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Infection and Immunity, July 2008, p. 2852-2861, Vol. 76, No. 7
0019-9567/08/$08.00+0     doi:10.1128/IAI.01726-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Type IV Pili in Francisella tularensis: Roles of pilF and pilT in Fiber Assembly, Host Cell Adherence, and Virulence {triangledown}

Subhra Chakraborty,1 Michael Monfett,1 Tamara M. Maier,2 Jorge L. Benach,1 Dara W. Frank,2 and David G. Thanassi1*

Center for Infectious Diseases, Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, New York 11794-5120,1 Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, Wisconsin 532262

Received 21 December 2007/ Returned for modification 28 January 2008/ Accepted 6 April 2008


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ABSTRACT
 
Francisella tularensis, a highly virulent facultative intracellular bacterium, is the causative agent of tularemia. Genome sequencing of all F. tularensis subspecies revealed the presence of genes that could encode type IV pili (Tfp). The live vaccine strain (LVS) expresses surface fibers resembling Tfp, but it was not established whether these fibers were indeed Tfp encoded by the pil genes. We show here that deletion of the pilF putative Tfp assembly ATPase in the LVS resulted in a complete loss of surface fibers. Disruption of the pilT putative disassembly ATPase also caused a complete loss of pili, indicating that pilT functions differently in F. tularensis than in model Tfp systems such as those found in Pseudomonas aeruginosa and Neisseria spp. The LVS pilF and pilT mutants were attenuated for virulence in a mouse model of tularemia by the intradermal route. Furthermore, although absence of pili had no effect on the ability of the LVS to replicate intracellularly, the pilF and pilT mutants were defective for adherence to macrophages, pneumocytes, and hepatocytes. This work confirms that the surface fibers expressed by the LVS are encoded by the pil genes and provides evidence that the Francisella pili contribute to host cell adhesion and virulence.


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INTRODUCTION
 
Francisella tularensis is a highly virulent, gram-negative, facultative intracellular bacterium that causes the zoonotic disease tularemia (14). Human illness can range from an ulceroglandular form to more serious conditions, including pneumonic, typhoidal, and meningitic tularemia (48, 63). Due to its high infectivity and potential for airborne transmission, F. tularensis has been designated a category A agent of bioterrorism (12). There are four subspecies of F. tularensis: F. tularensis subsp. tularensis, F. tularensis subsp. holarctica, F. tularensis subsp. mediasiatica, and "F. tularensis subsp. novicida," the first two being clinically important (48). F. tularensis subsp. tularensis (the type A biovar) causes the most severe form of tularemia, whereas F. tularensis subsp. holarctica (the type B biovar) is less virulent and produces milder disease symptoms in humans. F. tularensis subsp. novicida is infectious only for immunocompromised individuals; however, it causes tularemia-like symptoms in mice. The live vaccine strain (LVS), which was developed in the former Soviet Union, belongs to F. tularensis subsp. holarctica (12) and is now widely used to study tularemia because it remains pathogenic for certain animals and causes a lethal infection in mice that closely mimics the human disease (2, 13).

F. tularensis is able to escape the endosomal-lysosomal trafficking pathway within phagocytes (32, 59) and evades host innate immune responses, a characteristic attributable in part to an unusual lipopolysaccharide structure (15, 58) and a capsule that protects the bacteria from serum-mediated lysis (57). A duplicated pathogenicity island and a pair of regulatory genes (mglA and mglB) play important roles in the organism's escape from the phagosome and replication within the cytosol of host cells (4, 32, 60). Examination of the available Francisella genome sequences reveals a lack of secreted proteins or secretion systems that are typically present in intracellular pathogens (31, 56). However, some potential pathways for the secretion of virulence factors have been identified. Orthologs of the TolC outer membrane protein required for multidrug efflux and protein secretion by the type I secretion pathway are present in Francisella strains, and tolC was shown to be a virulence determinant of the LVS (19). Recently, it was proposed that the Francisella pathogenicity island encodes a type VI secretion system (43). The type VI system is a newly identified secretion pathway that appears to be widespread among bacterial pathogens, including Pseudomonas aeruginosa and Vibrio cholerae (42, 52). F. tularensis strains also contain orthologs of pil genes required for the biogenesis of type IV pili (Tfp) (18, 31). Tfp are multifunctional, filamentous surface fibers expressed by a wide variety of bacteria, including P. aeruginosa, Neisseria spp., enteropathogenic Escherichia coli, and V. cholerae (6, 38, 45). Tfp functions include surface motility, microcolony and biofilm formation, host cell adhesion, and natural transformation (1, 26). For many gram-negative pathogens, disruption of Tfp assembly results in reduced virulence (25, 33). The LVS expresses surface fibers resembling Tfp, although the identity of these fibers was not confirmed (18). As discussed below, a subset of the pil genes of F. tularensis subsp. novicida were shown to participate in protein secretion into culture supernatant fractions (22), suggesting that these genes might function as a secretion system in addition to or instead of Tfp biogenesis.

