Characterization of a Francisella tularensis-Caenorhabditis elegans Pathosystem for the Evaluation of Therapeutic Compounds

Abstract

Francisella tularensis is a highly infectious Gram-negative intracellular pathogen that causes tularemia. Because of its potential as a bioterrorism agent, there is a need for new therapeutic agents. We therefore developed a whole-animal Caenorhabditis elegans-F. tularensis pathosystem for high-throughput screening to identify and characterize potential therapeutic compounds. We found that the C. elegans p38 mitogen-activate protein (MAP) kinase cascade is involved in the immune response to F. tularensis, and we developed a robust F. tularensis-mediated C. elegans killing assay with a Z' factor consistently of >0.5, which was then utilized to screen a library of FDA-approved compounds that included 1,760 small molecules. In addition to clinically used antibiotics, five FDA-approved drugs were also identified as potential hits, including the anti-inflammatory drug diflunisal that showed anti-F. tularensis activity in vitro Moreover, the nonsteroidal anti-inflammatory drug (NSAID) diflunisal, at 4× MIC, blocked the replication of an F. tularensis live vaccine strain (LVS) in primary human macrophages and nonphagocytic cells. Diflunisal was nontoxic to human erythrocytes and HepG2 human liver cells at concentrations of ≥32 μg/ml. Finally, diflunisal exhibited synergetic activity with the antibiotic ciprofloxacin in both a checkerboard assay and a macrophage infection assay. In conclusion, the liquid C. elegans-F. tularensis LVS assay described here allows screening for anti-F. tularensis compounds and suggests that diflunisal could potentially be repurposed for the management of tularemia.

Keywords: Caenorhabditis elegans; Francisella; antibiotic; diflunisal; drug repurposing; high-throughput screen; tularemia.

FIG 1

The MAP kinase pathway is essential for survival of C. elegans challenged with F. tularensis. Percent survival of wild-type N2 or mutant sek-1 (kinase involved in the p38 MAP kinase cascade) C. elegans worms grown on lawns of E. coli or F. tularensis LVS was determined. Data represent the means ± standard errors of the means (n = 3).

FIG 2

Development of a high-throughput assay for anti-Francisella compounds. (a) Flowchart representing the work flow of the C. elegans-F. tularensis high-throughput screening assay. (b) Optimization of F. tularensis LVS inoculum for the liquid killing assay. (c) C. elegans-F. tularensis liquid killing assay: Sytox-stained images of C. elegans with or without ciprofloxacin.

FIG 3

Checkerboard and time-to-kill assay for diflunisal with ciprofloxacin. (a) Diflunisal was adjusted to a final concentration between 0.25 and 16 μg/ml, in combination with ciprofloxacin, which was adjusted to a final concentration between 0.03 and 2 μg/ml. The FIC index was 0.5 for the diflunisal-ciprofloxacin combination. The scale represents the optical density of bacterial growth of F. tularensis LVS. (b) Time-to-kill curve shows that the efficacy of diflunisal (2 μg/ml) is enhanced in combination with ciprofloxacin (0.06 μg/ml) compared to that of diflunisal alone (8 μg/ml) or ciprofloxacin alone (0.25 μg/ml). Data are means ± standard errors of the means (n = 3).

FIG 4

Hemolysis and cytotoxicity testing for diflunisal. (a) Human erythrocytes were treated with serial dilutions of Triton X-100 (0.00 to 1%) or diflunisal (0.06 to 64 μg/ml). (b) The viability of HepG2 cells was measured after treatment with serially diluted concentrations (1 to 64 μg/ml) of diflunisal. Data represent the means ± standard errors of the means (n = 3).

FIG 5

Diflunisal inhibits intracellular growth of F. tularensis LVS in macrophages and 293T/17 cells. Macrophages (a) and 293T/17 (b) cells were infected with F. tularensis LVS bacteria for 2 h, treated with gentamicin to kill extracellular bacteria, and then cocultured with 4× MIC of diflunisal for 22 h. ***, P < 0.001, comparing results with diflunisal to those with the DMSO control at the 24-h time point using two-way ANOVA with a Bonferroni posttest correction.

FIG 6

Diflunisal and ciprofloxacin show synergy in macrophages. Macrophages were infected with F. tularensis LVS bacteria for 2 h, followed by gentamicin treatment to kill extracellular bacteria, and cocultured with ciprofloxacin (0.25 μg/ml), diflunisal (8 μg/ml), or a combination of diflunisal (2 μg/ml) and ciprofloxacin (0.06 μg/ml) for 22 h. Data shown are CFU counts of intracellular F. tularensis LVS in macrophages. ***, P < 0.001, comparing results with diflunisal to those with the DMSO control at the 24-h time point using two-way ANOVA with a Bonferroni posttest correction.