Functional analyses of pilin-like proteins from Francisella tularensis: complementation of type IV pilus phenotypes in Neisseria gonorrhoeae

The Gram-negative facultative intracellular bacterium Francisella tularensis causes the zoonotic disease tularaemia (Mörner, 1992; Tärnvik, 1989). There are at least four subspecies of F. tularensis, each with a unique geographical distribution (Petersen & Schriefer, 2005). F. tularensis subsp. holarctica (type B) can been found in most of Europe, Asia and North America, while F. tularensis subsp. tularensis (type A) is found exclusively in North America. F. tularensis subsp. mediasiatica has been identified in Central Asia and, finally, F. tularensis subsp. novicida has been found in numerous locations in North America and in Australia (Petersen & Schriefer, 2005; Whipp et al., 2003). Human tularaemia infections are usually caused by subsp. tularensis and subsp. holarctica, the former being associated with more severe disease than the latter (Tärnvik & Berglund, 2003). The significantly less pathogenic subsp. novicida has only been reported to cause infection in immunocompromised individuals on rare occasions (Hollis et al., 1989; Tärnvik, 1989). Due to its high infectivity and capacity to cause severe illness and death, F. tularensis is considered a Category A agent of bioterrorism (Dennis et al., 2001; Khan et al., 2000). Compared to other Category A bacterial pathogens, relatively little is known about the virulence factors of F. tularensis. However, recent comparative genomic efforts (Svensson et al., 2005) and the development of genetic tools (Golovliov et al., 2003) have paved the way for progress in this research field.

The genome sequence of the virulent F. tularensis subsp. tularensis strain SCHU S4 contains several gene clusters with potential roles in virulence (Larsson et al., 2005). Amongst these are those encoding proteins related to those required for assembly and secretion of type IV pili (Tfp) (Larsson et al., 2005). Tfp are complex filamentous appendages defined by their shared structural, biochemical, antigenic and morphological features which are expressed by many human pathogens, including Neisseria species, Pseudomonas aeruginosa and Vibrio cholerae (Fullner & Mekalanos, 1999; Mattick et al., 1996; Tønjum & Koomey, 1997). Tfp play central roles in the expression of many phenotypes, including multicellular behaviours, natural genetic transformation, biofilm formation, cell signalling, motility and host cell adherence (Källström et al., 1998; Mattick, 2002; O'Toole & Kolter, 1998). Tfp biogenesis and the elaboration of associated phenotypes require the concerted actions of many proteins that are structurally conserved across species and genus boundaries (Strom & Lory, 1993). The pilus fibre is mainly composed of the major pilin subunit (called PilA in P. aeruginosa), which is expressed as a prepilin that upon cleavage by the prepilin peptidase PilD allows for proper pilus assembly and function (Strom et al., 1993). Translocation of the pilus to the cell surface is then enabled by the secretin PilQ (Wolfgang et al., 2000). In many instances, multiple proteins sharing structural similarities with the major pilin subunit, so-called minor pilins, are required for proper Tfp function and/or assembly (Alm et al., 1996a, b; Alm & Mattick, 1996; Helaine et al., 2007; Winther-Larsen et al., 2001, 2005; Wolfgang et al., 1998b). However, their role in Tfp biogenesis and function is still not completely understood. Furthermore, Tfp are dynamic structures that undergo retraction events mediated by members of the PilT retraction ATPase family (Nudleman & Kaiser, 2004; Skerker & Berg, 2001; Whitchurch et al., 1991). Rounds of Tfp extension and retraction mediate so called twitching motility on solid and semisolid surfaces (Mattick, 2002), and susceptibility to pilus-specific phages (Bradley & Pitt, 1974; Waldor & Mekalanos, 1996).

