Small molecule control of virulence gene expression in Francisella tularensis

Abstract

In Francisella tularensis, the SspA protein family members MglA and SspA form a complex that associates with RNA polymerase (RNAP) to positively control the expression of virulence genes critical for the intramacrophage growth and survival of the organism. Although the association of the MglA-SspA complex with RNAP is evidently central to its role in controlling gene expression, the molecular details of how MglA and SspA exert their effects are not known. Here we show that in the live vaccine strain of F. tularensis (LVS), the MglA-SspA complex works in concert with a putative DNA-binding protein we have called PigR, together with the alarmone guanosine tetraphosphate (ppGpp), to regulate the expression of target genes. In particular, we present evidence that MglA, SspA, PigR and ppGpp regulate expression of the same set of genes, and show that mglA, sspA, pigR and ppGpp null mutants exhibit similar intramacrophage growth defects and are strongly attenuated for virulence in mice. We show further that PigR interacts directly with the MglA-SspA complex, suggesting that the central role of the MglA and SspA proteins in the control of virulence gene expression is to serve as a target for a transcription activator. Finally, we present evidence that ppGpp exerts its effects by promoting the interaction between PigR and the RNAP-associated MglA-SspA complex. Through its responsiveness to ppGpp, the contact between PigR and the MglA-SspA complex allows the integration of nutritional cues into the regulatory network governing virulence gene expression.

Figure 1. ppGpp controls the expression of MglA/SspA-regulated genes in F. tularensis.

(A) Analysis of ppGpp concentrations in cells of the indicated strains of LVS by thin layer chromatography. For these analyses ppGpp was isolated from cells following a shift to conditions of nutrient limitation (see Materials and Methods). (B) Quantitative RT-PCR analysis of iglA, pdpA, and FTL_1219 transcript abundance in wild-type (WT), ΔmglA, ΔrelA, ΔrelA ΔspoT, and ΔmglA ΔrelA ΔspoT mutant backgrounds. RNA was isolated from cells grown in MH to mid-log. Transcripts were normalized to those of tul4, whose expression is not influenced by MglA or ppGpp. (C) Complementation of the effects of the ΔrelA ΔspoT mutations on iglA expression by spoT provided in trans. Quantitative RT-PCR analysis of iglA transcript abundance in wild-type (WT), and ΔrelA ΔspoT mutant cells harboring the indicated plasmids. Transcripts were normalized to tul4. Plasmid pF2-SpoT directs the synthesis of F. tularensis SpoT, whereas plasmid pF2 served as an empty vector control. (D) Venn diagram representation of the overlap between genes controlled by MglA and ppGpp. Each circle represents those genes whose expression was decreased by a factor of 2.5 or more (p<0.05) in the indicated mutant background compared to wild-type and whose expression altered by a factor of 2 or more in the other mutant background, as determined by DNA-microarray.

Figure 2. Cells of a ΔrelA ΔspoT mutant are defective for intramacrophage growth and for virulence in mice.

(A) Survival of wild-type (WT) and the indicated mutant derivates of F. tularensis strain LVS within J774 cells. J774 murine macrophages were infected with cells of the indicated bacterial strains at a multiplicity of infection of ∼15. Cells were lysed and bacteria (colony forming units [CFU]) were plated for enumeration 24 hours post-infection. (B) Survival of BALB/cByJ mice following intradermal delivery of ∼10⁷ cells of each of the indicated strains of LVS. Eight mice were inoculated for each strain tested. Experiments were performed at least twice.

Figure 3. ppGpp does not influence the abundance of MglA or SspA, and does not influence the association between RNAP and the MglA-SspA complex.

(A) Western blot analysis of the effect of ppGpp on the abundance of MglA-TAP and SspA-TAP. Proteins from equivalent numbers of LVS MglA-TAP cells (lane 1), LVS ΔrelA ΔspoT MglA-TAP cells (lane 2), LVS SspA-TAP cells (lane 3), and LVS ΔrelA ΔspoT SspA-TAP cells (lane 4) were electrophoresed on a 4–12% Bis-Tris NuPAGE gel and analysed by Western blotting. Upper panel, immunoblot probed with anti-TAP. Lower panel, immunoblot probed with antibody against Tul4 serves as a control for sample loading. (B) SDS-PAGE analysis of proteins that co-purify with β′-TAP in a wild-type (WT, lane 1) or in a ΔrelA ΔspoT mutant background (lane 2). Protein complexes were tandem affinity purified, electrophoresed on a 4–12% Bis-Tris NuPAGE gel, and stained with silver. Lane 1, proteins purified from strain LVS β′-TAP. Lane 2, proteins purified from strain LVS ΔrelA ΔspoT β′-TAP. Molecular weights are indicated on the left. The identities of certain proteins are indicated on the right.

Figure 4. Genetic screen for positive regulators of iglA expression.

(A) Schematic of iglA reporter in strain LVS iglA::lacZ in which one of the copies of the iglA gene has been replaced with lacZ. (B) Location of transposon insertions (indicated by small arrows) in genes identified in the screen. Numbers below each gene refer to base pairs. (C) Quantitative RT-PCR analysis of iglA transcript abundance in wild-type (WT), ΔtrmE, ΔcaiC, ΔcphA, and ΔpigR mutant backgrounds. Transcripts were normalized to those of tul4.

Figure 5. PigR and TrmE are required for intramacrophage growth and for virulence in mice.

