Elevated AIM2-mediated pyroptosis triggered by hypercytotoxic Francisella mutant strains is attributed to increased intracellular bacteriolysis

Abstract

Intracellular bacterial pathogens Francisella novicida and the Live Vaccine Strain (LVS) are recognized in the macrophage cytosol by the AIM2 inflammasome, which leads to the activation of caspase-1 and the processing and secretion of active IL-1β, IL-18 and pyroptosis. Previous studies have reported that F. novicida and LVS mutants in specific genes (e.g. FTT0584, mviN and ripA) induce elevated inflammasome activation and hypercytotoxicity in host cells, leading to the proposal that F. novicida and LVS may have proteins that actively modulate inflammasome activation. However, there has been no direct evidence of such inflammasome evasion mechanisms. Here, we demonstrate for the first time that the above mutants, along with a wide range of F. novicida hypercytotoxic mutants that are deficient for membrane-associated proteins (ΔFTT0584, ΔmviN, ΔripA, ΔfopA and ΔFTN1217) or deficient for genes involved in O-antigen or LPS biosynthesis (ΔwbtA and ΔlpxH) lyse more intracellularly, thus activating increased levels of AIM2-dependent pyroptosis and other innate immune signalling pathways. This suggests that an inflammasome-specific evasion mechanism may not be present in F. novicida and LVS. Furthermore, future studies may need to consider increased bacterial lysis as a possible cause of elevated stimulation of multiple innate immune pathways when the protein composition or surface carbohydrates of the bacterial membrane is altered.

Figure 1. F. novicida ΔFTT0584, ΔmviN, ΔripA, ΔfopA, ΔFTN1217, ΔwbtA and ΔlpxH mutants are hypercytotoxic in macrophages

Cytotoxicity levels by infected wild type BMDMs at (A) indicated times post infection or (B) 10 hrs post infection. BMDMs were infected with the indicated bacterial strains at an effective MOI of 20. Data is representative of 3 independent experiments. * p value < 0.05 using the Student's t test.

Figure 2. Francisella hypercytotoxic mutants trigger elevated AIM2 inflammasome activation in macrophages

IL-1β secretion by infected wild type BMDMs at (A) indicated times post infection or (B) 10 h post infection. (C) AIM2 transcript levels (D) cytotoxicity levels and (E) IL-1β secretion by the indicated strains of infected BMDMs at 8 hr post infection. BMDMs were infected with the indicated bacterial strains at an effective MOI of 20. Data is representative of 3 independent experiments. * p value < 0.05 using the Student's t test.

Figure 3. Francisella hypercytotoxic mutants hyperstimulate type 1 interferon and proinflammatory cytokine signaling

(A) Type 1 IFN, (B) TNF-α and (B) IL-6 secretion by infected ASC BMDMs at 8 hours post infection. BMDMs were infected with the indicated bacterial strains at an effective MOI of 20. Data is representative of 3 independent experiments. * p value < 0.05 using the Student's t test.

Figure 4. Francisella hypercytotoxic mutants require phagosomal escape to induce AIM2-mediated pyroptosis

(A) Cell death and (B) IL-1β secretion by infected wild type BMDMs at 10 hrs post infection. BMDMs were infected with the indicated bacterial strains at an effective MOI of 20. Data is representative of 3 independent experiments. * p value < 0.05 using the Student's t test.

Figure 5. Francisella hypercytotoxic mutants exhibit increased lysis in the intracellular milieu as visualized via confocal microscopy

(A) Immunofluorescence microscopy of BMDMs infected with the indicated bacterial strains at 7 hours post infection. DIC, differential interference contrast. Arrowheads indicate examples of colocalization of bacterial DNA, AIM2 and bacteria. (B) Graph showing the number of AIM2 specks in 100 BMDMs from Figure 5A. Scale bars; 10 μm. Data is representative of 2 independent experiments. * p value < 0.05 using a student's t test.

Figure 6. Francisella hypercytotoxic mutants exhibit aberrant morphologies during growth in minimal media

Bacterial morphology as visualized via scanning electron micropscopy of bacterial strains grown to late exponential phase in (A) tryptic soy broth supplemented with cysteine or (B) Chamberlain's defined media. Scale bars, 2μm. Images are representative of 2 independent experiments.

Figure 7. Francisella hypercytotoxic mutants exhibit increased lysis in the intracellular milieu as determined quantitatively via luminescence production

Luminescence levels of BMDMs infected with the indicated bacterial strains containing the luciferase reporter construct at 7 hours post infection. Data is represented as the average of at least 3 independent experiments. * p value < 0.05 using a student's t test.