Small molecule inhibitors of LcrF, a Yersinia pseudotuberculosis transcription factor, attenuate virulence and limit infection in a murine pneumonia model

Abstract

LcrF (VirF), a transcription factor in the multiple adaptational response (MAR) family, regulates expression of the Yersinia type III secretion system (T3SS). Yersinia pseudotuberculosis lcrF-null mutants showed attenuated virulence in tissue culture and animal models of infection. Targeting of LcrF offers a novel, antivirulence strategy for preventing Yersinia infection. A small molecule library was screened for inhibition of LcrF-DNA binding in an in vitro assay. All of the compounds lacked intrinsic antibacterial activity and did not demonstrate toxicity against mammalian cells. A subset of these compounds inhibited T3SS-dependent cytotoxicity of Y. pseudotuberculosis toward macrophages in vitro. In a murine model of Y. pseudotuberculosis pneumonia, two compounds significantly reduced the bacterial burden in the lungs and afforded a dramatic survival advantage. The MAR family of transcription factors is well conserved, with members playing central roles in pathogenesis across bacterial genera; thus, the inhibitors could have broad applicability.

FIG. 1.

Expression of LcrF in trans restores Yop secretion and cytotoxicity to the ΔLcrF mutant. (A) Western blot of Y. pseudotuberculosis culture supernatants (S) or whole bacteria pelleted by centrifugation (P) probed with antisera to YopE or S2. The strains are as follows: lanes 1 and 2, YPIIIpIB1ΔlcrF plus pTrc99A; lanes 3 to 7, YPIIIpIB1ΔlcrF plus pTrc99A-LcrF; lane 8, blank; lanes 9 and 10, YPIIIpIB1 plus pTrc99A. The culture IPTG concentrations are as indicated, plus the following: lane 4, 12.5 μM; lane 5, 25 μM; lane 6, 50 μM. (B) Y. pseudotuberculosis YPIIIpIB1ΔlcrF (ΔLcrF) shows reduced cytotoxicity to J774A.1 macrophage cells compared to the level for Y. pseudotuberculosis YPIIIpIB1 (WT), as measured by released lactate dehydrogenase (LDH) activity. Also shown is YPIIIpIB1⁻, which lacks the entire pIB1 plasmid. Percent cytotoxicity represents the mean LDH activity compared to the mean total LDH activity from uninfected J774A.1 cells lysed with detergent (100% cytotoxicity). Error bars indicate standard deviations. For lysed and uninfected cells, n = 4 wells; for the WT, ΔLcrF, and pIB1⁻ strains, n = 16 wells. (C) Cytotoxicity of the YPIIIpIB1-plus-pTrc99A (WT), YPIIIpIB1ΔlcrF-plus-pTrc99A, and YPIIIpIB1ΔlcrF-plus-pTrc99A-LcrF strains to J774.1 cells ± 100 μM IPTG. Percent cytotoxicity represents the mean LDH activity compared to the mean total LDH activity from uninfected J774A.1 cells lysed with detergent (100% cytotoxicity). Error bars indicate standard deviations. For lysed and uninfected cells, n = 4 wells; for the WT-plus-pTrc99A, ΔLcrF-plus-pTrc99A, and ΔLcrF-plus-pTrc99A-LcrF strains, n = 12 wells.

FIG. 2.

Deletion of the lcrF gene attenuates Y. pseudotuberculosis virulence. (A) Survival of BALB/c mice infected i.n. with the indicated numbers of CFU of wild-type Y. pseudotuberculosis IP2666pIB1 (WT) (n = 4 mice per group). **, P < 0.01 by the Kaplan-Meier survival analysis log rank test for 90 or 400 CFU compared to 1 CFU. (B) Survival of BALB/c mice infected i.n. with the indicated numbers of CFU of Y. pseudotuberculosis IP2666pIB1ΔlcrF (ΔLcrF) (n = 4 mice per group). **, P < 0.01 by the Kaplan-Meier survival analysis log rank test for 2,200 CFU compared to 2 CFU. Curves with open symbols indicate the apparent LD₅₀s.

FIG. 3.

Compounds A and B inhibit LcrF binding to the virC promoter in vitro. Percent inhibition of binding of purified 6His-LcrF protein to virC promoter DNA in a cell-free assay. The chemical structure and titration of LcrF inhibition by compound A (A) and compound B (B) are shown. Solid lines represent inhibition curve fits, and dotted lines represent the IC₅₀s calculated by the software program XLfit. Data are from single representative experiments.

FIG. 4.

Compounds A and B inhibit LcrF-dependent Y. pseudotuberculosis cytotoxicity. (A) Compounds A and B were added at the indicated concentrations to J774A.1 cells during infection with wild-type Y. pseudotuberculosis YPIIIpIB1 (WT). The percent WT cytotoxicity indicates the amount of LDH activity released compared to the level for J774A.1 cells infected with WT Y. pseudotuberculosis and receiving vehicle (Fig. 1). Bars indicate mean percentages of WT cytotoxicity, and error bars indicate standard deviations. For WT- and YPIIIpIB1ΔlcrF (ΔLcrF)-infected wells with vehicle, n = 8. For WT-infected wells with compound A or B, n = 4. (B) J774A.1 cells were infected with WT Y. pseudotuberculosis, and compound A, compound B, or vehicle was added to infected wells 5 min prior to sample collection at the end of the assay (n = 8). (C) Compound A, compound B, or vehicle was incubated with J774A.1 cells in the absence of bacteria for the same duration as that for an infection experiment. Half the wells were then lysed with detergent, and the LDH activity was compared to that of lysed, untreated cells (100% cytotoxicity) (n = 8).

FIG. 5.

Compounds A and B reduce the bacterial burden in the lungs in a murine model of Y. pseudotuberculosis pneumonia. Groups of 5 CD1 mice were dosed s.c. with vehicle or 25 mg/kg of compound A (A) or compound B (B) prior to and following i.n. infection with wild-type Y. pseudotuberculosis IP2666pIB1 (WT). Control groups of mice were dosed with vehicle and infected with Y. pseudotuberculosis IP2666pIB1ΔlcrF (ΔLcrF). Mice were sacrificed 4 days following infection, and the bacterial burden was determined in terms of number of CFU/gram lung tissue. Symbols represent output for individual animals, and bars represent the geometric mean for the group. P values were determined by Kruskal-Wallis one-way analysis of variance and a chi-square approximation. **, P < 0.01 for comparison to the WT; *, P < 0.05 for comparison to the WT. In panel B, for ΔLcrF, n = 4, and for all other groups, n = 5. Data represent the outcome from one of three experiments conducted for each compound.

FIG. 6.

Compounds A and B improve survival in a murine model of Y. pseudotuberculosis pneumonia. Symbols represent the survival of groups of 4 CD1 mice dosed s.c. with 25 mg/kg of the indicated compound or vehicle prior to and following i.n. infection with wild-type Y. pseudotuberculosis IP2666pIB1 (WT). A control group of mice was dosed with vehicle as described above and infected with Y. pseudotuberculosis IP2666pIB1ΔlcrF (ΔLcrF). P values were determined by the Kaplan-Meier survival analysis log rank test. **, P < 0.01 for comparison to the WT; *, P < 0.05 for comparison to the WT. Data represent the outcome from one of two experiments conducted for each compound.