data presented in A are derived from quadruplicate samples and are cumulative of five independent experiments and are represented as the mean ± S.E. The p values were determined by one-way ANOVA.