Identification of fevR, a novel regulator of virulence gene expression in Francisella novicida

Abstract

Francisella tularensis infects wild animals and humans to cause tularemia. This pathogen targets the cytosol of macrophages, where it replicates using the genes in the Francisella pathogenicity island (FPI). Virulence gene regulation in Francisella is complex, but transcriptional regulators MglA and SspA have been shown to regulate the expression of approximately 100 genes, including the entire FPI. We utilized a Francisella novicida transposon mutant library to identify additional regulatory factors and identified five additional genes that are essential for virulence gene expression. One regulatory gene, FTN_0480 (fevR, Francisella effector of virulence regulation), present in all Francisella species, is required for expression of the FPI genes and other genes in the MglA/SspA regulon. The expression of fevR is positively regulated by MglA. However, constitutive expression of fevR in an mglA mutant strain did not restore expression of the MglA/SspA regulon, demonstrating that mglA and fevR act in parallel to positively regulate virulence gene expression. Virulence studies revealed that fevR is essential for bacterial replication in macrophages and in mice, where we additionally show that fevR is required for the expression of genes in the MglA/SspA regulon in vivo. Thus, fevR is a crucial virulence gene in Francisella, required for the expression of virulence factors known to be essential for this pathogen's subversion of host defenses and pathogenesis in vivo.

FIG. 1.

Screen for F. novicida transposon mutants that regulate pepO. (A and B) RNA was isolated from transposon mutants grown in broth at 37°C to early stationary phase. pepO mRNA levels were determined by quantitative RT-PCR and normalized to that for the uvrD transcript. Thirteen of 24 transposon mutants had significantly lower levels of pepO transcript than the wild type (WT). Samples were obtained in triplicate. Experiments were performed at least three times. Means and standard deviations from a representative experiment are shown. *, P < 0.05; **, P < 0.01.

FIG. 2.

The MglA/SspA regulon is not expressed in mglA and ΔfevR mutants grown in broth. RNA was isolated from wild-type, mglA, and ΔfevR mutants during growth in TSB at 37°C to compare transcriptomes by microarray. The levels of the gene transcripts of the previously published MglA regulon (6) are shown. The levels of these transcripts in mglA and fevR mutants are identical and demonstrate that these genes contribute to MglA-regulated gene expression in broth. Columns represent individual time points increasing from left to right during the time course at 1, 3, 5, 7, and 9 h. Rows represent individual genes.

FIG. 3.

Constitutive expression of an fevR transcript does not rescue expression of the MglA/SspA regulon in an mglA mutant. (A) The fevR promoter was replaced with the groES promoter and the resulting gene was inserted into the chromosome of wild-type (wt) and mglA and ΔfevR mutant backgrounds to create wild-type fevR(Con) (fevR^C), mglA fevR(Con), and ΔfevR fevR(Con) mutants. Strains were grown in TSB at 37°C, and RNA was isolated at 2, 4, 6, and 8 h. Quantitative RT-PCR of fevR (B) and pepO (C) mRNA was performed at the 8-h time point, and levels were normalized to that for the uvrD control transcript. Although the mglA mutant containing the fevR(Con) construct expresses the fevR transcript, pepO expression is not rescued. Sampling was performed in triplicate. Means and standard deviations from a representative experiment are shown. (D) Microarray analysis of MglA regulon expression throughout the growth curves confirms the quantitative RT-PCR findings that both mglA and fevR are necessary for MglA/SspA regulon expression.

FIG. 4.

MglA and SspA do not coprecipitate with FevR. MglA-HA/SspA-His-, MglA-HA/FevR-GST-, and SspA-His/FevR-GST-tagged strains were created by placing the corresponding epitope tag at the C termini of the endogenous copies of products of mglA, sspA, and fevR. Cell extracts were prepared from double-tagged strains grown to late exponential phase. Each cell extract was used in coprecipitations with nickel beads, anti-HA antibody/protein G resin, or glutathione beads and immunoblotted to detect His, HA, and GST tags. The anti-GST antibody stained two bands, a high-molecular-weight band representing FevR-GST and a low-molecular-weight band likely to be a GST cleavage product.

FIG. 5.

mglA and fevR contribute to replication in macrophages. Bone marrow-derived macrophages were infected with either the wild-type, mglA, ΔfevR, or ΔfevR complemented (fevR-C) bacterial strain at a multiplicity of infection of 20 bacteria per macrophage. Macrophages were washed at 30 min postinfection, lysed at 2, 4, 8, and 12 h postinfection, and plated to enumerate the number of CFU. The means from a single experiment are shown and are representative of three independent experiments. The standard deviations are shown and are less than 10%.

FIG. 6.

mglA and fevR are necessary for virulence gene expression and survival in vivo. (A and B) Mice were infected subcutaneously with 5 × 10⁴ CFU of wild-type bacteria and 5 × 10⁴ CFU of mglA (point mutant), ΔfevR, ΔfevR complemented (fevR-C), and FPI deletion mutant bacteria, for a total of 10⁵ bacteria/mouse. After 2 days, the skin (A) and the spleens (B) were taken for counts and the CI was calculated. Each dot represents the CI value for one mouse. Bars represent the geometric means. (C and D) Mice were infected subcutaneously in two different spots on the abdomen with 10⁷ CFU of wild-type (wt) bacteria or mglA or ΔfevR mutants. After 24 h, one injection site was taken for counts, while the other was taken to isolate total RNA. Transcript levels of pepO (C) and mglA (D) were determined by quantitative RT-PCR and normalized to that for control transcript uvrD. Since the mglA mutant is a point mutant, the mglA transcript is still expressed in this strain. Each dot represents the average of quantitative PCR values from experiments performed triplicate for one mouse. Bars represent the geometric means.

FIG. 7.

Model for the MglA/SspA pathway. MglA and SspA positively regulate fevR expression, and fevR regulates expression of the MglA/SspA regulon. Constitutive expression of the fevR transcript in an mglA mutant did not rescue MglA/SspA regulon expression, suggesting that MglA/SspA and FevR work in parallel to activate downstream genes. Furthermore, while MglA and SspA copurify with each other, neither copurifies with FevR-GST. These data suggest that the MglA/SspA regulon is under the control of a common network motif called an FFL.