The biogenesis and regulation of Tfp is complex, involving a large number of proteins that are related to the type II secretion system of gram-negative bacteria and filamentous phage assembly (11, 38, 45). We use here the nomenclature originally proposed for the F. tularensis Tfp components (18), which corresponds to that used for Neisseria. Alternate designations will be indicated in parenthesis where appropriate. The F. tularensis pil genes are distributed in clusters or individually at different locations in the genome (18, 31). One F. tularensis cluster contains the genes pilNOPQ. PilQ is a member of a large family of proteins termed secretins, which forms channels in the bacterial outer membrane for secretion of the pilus fiber. A second cluster contains the genes pilFG, in which PilF (PilB) is a nucleotide-binding protein required for energizing pilus assembly and secretion, and PilG (PilC) is an integral cytoplasmic membrane protein of the pilus biosynthetic machinery (39, 65). The genes pilT and pilD occur at separate locations in the genome. PilD is the cytoplasmic membrane peptidase responsible for processing the prepilin subunits (39, 65). PilT proteins form a subgroup of bacterial type II secretion ATPases and belong to the RecA superfamily of hexameric ATPases (69). PilT functions as an antagonist of pilus assembly and is critically involved in twitching motility and pilus retraction, mediating bacterial surface translocation, and interactions between the bacteria and host tissues (41). ATP hydrolysis by PilT is proposed to mediate pilus disassembly from the fiber base at the cytoplasmic membrane, causing retraction (69). A Pseudomonas pilT mutant strain is hyperpiliated due to loss of pilus retraction but significantly less infectious than the wild-type strain in mouse models of corneal infection and acute pneumonia (10, 73). Interestingly, the F. tularensis LVS pilT gene contains a point mutation that introduces a premature stop codon, dividing the gene into two open reading frames (ORFs) (Fig. 1) (18). This nonsense mutation appears to be common to other F. tularensis subsp. holarctica strains, including FSC352, FSC354, and FSC200 (16, 50, 55). In contrast, the pilT sequences in F. tularensis subsp. novicida (U112) and F. tularensis subsp. tularensis (SchuS4) are intact and lack similar mutations (Fig. 1).


Figure 1
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  		FIG. 1. Analysis of pilT ORFs from F. tularensis SchuS4 (F. tularensis subsp. tularensis; FTT0088), U112 (F. tularensis subsp. novicida; FTN_1622), and LVS (F. tularensis subsp. holarctica; FTL_1770 and FTL_1771). The amino acid sequences of the ORFs are aligned, with nonconserved residues boxed in gray. The red triangle marks the location of the stop codon mutation in the LVS. The stop codon is followed by a stretch of 20 residues predicted not to be translated (boxed in red), which is followed by the predicted start site for the second ORF, indicated by the green arrow. The locations of the Walker box A (WBA), aspartate box (Asp), Walker box B (WBB), histidine box (His), and AIRNLIRE motifs are indicated. The motifs were identified by visual inspection of the PilT sequences and manual alignment with the motifs as previously defined in related family members (3, 54).

Gil et al. identified five genes in SchuS4 that could code for the major pilus subunit protein (pilE1 to pilE5) (18). In the LVS, pilE1 (pilA), pilE2 (pilE), and pilE3 (pilV) are not likely to be functional due to mutations and deletions within these genes (16, 18). However, the pilE4 and pilE5 genes of the LVS appear to be intact. Forslund et al. demonstrated that PilE1 (PilA) is an important virulence determinant of F. tularensis subsp. holarctica strains by the subcutaneous route of infection in mice (16). In a recent study by Hager et al. (22), seven proteins, including a protease (PepO), two chitinases (ChiA and ChiB), a chitin-binding protein (CbpA), a predicted β-glucosidase (BglX), and two proteins with unknown functions, were detected in culture supernatant fractions of F. tularensis subsp. novicida. Mutations in the Tfp genes pilF (pilB), pilG (pilC), pilQ, and pilE1 (pilA) abolished secretion of these proteins by F. tularensis subsp. novicida, whereas mutation of pilT did not affect secretion (22). The Hager et al. study revealed that the F. tularensis pil genes function as a type II-like system for the secretion of soluble proteins.

We show here that pilF and pilT are required for expression of the surface fibers by F. tularensis LVS, confirming that these fibers are encoded by the pil genes. The finding that pilT is required for pilus assembly in the LVS implies that pilT is functional despite being disrupted by mutation and indicates a role distinct from or in addition to pilus retraction. We demonstrate that the F. tularensis pili contribute to host cell adherence and are virulence determinants that are important for pathogenesis in mice by the intradermal route.


