A tolC mutant of Francisella tularensis is hypercytotoxic compared to the wild type and elicits increased proinflammatory responses from host cells

Abstract

The highly infectious bacterium Francisella tularensis is a facultative intracellular pathogen and the causative agent of tularemia. TolC, which is an outer membrane protein involved in drug efflux and type I protein secretion, is required for the virulence of the F. tularensis live vaccine strain (LVS) in mice. Here, we show that an LVS DeltatolC mutant colonizes livers, spleens, and lungs of mice infected intradermally or intranasally, but it is present at lower numbers in these organs than in those infected with the parental LVS. For both routes of infection, colonization by the DeltatolC mutant is most severely affected in the lungs, suggesting that TolC function is particularly important in this organ. The DeltatolC mutant is hypercytotoxic to murine and human macrophages compared to the wild-type LVS, and it elicits the increased secretion of proinflammatory chemokines from human macrophages and endothelial cells. Taken together, these data suggest that TolC function is required for F. tularensis to inhibit host cell death and dampen host immune responses. We propose that, in the absence of TolC, F. tularensis induces excessive host cell death, causing the bacterium to lose its intracellular replicative niche. This results in lower bacterial numbers, which then are cleared by the increased innate immune response of the host.

FIG. 1.

LVS ΔtolC is defective for colonizing the organs of mice inoculated by the intradermal route. C3H/HeN mice were infected intradermally with a sublethal dose (10⁵ CFU) of the LVS or ΔtolC mutant. Organs were harvested on days 2, 4, and 7 p.i., and CFU/g of organ weight were determined for the lung (A), liver (B), and spleen (C). The limit of detection per organ was 10 CFU. Two independent experiments with 5 mice per time point per bacterial strain were performed, and these data were combined. The bars indicate mean CFU values. Colonization by the ΔtolC mutant was significantly decreased compared to that of the wild-type LVS at each time point in each organ (P < 0.01). (D) Hematoxylin- and eosin-stained sections obtained from infected livers harvested at day 4 p.i. show that the wild-type LVS and ΔtolC mutant cause similar pathology. Arrows indicate granuloma-like lesions. Scale bars = 100 μm.

FIG. 2.

TolC is required for virulence of the LVS in mice by the intranasal route. C3H/HeN mice were infected intranasally with a lethal dose (1 × 10⁵ CFU) of the LVS, ΔtolC, or complemented strain (ΔtolC tolC⁺). The animals were monitored for survival for 18 days. Two independent experiments with 5 mice per bacterial strain were performed, and the data were combined. The ΔtolC mutant was significantly attenuated compared to the parental LVS (P < 0.0001), and complementation significantly restored virulence to the ΔtolC mutant (P < 0.01).

FIG. 3.

LVS ΔtolC is defective for colonizing the organs of mice inoculated by the intranasal route. C3H/HeN mice were intranasally infected with a sublethal dose (5 × 10³ CFU) of the LVS or ΔtolC mutant. Organs were harvested on days 3, 5, and 9 p.i., and CFU/g of organ weight were determined for the lung (A), liver (B), and spleen (C). The limit of detection per organ was 10 CFU. Two independent experiments with 5 mice per time point per bacterial strain were performed, and the data were combined. The bars indicate mean CFU values. Colonization by the ΔtolC mutant was significantly decreased compared to that of the wild-type LVS at each time point in each organ (P < 0.05 for lung day 3 p.i.; P < 0.0001 for all other comparisons).

FIG. 4.

LVS ΔtolC is hypercytotoxic to murine macrophages. muBMDM cultured from C3H/HeN mice were infected with the LVS, ΔtolC mutant, or complemented strain (ΔtolC tolC⁺) at an MOI of 50. Cytotoxicity was quantified by measuring LDH release at 17 (A) or 24 h p.i. (B). Bars represent means ± standard errors of the means (SEM) of three independent experiments. The ΔtolC mutant caused significantly increased LDH release compared to that of the wild-type LVS (P < 0.05). (C) muBMDM infected as described for panel B were assayed for apoptosis at 17 h p.i. by TUNEL staining. The images show overlays of TUNEL-positive cells (red) and corresponding phase-contrast images. The percentages of TUNEL-positive cells ± SEM were calculated from 10 separate fields and represent the averages from three independent experiments. The ΔtolC mutant caused significantly increased TUNEL staining compared to that of the wild-type LVS (P < 0.05). Scale bars = 50 μm.

FIG. 5.

Hypercytotoxicity of the LVS ΔtolC is independent of caspase-1 and involves caspase-3. (A) muBMDM isolated from wild-type or caspase-1-deficient C57BL/6 mice were infected with the LVS or ΔtolC mutant at an MOI of 50. Cytotoxicity was quantified by measuring LDH release at 24 h p.i. The ΔtolC mutant caused significantly increased LDH release compared to that of the wild-type LVS for the caspase-1-deficient muBMDM (P < 0.05). Bars represent means ± SEM from three replicate samples. A representative experiment is shown. The experiment was repeated with similar results. (B) muBMDM isolated from C3H/HeN mice were uninfected or were infected with the LVS, ΔtolC mutant, or complemented strain (ΔtolC tolC⁺) at an MOI of 50. Activated caspase-3/-7 was measured at 17 h p.i. using a luminescence-based assay. Infection with the ΔtolC mutant caused a significant increase in caspase-3/-7 activity compared to that of the wild-type LVS (P < 0.05). Bars represent means ± SEM from three replicate samples. A representative experiment is shown. The experiment was repeated twice more with similar results. (C) muBMDM from C3H/HeN mice were infected as described for panel B and probed with an antibody against mature caspase-3 followed by a TRITC-conjugated secondary antibody. The images show overlays of caspase-3-positive cells (red) and corresponding phase-contrast images. The percentages of caspase-3-positive cells ± SEM were calculated from 10 separate fields and represent the averages from three independent experiments. The ΔtolC mutant caused significantly increased staining compared to that of the wild-type LVS (P < 0.05). Bars = 50 μm.

FIG. 6.

LVS ΔtolC is hypercytotoxic to human macrophages and elicits increased secretion of IL-1β. huMDM were infected with the LVS, ΔtolC mutant, or complemented strain (ΔtolC tolC⁺) at an MOI of 50. Cytotoxicity was quantified by measuring LDH release at 17 (A) or 24 h p.i. (B). Bars represent means ± SEM from three independent experiments. The ΔtolC mutant caused significantly increased LDH release compared to that of the wild-type LVS at 24 h p.i. (P < 0.05). (C) huMDM were infected with the LVS or ΔtolC mutant at an MOI of 25. IL-1β release was quantified by ELISA of conditioned media at 24 h p.i. Bars represent means ± SEM from three independent experiments. The ΔtolC mutant caused a significant increase in IL-1ß secretion compared to the wild-type LVS (P < 0.05).

FIG. 7.

LVS ΔtolC elicits increased secretion of proinflammatory chemokines from human cells. huMDM were uninfected or infected with the LVS or ΔtolC mutant at an MOI of 25. The secretion of CCL2 (A) or CXCL8 (B) was quantified by the ELISA of conditioned medium at 24 h p.i. (C) HUVEC were incubated with medium alone or with the LVS or ΔtolC mutant at an MOI of 75. The secretion of CXCL8 was quantified by the ELISA of conditioned medium at 24 h p.i. The ΔtolC mutant caused the significantly increased secretion of the proinflammatory chemokines compared to that of the wild-type LVS (P < 0.05). Bars represent means ± SEM from three replicate samples. Each graph shows a representative experiment. Each experiment was repeated twice more with similar results.