Many components part of the type II protein secretion system (T2SS) share remarkable similarities with those involved in Tfp expression (Peabody et al., 2003) and, if overexpressed, T2SS pilin-like molecules can polymerize into Tfp-like structures (Durand et al., 2003; Vignon et al., 2003). Despite these similarities, functional interactions between these two systems have only been reported in one instance (Lu et al., 1997). Nonetheless, Tfp systems in V. cholerae and Dichelobacter nodosus have been implicated in the translocation of folded proteins from the periplasm to the extracellular milieu (Han et al., 2008; Kirn et al., 2003).

A number of findings suggest that Francisella Tfp-associated genes are involved in pathogenesis. A spontaneous mutant of F. tularensis subsp. holarctica with a deletion within a pilin-like gene locus showed reduced virulence in mice, and a similar rearrangement at this particular locus is present in the subsp. holarctica live vaccine strain (LVS) (Forslund et al., 2006). Interestingly, secretion of multiple proteins in F. tularensis subsp. novicida was abolished by mutations in genes predicted to encode Tfp and ancillary factors (Hager et al., 2006), suggesting that Tfp in this subspecies might also be associated with a T2SS as in D. nodosus and V. cholerae (Han et al., 2008; Kirn et al., 2003). Surprisingly, these mutants revealed increased virulence in an animal model (Hager et al., 2006). These perplexing results were reconciled by the observation that PepO, one of the secreted substrates, is a zinc protease that promotes increased vasoconstriction and potentially inhibits tissue spread and dissemination. Interestingly, PepO appears to be absent from the human-virulent Francisella lineages.

Central questions yet to be resolved for Francisella species are what components encompass the pilus machinery and which protein constitutes the major structural subunit. For the LVS, one report concluded that it expresses pilus-like appendages, and many of the genes whose products are predicted to be involved in Tfp biogenesis were shown to be transcribed (Gil et al., 2004). More recently, null mutations in genes encoding a putative Tfp assembly ATPase (PilB, P. aeruginosa nomenclature) and a homologue for PilT were shown to prevent the formation of Tfp-like organelles in the LVS (Chakraborty et al., 2008). Importantly, however, studies using the LVS are complicated by the fact that it carries mutations in genes encoding strong candidates of Tfp pilin subunits (based on structural similarities and synteny) (Forslund et al., 2006). Tfp-like appendages have also been observed in F. tularensis subsp. novicida, and their expression was shown to depend on the putative assembly and retraction ATPase genes (Zogaj et al., 2008). Intriguingly, mutants lacking homologues of components (PilC and PilQ, P. aeruginosa nomenclature) critical to surface expression of Tfp in other pathogens such as N. gonorrhoeae and P. aeruginosa were unaltered in organelle expression, but defective in protein secretion and virulence. Conversely, a mutant defective in expression of the pilin-like protein termed PilE4 retained protein secretion and virulence, but lacked Tfp-like organelles (Zogaj et al., 2008). The authors concluded that Tfp expression and protein secretion can be dissociated in subsp. novicida and that PilE4 is probably the major subunit of Tfp. Still, conclusive evidence as to which protein constitutes the major structural subunit has yet to be presented.

In order to address some of these issues, we employed a strategy in which pilin genes from F. tularensis were expressed in N. gonorrhoeae. Based on the extensive similarity of pilins from different species and the highly conserved nature of the assembly machineries, trans-species complementation has been achieved (Elleman et al., 1986; Hoyne et al., 1992; Sauvonnet et al., 2000; Winther-Larsen et al., 2007). Our results strongly suggest that one particular class of pilin protein from subsp. tularensis and its counterpart from subsp. novicida is capable of forming Tfp-like appendages and complementing the Tfp-dependent phenotype of natural genetic transformation in a N. gonorrhoeae pilin subunit mutant.