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MATERIALS AND METHODS
 
Strains and plasmids. The strains and plasmids used in the present study are described in Table 1. Unless otherwise noted, the LVS was grown on Mueller-Hinton II chocolate agar plates (MHC; BD Biosciences) containing 1% hemoglobin and 1% IsoVitaleX (BD Biosciences) or in Mueller-Hinton broth (MHB; BD Biosciences) supplemented with 0.1% glucose, 2% IsoVitaleX, 0.025% ferric pyrophosphate, 0.625 mM CaCl2, and 0.530 mM MgCl2 and were incubated at 37°C with 5% CO2 as described previously (18, 19). E. coli strains were grown on Luria-Bertani (LB) agar plates or in LB broth supplemented with 10 µg of chloramphenicol/ml, 100 µg of ampicillin/ml, or 50 µg of kanamycin/ml as appropriate. F. tularensis deletion mutants and complementation plasmids were constructed as described previously (19, 21). Strain DTB3 was derived from the LVS and contains a deletion of the ftlC gene (19). Due to its greater antibiotic sensitivity, strain DTB3 was used in the present study to facilitate creation of the pilF deletion mutant. Briefly, we used an allelic exchange protocol (19, 21) to generate the pilF deletion mutant. Regions upstream and downstream of pilF were PCR amplified and ligated together into the pPV suicide vector (21), which contains markers conferring chloramphenicol resistance and sucrose sensitivity. E. coli S17-1 was used to conjugate the suicide plasmid into strain DTB3, and chloramphenicol-resistant, sucrose-sensitive colonies were screened by PCR using primer pairs both internal and external to pilF (Table 2) to verify integration of the suicide plasmid into the target gene (Fig. 2A). Colonies were then grown on medium containing sucrose to select for the second recombination event and elimination of the vector sequences. Sucrose-resistant, chloramphenicol-sensitive colonies were again screened by PCR to verify deletion of the target gene and elimination of the vector sequences (Fig. 2A). We also verified that all intermediate and final strains were F. tularensis and not contaminants by using PCR to detect the F. tularensis pilT gene. The final {Delta}pilF strain was named DTLB. The pilT::HimarFT and pilF::HimarFT transposon mutants were isolated as described in Maier et al. (37). HimarFT-based mutagenesis of F. tularensis LVS resulted in random, single, stable insertions at high efficiency.


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  		TABLE 1. Strains and plasmids used in this study


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  		TABLE 2. Primers used in this study


Figure 2
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  		FIG. 2. LVS pilF and pilT mutants. (A) Construction and validation of the DTLB ({Delta}pilF) mutant. PCR was performed with primers internal or external to the pilF gene. Lanes: 1, intermediate strain; 2, final deletion strain DTLB; 3, allelic replacement plasmid pPVLB; 4, LVS. (B and C) Schematic representations of the pilF and pilT transposon insertion mutations. (B) The HimarFT transposon inserted at bp 1169 of pilF. (C) The HimarFT transposon inserted at bp 127 of the first pilT ORF (FTL_1771). The aphA-2 gene for kanamycin resistance in the transposon is under control of the groE promoter, which could drive expression of downstream genes.

Plasmid pGPB, expressing pilF, was constructed by PCR, amplifying the gene from the LVS and cloning it into vector pFNLTP6-gro-gfp (36). This placed pilF under control of the F. tularensis groE promoter. The plasmid was transformed into DTLB by electroporation. PCR primers are described in Table 2. The PCR amplicons were purified by using a Qiagen (Valencia, CA) gel purification kit and sequenced by using a BigDye terminator V3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA). Sequences were analyzed by using the MacVector software program (Oxford Molecular, Madison, WI). The complementation plasmid p633TL, expressing pilT from the LVS, was constructed by PCR amplification of the pilT gene from the LVS (including both ORFs 1771 and 1770) with primer pairs PilTFCOM and PilTRCOM (Table 2). The PCR product was ligated into the pGEM-T Easy vector (Promega) and subsequently subcloned into vector pMP633 (35) using EcoRV and MluI. Plasmid p633TL was transformed into LVS pilT::HimarFT by electroporation. The complementation plasmid p633TN, expressing pilT from F. tularensis subsp. novicida, was constructed following the same strategy, except the PilTFCOM and PilTRCOM primers were used to amplify the pilT gene from F. tularensis subsp. novicida strain U112.

Cell lines and media. Murine bone marrow-derived macrophages (muBMDM) were obtained as described previously (7) and resuspended in bone marrow medium (Dulbecco modified Eagle medium [Invitrogen, Carlsbad, CA] containing 2 mM L-glutamine, 1 mM sodium pyruvate, 20% heat-inactivated fetal bovine serum [FBS; HyClone, Logan, UT], and 30% medium previously conditioned by L929 cells). The conditioned medium was obtained by plating 2 x 105 L929 cells in 75-cm2 culture flasks in minimum essential medium (Invitrogen) containing 2 mM L-glutamine, 1 mM sodium pyruvate, 1 mM nonessential amino acids (Invitrogen), and 10% FBS and collecting the medium after 10 days. The A549 human lung carcinoma cell line was purchased from the American Type Culture Collection (ATCC) and grown in DMEM plus 10% FBS. The murine hepatocyte cell line FL83B, derived from the normal liver of a 15- to 17-day-old fetal mouse, was purchased from the ATCC. The hepatocytes were grown in F12K medium (ATCC) plus 10% FBS.

TEM. Single bacterial colonies were inoculated into Chamberlain's defined medium, prepared as described previously (18). These cultures were incubated at 37°C in 5% CO2 with shaking at 100 rpm for 16 h. For transmission electron microscopy (TEM), bacteria were washed once with phosphate-buffered saline (PBS), adsorbed onto polyvinyl formal-carbon-coated grids (Ernest F. Fullam, Latham, NY) for 2 min, and fixed with 1% glutaraldehyde (Sigma-Aldrich, St. Louis, MO) for 1 min. The grids were washed twice with PBS and twice with water and then negatively stained with 0.5% phosphotungstic acid (Ted Pella, Inc., Redding, CA) for 35 s. The grids were viewed in a transmission electron microscope (FEI TECNAI 12 BioTwin G02) at 80-kV accelerating voltage, and images were obtained by using a AMT XR-60 charge-coupled device digital camera system.

