Contributions of TolC Orthologs to Francisella tularensis Schu S4 Multidrug Resistance, Modulation of Host Cell Responses, and Virulence

Abstract

Francisella tularensis is a Gram-negative, facultative intracellular pathogen and the causative agent of tularemia. Previous studies with the attenuated live vaccine strain (LVS) identified a role for the outer membrane protein TolC in modulation of host cell responses during infection and virulence in the mouse model of tularemia. TolC is an integral part of efflux pumps that export small molecules and type I secretion systems that export a range of bacterial virulence factors. In this study, we analyzed TolC and its two orthologs, FtlC and SilC, present in the fully virulent F. tularensis Schu S4 strain for their contributions to multidrug efflux, suppression of innate immune responses, and virulence. We found that each TolC ortholog participated in multidrug efflux, with overlapping substrate specificities for TolC and FtlC and a distinct substrate profile for SilC. In contrast to their shared roles in drug efflux, only TolC functioned in the modulation of macrophage apoptotic and proinflammatory responses to Schu S4 infection, consistent with a role in virulence factor delivery to host cells. In agreement with previous results with the LVS, the Schu S4 ΔtolC mutant was highly attenuated for virulence in mice by both the intranasal and intradermal routes of infection. Unexpectedly, FtlC was also critical for Schu S4 virulence, but only by the intradermal route. Our data demonstrate a conserved and critical role for TolC in modulation of host immune responses and Francisella virulence and also highlight strain- and route-dependent differences in the pathogenesis of tularemia.

Keywords: Francisella tularensis; Schu S4; TolC; tularemia; virulence.

FIG 1

Cytotoxicity of the Schu S4 ΔtolC, ΔftlC, and ΔsilC mutants toward murine and human macrophages. (A and B) BMDM were infected with either the WT Schu S4 or indicated mutant or complemented strain at an MOI of 500 for 2 h, treated with gentamicin for 1 h, and then incubated in gentamicin-free medium until the indicated time points. (A) At 24 h postinfection, BMDM culture supernatants were collected and assayed for levels of LDH release. (B) At 8, 24, or 48 h postinfection, BMDM culture supernatants were collected and assayed for levels of LDH release. (C) huMDM were infected with either the WT Schu S4 or indicated mutant strain at an MOI of 25. At 24 h postinfection, culture supernatants were collected and assayed for levels of LDH release. Data represent means ± SEMs from at least three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; calculated by one-way ANOVA with Tukey’s multiple-comparison posttest (A and C) or unpaired Student t test (B).

FIG 2

Intracellular replication of the Schu S4 ΔtolC, ΔftlC, and ΔsilC mutants. BMDM were infected as described for Fig. 1 with either the WT Schu S4 or indicated mutant or complemented strain at an MOI of 500. At 3 or 24 h postinfection, cells were washed and then lysed in DMEM plus 0.1% DOC and serially diluted to determine levels of intracellular bacteria by plating for CFU. Data represent means ± SEMs from at least 3 independent experiments. *, P < 0.05, calculated by one-way ANOVA with Tukey’s multiple-comparison posttest.

FIG 3

Analysis of apoptotic and pyroptotic markers in response to Schu S4 ΔtolC infection of BMDM. BMDM were infected with either WT Schu S4 or the ΔtolC mutant at an MOI of 500. At 24 h postinfection, cells were collected and lysed. Activation of either caspase-1 (A) or caspase-3 and PARP (B) was examined via SDS-PAGE and immunoblotting. As a positive control for apoptosis, cells were treated with staurosporine (STS; 125 ng/ml) for 5 h. The black arrowhead in panel B indicates the 89-kDa PARP cleavage fragment. Results are representative of those from 2 independent experiments.

FIG 4

Coinfection of BMDM with WT and ΔtolC Schu S4. BMDM were infected singly with either WT Schu S4 or the ΔtolC mutant at an MOI of 500 or coinfected with the indicated MOI of WT Schu S4 and the ΔtolC mutant. At 24 h postinfection, culture supernatants were collected and assayed for the levels of LDH release. Data represent means ± SEMs from at least three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; calculated by one-way ANOVA with Tukey’s multiple-comparison posttest.

FIG 5

Proinflammatory responses elicited by the Schu S4 ΔtolC mutant during infection of BMDM. BMDM were infected with either WT Schu S4 or the ΔtolC mutant at an MOI of 500 for 24 h. Culture supernatants were collected and assayed for levels of the indicted cytokines. Data represent means ± SEMs from at least three independent experiments. **, P < 0.01; ****, P < 0.0001; calculated by one-way ANOVA with Tukey’s multiple-comparison posttest (for CXCL1) or unpaired Student t test (for IL-6 and CXCL2, comparing the WT with the ΔtolC mutant).

FIG 6

Contributions of TolC orthologs to F. tularensis Schu S4 virulence in mice via the intranasal route. Mice were infected intranasally with the indicated doses of either WT Schu S4 or the ΔtolC mutant (A), ΔftlC mutant (B), or ΔsilC mutant (C) and monitored for survival for 21 days (for all strains, n = 10 mice). (D) Mice were infected with 50 CFU of the WT, the ΔtolC mutant, or the complemented Schu S4 strain and monitored for survival for 21 days (for WT-infected mice, n = 3; for all others, n = 5). **, P < 0.01; ***, P < 0.001; calculated by log rank test.

FIG 7

Contributions of TolC orthologs to F. tularensis Schu S4 virulence in mice via the intradermal route. Mice were infected intradermally with the indicated doses of either WT Schu S4 or the ΔtolC mutant (A), ΔftlC mutant (B), or ΔsilC mutant (C) and monitored for survival for 21 days (for all strains, n = 10 mice). (D) Mice were infected with 50 CFU of WT Schu S4 or the indicated mutant or complemented strain and monitored for survival for 21 days (for WT-infected mice, n = 3; for all others, n = 5). ***, P < 0.001; calculated by log rank test.