Hfq, a novel pleiotropic regulator of virulence-associated genes in Francisella tularensis

Abstract

Francisella tularensis is a highly infectious pathogen that infects animals and humans, causing tularemia. The ability to replicate within macrophages is central for virulence and relies on expression of genes located in the Francisella pathogenicity island (FPI), as well as expression of other genes. Regulation of FPI-encoded virulence gene expression in F. tularensis involves at least four regulatory proteins and is not fully understood. Here we studied the RNA-binding protein Hfq in F. tularensis and particularly the role that it plays as a global regulator of gene expression in stress tolerance and pathogenesis. We demonstrate that Hfq promotes resistance to several cellular stresses (including osmotic and membrane stresses). Furthermore, we show that Hfq is important for the ability of the F. tularensis vaccine strain LVS to induce disease and persist in organs of infected mice. We also demonstrate that Hfq is important for stress tolerance and full virulence in a virulent clinical isolate of F. tularensis, FSC200. Finally, microarray analyses revealed that Hfq regulates expression of numerous genes, including genes located in the FPI. Strikingly, Hfq negatively regulates only one of two divergently expressed putative operons in the FPI, in contrast to the other known regulators, which regulate the entire FPI. Hfq thus appears to be a new pleiotropic regulator of virulence in F. tularensis, acting mostly as a repressor, in contrast to the other regulators identified so far. Moreover, the results obtained suggest a novel regulatory mechanism for a subset of FPI genes.

FIG. 1.

Francisella Hfq protein and hfq locus. (A) Alignment of the Hfq proteins of F. tularensis strains LVS, FSC200, and Schu S4 and Hfq of E. coli performed using the ClustalW program. Residues that are identical in all strains are indicated by asterisks, conserved substitutions are indicated by colons, and semiconserved substitutions are indicated by periods. Amino acids which have been implicated in binding of RNA (5) are enclosed in boxes. (B) Genetic organization of the hfq locus in F. tularensis. miaA encodes tRNA delta(2)-isopentenylpyrophosphate transferase, and hflX encodes a GTP-binding protein. Arrows indicate fragments A, B, C, and D amplified in PCR as shown in panels C and D. (C) RT-PCR of the hfq locus. PCR to amplify fragments A, B, and C (see panel B) was performed with genomic DNA from LVS (DNA), with RNA from LVS after RT (RNA), or with RNA from LVS without RT (−RT) as a control. (D) Deletion in hfq has no polar effect on the downstream gene hflX. RT-PCR to amplify fragment D (see panel B) was performed with RNA isolated from either LVS or the hfq mutant after RT (RNA) or with RNA without RT (−RT). A control reaction was performed with DNA isolated from LVS (DNA). (E) Sequence of the promoter region of hfq. The transcription start site (+1) as determined by 5′ RACE is indicated by bold type, and putative −10 and −35 sequences are underlined. SD, Shine-Delgarno sequence. (F) Immunoblotting of bacterial lysates from strain LVS and the hfq mutant using anti-Hfq antiserum. The same amount of bacterial material, based on the optical density of the culture, was deposited in each lane for bacteria at different growth phases, including early exponential phase (OD₆₀₀, 0.2), exponential phase (OD₆₀₀, 0.4), and early stationary phase (OD₆₀₀, 0.8).

FIG. 2.

Growth characteristics of the LVS hfq mutant. (A) Colonies of wild-type F. tularensis LVS, the hfq mutant, and the hfq mutant containing a plasmid containing hfq on chocolate plates after 72 h of growth at 37°C. (B) Growth of F. tularensis LVS and the hfq mutant in Schaedler K3 broth at 37°C. (C) Transmission electron micrographs of LVS and the hfq mutant after growth on solid medium.

FIG. 3.

The hfq mutant is more sensitive to stress than the LVS parent strain. (A) Serial fivefold dilutions of stationary-phase cultures were spotted onto chocolate agar plates and incubated at 37°C or 40°C. (B) Growth of LVS and the hfq mutant in complex broth supplemented with 0%, 1%, or 2% NaCl. (C) Serial 10-fold dilutions of stationary-phase cultures of LVS, the hfq mutant, and the hfq mutant containing a plasmid with hfq were spotted onto chocolate agar plates containing 0.2% SDS, 2% NaCl, or H₂O (control) and incubated at 37°C.

FIG. 4.

Intracellular multiplication of LVS and the hfq mutant in murine macrophage cell lines J774 and RAW and in murine BMM. Macrophages were infected by LVS or hfq mutant bacteria at a multiplicity of infection of ∼100, and the number of intracellular bacteria was determined after 0, 4, 24, and 48 h of infection. The results are expressed as average log₁₀ (CFU/well) ± standard deviation for two experiments, each performed in triplicate.

FIG. 5.

The LVS hfq mutant is attenuated for virulence in mice. (A) Survival of mice after infection with LVS or the hfq mutant for 9 days after infection. Groups of five mice were infected by the i.p. route with different numbers of bacteria. The numbers of bacteria (in log₁₀ CFU) used for infection are indicated on the right. (B) Multiplication of LVS and the hfq mutant in target organs of mice after i.p. infection with ∼3 × 10⁴ CFU. Five mice were sacrificed after 2, 3, 4, and 7 days of infection, and the bacterial burdens in the spleen and liver were determined. Only one mouse survived after infection by LVS to day 7, and therefore no data for LVS at day 7 are shown.

FIG. 6.

Growth characteristics and stress resistance of the FSC200 hfq mutant. (A) Growth of FSC200 and FSC768 in defined medium with additional 0%, 1%, or 2% NaCl. (B) Serial fivefold dilutions of stationary-phase cultures were spotted onto McLeod agar plates and incubated at 37°C or 40°C.

FIG. 7.

Virulence of FSC200 is affected by the hfq mutation. (A) Intracellular replication in the J774 murine macrophage cell line. (B) Survival of mice after infection with FSC200 or FSC768 for 11 days after infection. Groups of five mice were infected by the i.p. or subcutaneous route with different numbers of bacteria. The numbers of bacteria (in log₁₀ CFU) used for infection are indicated in parentheses after the strain designation. (C) Five mice were inoculated by the subcutaneous route with approximately 100 bacteria from a 1:1 mixture of FSC200 and FSC768. Spleens were homogenized after 5 days of infection, and the numbers of wild-type and mutant bacteria were determined. The results are expressed as the CI, which was calculated using the ratio of mutant bacteria to wild-type bacteria. Each symbol represents data for a single mouse, and the average CI is indicated by a solid line.

FIG. 8.

Schematic diagram of the FPI. Genes are numbered according to the LVS strain nomenclature for one of the two copies of the FPI. The two proposed divergent operons are indicated at the bottom.

FIG. 9.

Quantification of transcription of selected genes by real-time RT-PCR. Transcript levels were normalized to the level of DNA helicase (FTL_1656), and the differences (relative to LVS) and standard deviations are indicated for eight genes. RNAs from strains grown under two different growth conditions (defined medium and solid medium) were used for the analysis. The data are data for triplicate samples analyzed at the same time. The difference between the transcript levels of the LVS and hfq strains was significant (P < 0.05) for both growth conditions for pdpB, FTL_0120, FTL_0994, katG, FTL_0317, and iglC, as determined using Student's t test.