DNA microarray. The wild-type LVS, strain DTB3 ({Delta}pilF), and the pilT::HimarFT mutant were grown to early logarithmic phase (optical density at 600 nm [OD600], ~0.2) in MHB at 37°C with 5% CO2. The cultures were harvested by centrifugation at 6,000 x g for 15 min at 4°C. Total RNA was isolated with the RNeasy Midi kit (Qiagen, Valencia, CA) by following the manufacturer's protocol. During RNA extraction, treatment with the RNase-free DNase set (Qiagen) was used to remove any DNA contamination. RNA concentration was determined by spectrophotometry (OD260), and RNA integrity was verified by agarose gel electrophoresis.

70-mer oligonucleotide microarrays representing all ORFs from SchuS4 (F. tularensis subsp. tularensis), LVS (F. tularensis subsp. holarctica), and F6168 (F. tularensis subsp. novicida) were obtained from The Institute for Genomic Research (TIGR). The microarray slides were prehybridized, washed, and dried immediately before hybridization using the protocol recommended by TIGR (http://www.tigr.org/tdb/microarray/protocolsTIGR.shtml). For hybridization, cDNA with 150 pmol Cy3 and cDNA with 150 pmol Cy5 were included in a 55 µl of hybridization solution containing 25% (vol/vol) formamide, 5x SSC buffer (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 0.1% sodium dodecyl sulfate (SDS), and 100 µg of sonicated salmon sperm DNA/ml. Hybridization was performed under lifter coverslips (25 by 60 mm; Erie Scientific) at 50°C in a humidified chamber for 16 to 20 h. Hybridized arrays were washed with gentle shaking as follows: twice briefly with 2x SSC-0.1% SDS at 50°C, twice for 10 min with 2x SSC-0.1% SDS at 50°C, twice briefly with 0.1x SSC-0.1% SDS at 50°C, twice for 10 min with 0.1x SSC-0.1% SDS, and four times briefly with 0.1x SSC at room temperature. Arrays were dried by placing them into a 50-ml Falcon tube containing tissue paper in the bottom and were centrifuged at 1,300 x g for 2 min at room temperature. Arrays were scanned by using an Axon 4000B scanner that was controlled using GenePix Pro 6 software with a pixel size of 10 µm and two-pass sequential line averaging. The laser power was set to 100%, and PMT gains were subjectively adjusted during prescanning to maximize the effective dynamic range and to limit image saturation. Lossless image files were stored for later analysis.

Microarray data was analyzed using the Limma module of the Bioconductor package for the R statistical environment (17, 62). The "normexp" method was used for background correction, followed by print tip loss normalization and between-array normalization of intensities. The microarray data for each gene was fitted to a linear model, and statistics were generated by using the lmFit and eBayes functions (17). The P values were adjusted for multiple testing using the Benjamini and Hochberg method within Limma. Genes with P values of <0.05 were considered differentially regulated. Annotations for microarray data were derived from TIGR gene array list files.

Intracellular infection experiments. muBMDM, A549, and FL83B cells were seeded in 24-well plates at concentrations of 1.5 x 105, 1 x 105, and 1 x 105 cells per well, respectively, and were used for experiments the next day. For each experiment, F. tularensis strains were streaked from frozen stocks to MHC, and a single colony was grown in Chamberlain's defined medium for 16 to 18 h at 37°C with shaking at 100 rpm in a 5% CO2 atmosphere. Aliquots of the bacterial cultures were centrifuged, resuspended in the appropriate cell culture medium, and added at a multiplicity of infection of 50, 400, or 1,000 to the muBMDM, A549, or FL83B cells, respectively. Bacterial concentrations were initially estimated by determining the OD600 of the suspension culture, and actual numbers of viable bacteria were determined by CFU counts on MHC plates. Cell culture plates were centrifuged for 5 min at 800 rpm to facilitate contact between the cells and bacteria. After 2 h of coculture at 37°C, the cells were washed extensively and incubated with 5 µg of gentamicin/ml for 1 h to kill any remaining extracellular bacteria. To measure viable intracellular bacteria, a set of wells was lysed with water at 4°C for 10 min, and serial dilutions were plated to determine the CFU. The remaining wells were incubated for a total of 24 h in the appropriate cell culture medium before determination of CFU.

Cell adherence assay. muBMDM, A549, and FL83B cells were seeded in 24-well plates as for the intracellular infection experiments. The next day, the cells were treated with 1 µg of cytochalasin D (Sigma)/ml for 1 h to inhibit the internalization of bacteria inside the cells and then thoroughly washed with PBS. Aliquots of bacterial cultures were centrifuged, resuspended in the appropriate cell culture medium, and added at a multiplicity of infection of 100, 800, or 2,000 to the muBMDM, A549, or FL83B cells, respectively. Bacterial concentrations were initially estimated by determining the OD600 of the suspension culture, and actual numbers of viable bacteria were determined by CFU counts on MHC plates. Cell culture plates were centrifuged for 5 min at 800 rpm to facilitate contact between the cells and bacteria. After 2 h of coculture at 37°C, the cells were washed extensively and lysed with water at 4°C for 10 min, and serial dilutions were plated to determine CFU.