(A) Survival of wild-type (WT) cells, and cells of the indicated mutant derivates of F. tularensis strain LVS within J774 cells. J774 murine macrophages were infected with cells of the indicated bacterial strains at a multiplicity of infection of ∼15. Cells were lysed and bacteria (colony forming units [CFU]) were plated for enumeration 24 hours post-infection. (B) Survival of BALB/cByJ mice following intradermal delivery of ∼10⁷ cells of each of the indicated strains of LVS. Eight mice were inoculated for each strain tested. Experiments were performed at least twice.

Figure 6. Ectopic expression of pigR partially complements a ΔrelA ΔspoT mutant but fails to complement a ΔmglA mutant.

(A) Expression of pigR is positively regulated by MglA and by ppGpp. Quantitative RT-PCR analysis of iglA transcript abundance in wild-type (WT), ΔmglA, and ΔrelA ΔspoT mutant backgrounds. Transcripts were normalized to those of tul4. (B) Quantitative RT-PCR analysis of iglA, pdpA, and FTL_1219 transcript abundance in wild-type (WT), ΔpigR, ΔmglA, and ΔrelA ΔspoT mutant cells containing the indicated plasmids. Plasmid pF-PigR directs the synthesis of PigR and plasmid pF serves as an empty vector control. Transcripts were normalized to those of tul4.

Figure 7. The pigR gene is positively autoregulated.

(A) Schematic of lacZ reporter in strain LVS pigR::lacZ. In this strain the native pigR gene was replaced with lacZ, thus placing lacZ under the control of the pigR promoter (PpigR) on the LVS chromosome. (B) Quantification of lacZ expression in strains LVS pigR::lacZ, LVS ΔmglA pigR::lacZ, and LVS ΔrelA ΔspoT pigR::lacZ containing the indicated plasmids. Plasmids pF-PigR, pF-PigR+MglA, pF-PigR+SpoT, and pF2-SpoT direct the synthesis of PigR, PigR and MglA, PigR and SpoT, and SpoT, respectively. Plasmid pF served as the empty control vector for plasmids pF-PigR, pF-PigR+MglA, and pF-PigR+SpoT, whereas plasmid pF2 served as the empty control vector for plasmid pF2-SpoT.

Figure 8. PigR interacts with the MglA-SspA complex.

(A) Schematic representation of the bacterial bridge-hybrid system used to detect an interaction between PigR and the MglA-SspA complex. In this system F. tularensis SspA interacts with the MglA-ω fusion protein to form a heteromeric complex that associates with E. coli RNAP. Contact between the heteromeric MglA-SspA complex displayed on RNAP and the DNA-bound PigR-Zif fusion activates transcription from the test promoter driving expression of lacZ. The test promoter placZif1-61 is present on an F′ episome in E. coli strain KDZif1ΔZ and bears a Zif binding site centered 61 bp upstream of the transcription start site of the lac core promoter (whose −10 and −35 elements are indicated). (B) Transcription activation by PigR-Zif in the presence of F. tularensis SspA and the MglA-ω fusion protein. Assays were performed with cells of the E. coli reporter strain KDZif1ΔZ containing compatible plasmids that directed the IPTG-controlled synthesis of the indicated proteins. Cells were grown in the presence of different concentrations of IPTG and assayed for β-galactosidase activity.

Figure 9. ppGpp promotes the interaction between PigR and the MglA-SspA complex.

PigR-V was ectopically expressed in wild-type (WT) or in ΔrelA ΔspoT mutant cells producing MglA-TAP, or β′-TAP. Cells were grown to mid-log and formaldehyde was added to crosslink PigR-V to associated proteins. Following TAP and reversal of the crosslinks, proteins that co-purified with MglA-TAP, or β′-TAP were separated on a 4–12% Bis-Tris NuPAGE gel and the presence of PigR-V was analyzed by Western blotting. Proteins present in cell lysates prior to TAP were separated and analyzed in the same manner. Upper panels: PigR-V present following TAP (top), and PigR-V present in the cell lysate prior to TAP (bottom). Lower panels: Calmodulin binding peptide (CBP)-tagged proteins present following TAP (top), and TAP-tagged proteins present in the cell lysate prior to TAP (bottom). Note that because the protein A moieties of the TAP-tag are removed during the tandem affinity purification procedure, the CBP portion of the TAP-tag is all that remains following TAP. The anti-CBP antibody is therefore used here to detect proteins that initially contained the TAP-tag. Note also that the MglA and β′ species are different sizes and so different portions of the corresponding Western blot are shown. Proteins were from the following cells: Lane 1, LVS synthesizing PigR-V. Lane 2, LVS MglA-TAP. Lane 3, LVS MglA-TAP synthesizing PigR-V. Lane 4, LVS MglA-TAP ΔrelA ΔspoT synthesizing PigR-V. Lane 5, LVS β′-TAP. Lane 6, LVS β′-TAP synthesizing PigR-V. Lane 7, LVS β′-TAP ΔrelA ΔspoT synthesizing PigR-V.

Figure 10. Model for how the MglA-SspA complex, ppGpp, and PigR, positively control the expression of target genes.

According to our model PigR interacts directly with the RNAP-associated MglA-SspA complex and ppGpp promotes this interaction (either by interacting directly with PigR and/or the MglA-SspA complex, or by interacting directly with RNAP). Although the MglA-SspA complex is depicted as interacting with the α subunit of RNAP (for convenience), it is not known which RNAP subunit(s) serve as a contact site for the MglA-SspA complex.