Genome-wide screen in Francisella novicida for genes required for pulmonary and systemic infection in mice

Abstract

Francisella tularensis is a gram-negative, highly infectious, aerosolizable facultative intracellular pathogen that causes the potentially life-threatening disease tularemia. To date there is no approved vaccine available, and little is known about the molecular mechanisms important for infection, survival, and dissemination at different times of infection. We report the first whole-genome screen using an inhalation mouse model to monitor infection in the lung and dissemination to the liver and spleen. We queried a comprehensive library of 2,998 sequence-defined transposon insertion mutants in Francisella novicida strain U112 using a microarray-based negative-selection screen. We were able to track the behavior of 1,029 annotated genes, equivalent to a detection rate of 75% and corresponding to approximately 57% of the entire F. novicida genome. As expected, most transposon mutants retained the ability to colonize, but 125 candidate virulence genes (12%) could not be detected in at least one of the three organs. They fell into a variety of functional categories, with one-third having no annotated function and a statistically significant enrichment of genes involved in transcription. Based on the observation that behavior during complex pool infections correlated with the degree of attenuation during single-strain infection we identified nine genes expected to strongly contribute to infection. These included two genes, those for ATP synthase C (FTN_1645) and thioredoxin (FTN_1415), that when mutated allowed increased host survival and conferred protection in vaccination experiments.

FIG. 1.

Wild-type or mutant pool total bacterial load for lung, liver, and spleen. Lung, liver, and spleen data were averaged across all four mice per time point and across all 11 pools per organ. Mice were infected with pools of ∼282 mutants per pool, and depositions ranging from 1.6 × 10³ to 2 × 10⁴ CFU/lung across all 11 pools show similar overall growth and dissemination rates as high-dose (1.7 × 10⁴ CFU) single wild-type strain infections.

FIG. 2.

Schematic of negative-selection screen for identification of genes essential for colonization and dissemination in mouse inhalation infection model. Separate infections by inhalation were performed for each of the 11 mutant pools. Four mice per time point were sacrificed at 0 h (deposition) and at 24 h, 48 h, and 72 h (different outputs) and genomic DNA isolated from lung, liver, and spleen for each mouse separately at all time points. After combining DNA from all four mice per time point and organ, DNAs next to the transposon insertions were amplified in a two-step PCR to label deposition DNAs with Cy3 and output DNAs with Cy5. The resulting log₂ (Cy5/Cy3) provided an indication of the representation of individual mutants in the output pool compared to the input pool.

FIG. 3.

Enrichment of functional categories in aerosol infection mutants. The percentage of genes in each category relative to genes with an infection phenotype (gray) or in the Francisella genome (black) is shown. *, significant enrichment (P = 0.0017 by chi-square test with Bonferroni correction for multiple comparisons). Abbreviations: IC, intracellular; trans, translation; mod, modification; t.o., turnover; sec, secondary; metab, metabolism; biosynth, biosynthesis; catab, catabolism.

FIG. 4.

Heat map of genes validated by single-strain infections in vivo. Results [log₂ (output/input)] from microarray experiments for eight mutants tested in vivo in separate infections for the primary screen (labeling both the forward and reverse sides of the transposon) and the secondary screen (labeling only the reverse side of the transposon) are shown. Results from transposon mutants included in different pools are shown separately. Color code for heat map: gray, no data obtained; blue, overrepresentation of output compared to input signal, log R > 1; black, same signal for output and input, log R = 0 (mutant present in output and input pools); yellow, overrepresentation of input compared to output signal, log R < −1 (negatively selected, mutant lost in output pool).

FIG. 5.

Severely attenuated mutants show normal growth in broth (TSB-C) (A) and a macrophage cell line (MH-S) (B). (A) Growth in broth of FTN_1415 (▪) and FTN_1645 (▴) in comparison to wild type (U112, ♦). (B) Growth in MH-S mouse alveolar macrophages of FTN_1415 and FTN_1645 compared to wild type. Results are representative of three independent experiments; error bars are standard errors of the means.

FIG. 6.

Bacterial burdens for most strongly attenuated mutants and the wild type in lung (A), liver (B), and spleen (C). C57BL/6 mice were infected by aerosol with the wild type (142 CFU), FTN_1415 (4 CFU), and FTN_1645 (9 CFU) in individual infections, and four mice were sacrificed at each time point. The data presented are the mean bacterial loads, and error bars show standard errors of the means. All mice infected with the wild type died at day 3. One mouse infected with FTN_1415 mutant died at day 11.