Mouse infection experiments. For virulence studies, groups of five 6- to 8-week-old C3H/HeN mice (Charles River) were used, and four independent experiments were performed. Mice were housed under conventional conditions and allowed to acclimatize for at least 7 days before infection. LVS, DTB3, DTLB, pilT::HimarFT, and DTLB/pGPB were grown in MHB overnight to an OD600 of 0.2 and diluted in PBS, and 100 µl was injected intradermally to give infectious doses of 107 or 106 CFU per mouse. The actual infectious doses were determined by viable count. The animals were monitored for 14 days.

All animal research protocols were approved by the Institutional Animal Care and Use Committee of Stony Brook University.


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RESULTS
 
LVS pilF and pilT mutants. To investigate whether the Tfp genes in F. tularensis are involved in expression of the surface fibers observed by Gil et al. (18), we used an allelic replacement technique (21) to construct a deletion of the pilF (pilB) gene in the LVS and examined previously isolated pilT::HimarFT and pilF::HimarFT transposon mutants in the LVS (37). In characterized Tfp systems, pilF is required for pilus assembly, whereas pilT drives pilus retraction (3). To construct the pilF deletion mutant, we used strain DTB3, which is a derivative of the LVS containing a deletion of the ftlC gene (19). DTB3 is more sensitive to chloramphenicol due to reduced drug efflux, facilitating construction of the mutant. Importantly, DTB3 is not impaired for virulence in the mouse model of tularemia (see Fig. 5) and is not defective for replication in macrophages or other cell types (19) (G. J. Platz and D. G. Thanassi, unpublished data). Strain DTB3 was tested alongside the wild-type LVS in all assays used in the present study and exhibited no defects relative to the wild-type LVS. For clarity of the text, we only show results for the wild-type LVS, except for the mouse infection studies (see Fig. 5). Using strain DTB3, we were able to successfully delete pilF; this strain was designated DTLB. Proper construction of strain DTLB was validated both phenotypically and genotypically as described in Materials and Methods. Figure 2A shows the PCR verification of the deletion of pilF in DTLB. The LVS pilF::HimarFT and pilT::HimarFT transposon mutants were isolated and confirmed as described by Maier et al. (37). None of the mutant strains exhibited growth defects on solid or in liquid media (data not shown).


Figure 5
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  		FIG. 5. pilF and pilT are virulence determinants of F. tularensis. C3H/HeN mice were infected intradermally with 107 CFU (A) or 106 CFU (B) of the LVS, DTB3 ({Delta}ftlC), DTLB ({Delta}pilF), pilT::HimarFT, or DTLB complemented with pilF ({Delta}pilF/pGPB). The mice were monitored for survival for 14 days. A total of 20 mice were used for each strain and each dose.

The transposon insertion locations in pilF and pilT are shown in Fig. 2. In pilF::HimarFT, the transposon was inserted toward the end of the gene, at bp 1169 of 1782 (Fig. 2B). In pilT::HimarFT, the transposon was inserted near the beginning of the gene, at bp 127 of 1029 (Fig. 2C). PilT is thought to be composed of two major structural domains. The N-terminal domain, containing ca. 100 to 115 amino acids, is required for membrane association and polar localization of PilT (8). The C-terminal domain, ~240 amino acids, contains sequences commonly associated with NTPase activity, including the Walker A phosphate-binding (P) loop and a loosely defined Walker B box (Fig. 1) (28). The C-terminal domain also contains Asp and His boxes defined for type II and type IV secretion ATPases (28, 51, 54). PilT has low NTPase activity in vitro (24, 46), but this activity is likely important for PilT function, since mutation of the P-loop lysine prevents twitching motility (3). Beyond the ~170 amino acids that make up the ATPase core, the C-terminal ~70 amino acids also contain a well-conserved PilT-specific AIRNLIRE motif which is required for pilus retraction but not ATPase activity (Fig. 1) (3). The nonsense mutation in the LVS pilT introduces a stop codon that divides pilT into two ORFs: FTL_1771 and FTL_1770 (Fig. 1). The stop codon is followed by 60 nucleotides, which is followed by the start codon of the second predicted pilT ORF (FTL_1770). Whether this second ORF is expressed is not known, since it contains a weaker TTG start codon and no obvious preceding Shine-Dalgarno sequence. Two virulent F. tularensis subsp. holarctica strains, OSU18 and FSC200, also share this same nonsense mutation in pilT (16, 50, 55). The intergenic region between the two ORFs of pilT includes the Walker A box (Fig. 1). However, the Walker Box B, the conserved Asp and His regions, and the AIRNLIRE motif are present in the second pilT ORF. Nevertheless, loss of the Walker A box would be expected to disable the NTPase activity of the protein (5, 8).

