Cutting edge: mutation of Francisella tularensis mviN leads to increased macrophage absent in melanoma 2 inflammasome activation and a loss of virulence

Abstract

The mechanisms by which the intracellular pathogen Francisella tularensis evades innate immunity are not well defined. We have identified a gene with homology to Escherichia coli mviN, a putative lipid II flippase, which F. tularensis uses to evade activation of innate immune pathways. Infection of mice with a F. tularensis mviN mutant resulted in improved survival and decreased bacterial burdens compared to infection with wild-type F. tularensis. The mviN mutant also induced increased absent in melanoma 2 inflammasome-dependent IL-1beta secretion and cytotoxicity in macrophages. The compromised in vivo virulence of the mviN mutant depended upon inflammasome activation, as caspase 1- and apoptosis-associated speck-like protein containing a caspase recruitment domain-deficient mice did not exhibit preferential survival following infection. This study demonstrates that mviN limits F. tularensis-induced absent in melanoma 2 inflammasome activation, which is critical for its virulence in vivo.

Figure 1

F. tularensis LVS mviN mutant is attenuated in vivo. (A) WT (n=10 per group) mice were injected i.p. with the indicated amount of F. tularensis LVS (LVS) or the mviN mutant (mviN::Tn5) and survival monitored. Data are representative of 5 independent experiments at the 3 × 10⁴ infective dose, each involving a minimum of 5 mice per group. (B) 3 d p.i. with 3 × 10⁴ CFU of LVS or the mviN mutant organs were harvested, homogenized and dilutions plated for enumeration of CFU (n=3–5 mice per group). (C) Number of lesions per 200 × field of H&E-stained liver sections from WT mice 3 d p.i. with either LVS or the mviN mutant were scored; n=5 mice per group (3 random fields were examined per mouse); *, p = 0.016. (D) Representative H&E-stained sections of liver from WT mice 3 d p.i. with either F. tularensis LVS or the mviN mutant. Arrows indicate necropurulent foci.

Figure 2

Mutation of F. tularensis LVS mviN results in enhanced Mφ cytotoxicity and caspase-1 activation. (A–E) LPS-primed WT Mφ were infected with F. tularensis LVS (LVS), the mviN mutant (mviN::Tn5) or the complemented mviN mutant (mviN::Tn5 + mviN) at an MOI of 50:1. Supernatants were collected 9 h after infection (B, D, and E) or as indicated (A and C). Cytotoxicity was measured by LDH release and expressed as a percentage of LDH release by Triton X-100 detergent (A and B). IL-1β secreted into supernatants was measured by ELISA (C-E). (F, G) Unprimed WT Mφ were infected with LVS or the mviN mutant for 9 h; IL-6 and TNFα released into the supernatant was measured by ELISA. (H) Lysates from LPS-primed WT and caspase-1-deficient Mφ (lane with caspase-1-/- Mφ lysate is marked *) infected with LVS or the mviN mutant (50:1 MOI) for the indicated times were immunoblotted with antibodies against the p10 subunit of caspase-1 or GAPDH. Results are representative of two (B, F, G) and three (A, C-E, H) separate experiments.

Figure 3

MviN limits Francisella-induced AIM2 inflammasome activation. (A, B) LPS-primed Mφ from WT, caspase-1-, ASC-, NLRP3-, NLRC4, and AIM2-deficient mice were infected with either F. tularensis LVS (LVS) or the mviN mutant (mviN::Tn5) at a 50:1 MOI, or challenged with Alum (500 μg/ml). Supernatants were collected at 9 h and IL-1β release measured by ELISA. Results are representative of two (A) and five (B) separate experiments. (C) Lysates from LPS-primed WT and AIM2-deficient Mφ infected with LVS or the mviN mutant for 9 h were immunoblotted with antibodies against the p10 subunit of caspase-1 or GAPDH. Results are representative of two separate experiments. (D, E) WT, caspase-1- or ASC-deficient mice (n=5 per group) were infected i.p. with 1 × 10⁵ CFUs of LVS or the mviN mutant and survival monitored.