Expression of Francisella Tfp requires pilF and pilT. Strains DTLB ({Delta}pilF), pilF::HimarFT, and pilT::HimarFT were examined for the expression of surface fibers using whole bacteria, negative-stain TEM. The LVS expressed pilus fibers as previously demonstrated (18) (Fig. 3A and B). In contrast, the {Delta}pilF strain DTLB did not express any piluslike structures (Fig. 3C and D). Complementation of DTLB with a plasmid-borne copy of pilF restored expression of the pili (Fig. 3E and F). This demonstrates that the LVS surface fibers require pilF for expression, as found in other Tfp systems (29), and suggests that the fibers are indeed Tfp. Similar to strain DTLB, pilus expression by the LVS pilF::HimarFT mutant was dramatically reduced, although a few surface fibers were occasionally observed (data not shown). This low expression of fibers suggests that the transposon insertion, which occurred toward the end of the pilF gene (Fig. 2B), did not completely disable PilF function.


Figure 3
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  		FIG. 3. Whole bacteria, negative-stain TEM of F. tularensis LVS. (A and B) The wild-type LVS expresses pilus fibers. (C and D) No fibers were detected on strain DTLB ({Delta}pilF). (E and F) Complementation of strain DTLB with plasmid pGPB (pilF+) restored the expression of pili. (G and H) The pilT::HimarFT mutant expresses no pilus fibers. (I through L) Complementation of the LVS pilT::HimarFT mutant with pilT from either the LVS (p633TL; panels I and J) or F. tularensis subsp. novicida (p633TN; panels K and L) restored the expression of pili.

We also found a complete lack of pilus fibers on the surface of the LVS pilT::HimarFT mutant (Fig. 3G and H). This was surprising for two reasons. First, in other Tfp systems, PilT is only required for pilus retraction, and mutation of pilT results in hyperpiliation rather than the loss of pili (70). Second, as discussed above, the pilT gene in the LVS already contains a premature stop codon and was expected to be nonfunctional. Thus, a transposon insertion into an already nonfunctional gene should not cause an additional loss of function. To determine whether the phenotypes of the pilT::HimarFT and the DTLB ({Delta}pilF) mutants were caused by downregulation of other pil genes or some other transcriptional effect, we performed DNA microarray analysis of the LVS, DTLB, and pilT::HimarFT strains using an array specific for the LVS and other strains of F. tularensis. None of the known Tfp structural, assembly, or regulatory genes was positively or negatively affected by the pilT or pilF mutations compared to the LVS (data not shown). As expected, message corresponding to the pilT gene in pilT::HimarFT and the pilF gene in DTLB was decreased (2.8- and 6.4-fold, respectively). Thus, the {Delta}pilF and pilT::HimarFT mutations are nonpolar, suggesting that the mutant phenotypes are directly caused by disruption of these genes. To provide additional support for the specificity of the LVS pilT::HimarFT mutation, we complemented this strain with either plasmid p633TL, containing the complete pilT gene from the LVS (both ORFs 1771 and 1770), or plasmid p633TN, containing the intact pilT gene from F. tularensis subsp. novicida strain U112. Complementation with either plasmid restored expression of the pilus fibers, as shown by negative-stain TEM (Fig. 3I through L). This confirms that pilT is required for expression of the LVS surface fibers and shows that the LVS pilT gene is functional.

Francisella Tfp contribute to host cell adherence but are not required for intracellular replication. Adherence to host epithelial tissues is an early step of bacterial pathogenesis that is required for successful colonization and subsequent infection (27, 47). Tfp serve as host cell adhesins for a number of bacterial pathogens (20, 66, 72). To investigate the role of Tfp in the interaction of F. tularensis with different cell types, we compared adhesion of the LVS, DTLB ({Delta}pilF), and the pilT::HimarFT strain to primary murine bone marrow-derived macrophages (muBMDM), A549 human lung epithelial cells, and FL83B murine hepatocytes. For these experiments, the cells were treated with cytochalasin D to inhibit internalization of the bacteria (see Materials and Methods). A recent study found that treatment of HEp-2 cells with cytochalasin D almost completely abrogated the uptake of F. tularensis (34). For each of the cell types tested, the {Delta}pilF and pilT::HimarFT mutants were significantly defective in adherence compared to the LVS (Fig. 4). Interestingly, the {Delta}pilF mutant consistently exhibited a greater defect in adherence compared to the pilT mutant, suggesting subtle differences in the phenotypes of these two mutants. Expression of pilF in trans restored the adherence of the strain DTLB back to levels similar to the wild-type LVS (Fig. 4), confirming that the adherence defect of this strain was specifically due to the the loss of pilF. These findings demonstrate that the Francisella pili contribute to host cell adherence and suggest they may mediate colonization of different tissues within the host.


Figure 4
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  		FIG. 4. pilF and pilT contribute to host cell adherence. Cells were treated with cytochalasin D to inhibit the internalization of bacteria and then infected with wild-type LVS (WT), DTLB ({Delta}pilF), DTLB complemented with pilF ({Delta}pilF/pGPB), or the pilT::HimarFT mutant. The graphs show the CFU of bacteria that adhered to murine bone marrow-derived macrophages (A), A549 human lung epithelial cells (B), and FL83B murine hepatocytes (C). Error bars indicate the standard deviation of triplicate samples. P values were calculated by using a paired t test, comparing DTLB or pilT:HimarFT with the wild-type LVS or comparing DTLB/pGPB with DTLB (*, P < 0.05; **, P < 0.02).

The ability to survive and replicate intracellularly constitutes a major virulence determinant of F. tularensis (32, 44), and we next addressed the question of whether pilF and pilT are required for growth within muBMDM, A549, or FL83B cells. Mutant strains DTLB ({Delta}pilF) and pilT::HimarFT exhibited no defects in intracellular survival and replication compared to the wild-type LVS for each of the cell types tested (data not shown). Therefore, in contrast to their role in host cell adhesion described above, the Francisella pili are not required for intracellular replication.

Tfp are virulence factors of F. tularensis. The contribution of Tfp to the virulence of Francisella was investigated using a mouse model of tularemia. Mice were inoculated intradermally with the LVS, DTB3 ({Delta}ftlC), DTLB ({Delta}pilF), or the pilT::HimarFT mutant, and the mice were monitored for survival for 14 days. In the experiment shown in Fig. 5A, mice inoculated with 107 CFU of the LVS or DTB3 (the parent strain of DTLB) began succumbing to the infection 2 to 3 days after inoculation, with no surviving mice by days 7 to 8. In comparison, DTLB ({Delta}pilF) was highly attenuated, with 70% survival on day 5 and 60% survival through day 14 (Fig. 5A). The pilT::HimarFT mutant was similarly attenuated, with 60% survival through day 14. Comparable results were obtained with an infectious dose of 106 CFU (Fig. 5B). At this dose, 90% of the mice inoculated with strain DTLB ({Delta}pilF) survived through day 14, whereas only 15 to 25% of mice inoculated with the wild-type LVS or strain DTB3 ({Delta}ftlC) survived for the duration of the study. The pilT::HimarFT mutant also exhibited attenuation at 106 CFU, with 70% survival through day 14. As noted above for the adhesion assay, subtle differences were observed between the {Delta}pilF and pilT mutants, with the {Delta}pilF mutant exhibiting a slightly higher level of attenuation, particularly at the lower infectious dose (Fig. 5B). Complementation of strain DTLB with plasmid pGPB (pilF) restored the virulence of this strain in the mouse infection model at both the 107 and the 106 CFU infectious doses (Fig. 5), confirming that the virulence defect was specifically due to loss of pilF. Overall, our results demonstrate that both pilF and pilT are important virulence determinants of the LVS and define a function for the pil genes in the pathogenesis of F. tularensis by the intradermal route.


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DISCUSSION
 
We show here, through analysis of pilF and pilT mutations in the LVS, that the surface fibers expressed by F. tularensis are encoded by the pil genes, suggesting that these fibers are Tfp. We provide evidence that the pili are important for adherence to different host cell types, and we demonstrate that the pil genes function as virulence determinants of F. tularensis.

Whole-bacteria, negative-stain TEM of the {Delta}pilF strain DTLB revealed a complete absence of surface fibers, which matches results found for Tfp systems in other bacteria upon loss of pilF (29). Thus, PilF likely serves as the Tfp assembly ATPase in F. tularensis. Similarly, the LVS pilF::HimarFT mutant had a dramatic, although not complete, loss of surface fibers. The Walker box A motif in PilF, located at residues 336 to 348, precedes the transposon insertion site, and the presence of this intact motif may explain the low activity retained in the pilF::HimarFT mutant. Taking the above findings together with the demonstration by Hager et al. that the pil genes function as a type II-like system for the secretion of soluble proteins in F. tularensis subsp. novicida (22), we propose that the F. tularensis pil genes perform dual roles, both assembling Tfp and serving as a protein secretion system. This dual function makes sense given that F. tularensis lacks many secretion systems found in other intracellular pathogens and would need to make maximum use of its limited secretion capability. The type II secretion and Tfp biogenesis machinery are closely related and share many features (49, 68). Indeed, a dual function for Tfp machinery has been established in other gram-negative pathogens. The toxin coregulated pilus of V. cholerae was shown to secrete a soluble virulence factor and the Tfp system in the ovine foot rot pathogen Dichelobacter nodosus was shown to secrete extracellular proteases (23, 30).

A striking observation made in the present study is the lack of any surface fibers in the LVS pilT::HimarFT mutant. This was surprising not only because mutation of pilT in other Tfp systems results in hyperpiliation rather than loss of pili (69) but also because pilT was already expected to be nonfunctional in the LVS due to disruption of its ORF by a premature stop codon. Nevertheless, because the pilT::HimarFT mutant failed to express Tfp and complementation with either the LVS or F. tularensis subsp. novicida pilT gene in trans restored pilus expression, it is likely that PilT is directly or indirectly required for pilus biogenesis. This paradox may be explained by several possibilities. The HimarFT transposon contains a groE promoter driving the aphA-2 gene for kanamycin resistance (Fig. 2), and this promoter could drive expression of downstream genes. However, pilT is not part of an operon, is not followed by an ORF in the same direction, and our microarray analysis found that none of the other pil genes were down- or upregulated in the pilT::HimarFT mutant. A second possibility is that the HimarFT transposon resulted in generation of a pilT protein fragment that acted in a dominant-negative manner, interacting with and titrating away other factors required for pilus biogenesis. The HimarFT insertion occurred in the first predicted pilT ORF in the LVS, at bp 127 (Fig. 2C). Thus, a protein fragment containing the region of pilT preceding the insertion could be synthesized. In addition, the aphA-2 gene in the HimarFT transposon could possibly drive expression of a fusion protein with the region of pilT downstream of the insertion. A third possibility, and one that we favor, is that pilT is functional in the LVS despite containing a premature stop codon. This possibility is supported by the fact that we could complement LVS pilT::HimarFT with the "mutated" LVS pilT gene. Thus, the LVS pilT gene is functional. The first pilT ORF (FTL_1771) in the LVS contains the N-terminal domain of PilT, which functions in membrane association and polar localization (8); this function may be important for pilus biogenesis in F. tularensis. Alternatively, there may be readthrough of the premature stop codon to produce a full-length PilT protein. We note that F. tularensis subsp. novicida contains an intact pilT gene (Fig. 1) and that the F. tularensis subsp. novicida pilT gene complemented the LVS pilT::HimarFT mutant, arguing that pilT function is preserved in the LVS despite the premature stop codon. Overall, our results show that the LVS pilT is required for pilus assembly and has evolved to perform a function distinct from or in addition to pilus retraction. Such a function for PilT implies that the paradigm of Tfp dynamics established in the P. aeruginosa and Neisseria spp. model systems may not be applicable to F. tularensis. Interestingly, a recent study in the gram-positive bacterium Clostridium perfringens also found that PilT is required for the assembly of Tfp (67).

Our studies with the LVS pilF and pilT mutants suggest that F. tularensis uses pili for adhesion to host cells. Both the {Delta}pilF and the pilT::HimarFT mutants were defective for adherence to macrophages, hepatocytes, and lung epithelial cells. In addition to adhesion, F. tularensis is capable of surviving and replicating inside these different cell types (9, 40, 53). However, the LVS pilF and pilT mutants did not exhibit any growth defects in the cell lines tested. Similar results were found for the intracellular pathogen Legionella pneumophila. The Tfp of L. pneumophila have a role in promoting adherence to mammalian and protozoan cells but are not involved in intracellular survival and replication (64). Significantly, we found that the LVS pilF and pilT mutants were attenuated for virulence in mice when inoculated by the intradermal route. These findings identify pilF and pilT as virulence factors of F. tularensis and support a role for the pili in pathogenesis. Interestingly, the LVS pilT::HimarFT mutant consistently exhibited milder adhesion and virulence phenotypes compared to the {Delta}pilF mutant, suggesting that these genes have overlapping but distinct functions. Hager et al. found that pilF (pilB), but not pilT, was required for protein secretion in F. tularensis subsp. novicida (22). Therefore, the LVS {Delta}pilF mutant may affect both Tfp expression and protein secretion, whereas the pilT::HimarFT mutant may only affect Tfp biogenesis. However, the PilE1 (PilA) potential pilin subunit was also required for protein secretion in F. tularensis subsp. novicida (22), but pilE1 is not intact in the LVS (18, 31). Thus, the protein secretion function of the pil genes may not be operational in the LVS. Future studies are needed to address the specific roles of pilF and pilT in pilus biogenesis and whether the pil genes also mediate protein secretion in the LVS.

In summary, we have established that the surface fibers expressed by the LVS are encoded by the pil genes and provided evidence that the Francisella pili play important roles in attachment to host cells and virulence in the mouse infection model. We found that, in addition to pilF, pilT was required for pilus assembly. This suggests that the pilus machinery of F. tularensis functions differently than in established model systems. Therefore, identification and characterization of the complete pilus assembly pathway in F. tularensis will impact our understanding of pilus biogenesis, as well as elucidate the molecular mechanisms that make Francisella such a successful pathogen.


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ACKNOWLEDGMENTS
 
We thank Patricio Mena and Gloria Monsalve (Stony Brook University, Stony Brook, NY) for assistance with the mouse experiments and Lance Palmer (Stony Brook University) for help with analysis of the microarray data. We thank Susan Van Horn (Stony Brook University) and the Central Microscopy Imaging Center for assistance with the TEM. We thank Martin S. Pavelka, Jr. (University of Rochester, Rochester, NY), for providing plasmid pMP633. We thank Karl Klose and Xhavit Zogaj (University of Texas San Antonio, San Antonio, TX) for critical reading of the manuscript and helpful discussions.

This study was supported by National Institutes of Health grant AI055621.


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FOOTNOTES
 
* Corresponding author. Mailing address: 242 Center for Infectious Diseases, Stony Brook University, Stony Brook, NY 11794-5120. Phone: (631) 632-4549. Fax: (631) 632-4294. E-mail: david.thanassi{at}stonybrook.edu Back

{triangledown} Published ahead of print on 21 April 2008. Back

Editor: A. Camilli


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Infection and Immunity, July 2008, p. 2852-2861, Vol. 76, No. 7
0019-9567/08/$08.00+0     doi:10.1128/IAI.01726-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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