Bacterial strains, plasmids and growth conditions.
The F. tularensis, Escherichia coli and N. gonorrhoeae strains used in this study are listed in Table 1. F. tularensis strains were grown on modified Thayer–Martin agar plates (Sandström et al., 1984) and N. gonorrhoeae strains in GC medium or on GC agar (Freitag et al., 1995) plates, both at 37 °C in a 5 % CO₂ atmosphere. E. coli strains were grown on blood agar base (BAB; Merck) plates or in Luria–Bertani broth (LB) at 37 °C. Where appropriate, antibiotics were used at the following concentrations; kanamycin 50 µg ml^–1, chloramphenicol 10 µg ml^–1 and erythromycin 8 µg ml^–1. Preparation of plasmid DNA, restriction enzyme digests, ligations and transformations into E. coli were performed essentially as described by Sambrook et al. (1989).

Table 1. Strains and plasmids used in this study

Construction of translational fusions.
The gonococcal pilE gene (also denoted pilE_GC) was fused in-frame to the Francisella pilin genes (denoted by subscript Ft for F. tularensis subsp. tularensis and Fn for F. tularensis subsp. novicida) pilA_Ft, pilE_Ft, pilV_Ft, FTT0861_Ft, FTT0230_Ft, FTT1314_Ft and pilA_Fn at the glycine–phenylalanine junction by overlap extension (SOEing) PCR using primers pilE_GC_F, pilE_GC_R1-7, pilA_Ft/pilE_Ft/pilV_Ft/FTT0861_Ft/FTT0230_Ft/FTT1314_Ft/pilA_Fn_F2 and pilA_Ft/pilE_Ft/pilV_Ft/FTT0861_Ft/FTT0230_Ft/FTT1314_Ft/pilA_Fn_R1, where pilE_GC_F and pilA_Ft/pilE_Ft/pilV_Ft/FTT0861_Ft/FTT0230_Ft/FTT1314_Ft/pilA_Fn_R1 have complementary sequences (see Supplementary Table S1, available with the online version of this paper). For N. gonorrhoeae, pilE_GC plasmid pPilE2 was used as template and for Francisella, strain FSC237 (subsp. tularensis, SCHU S4) or FSC040 (subsp. novicida, U112). The fusion proteins carry the first seven residues of gonococcal PilE_GC while the remaining protein is Francisella derived. The amplified fragments were digested with Bsu36I and StuI and subcloned into p2/16/1, generating plasmids pEMS25–31. The plasmids were then introduced into gonococcal strains MW24, KS101, GP111 and KS79 by transformation and selection on GC agar plates containing erythromycin as previously described (Freitag et al., 1995). Direct sequencing of PCR products derived from transformants was performed using the primers p2/16/1_5' and p2/16/1_3' to ensure correct sequences (Eurofins MVG Operon).

Construction of T2SS pseudopilin transcriptional and translational fusions.
The gonococcal pilE_GC gene was fused in-frame to the Klebsiella oxytoca pulG gene and the P. aeruginosa PAK xcpT gene at the translational start site or at the pilin peptidase cleavage site by SOEing PCR. The upstream pilE_GC region was amplified from plasmid pPilE2 using primer 266 (5'-pilE_Sac/Bam) together with HC2 (pilE_GFpulG), HC7 (pilE_ATGpulG), HC12 (pilE_GFxcpT) and HC21 (pilE_ATGxcpT) (Table S1). The downstream region was amplified from plasmid pCHAP231 using primers HC4 (pilE_GFpulG) and HC6 (pilE_ATGpulG) together with primer HC9. Primers HC2 and HC7 had overlapping sequences with primers HC4 and HC5, respectively. The two PCR products were joined together using the flanking primers 266 and HC10. For xcpT, the downstream region was amplified from plasmid pMTWT using primers HC13 (pilE_GFxcpT) and HC19 (pilE_ATGxcpT) together with HC24. Primers HC12 and HC21 had overlapping sequences with HC13 and HC19, respectively. The two PCR products were joined together using the flanking primers 266 and HC23. The amplified products were cloned into pCRII-TOPO (Invitrogen) giving rise to plasmids pHW10–13. Plasmids pHW10–13 were digested with SacI, and the DNA fragments containing the pilE : : pulG or pilE : : xcpT fusions were cloned into plasmid p2/16/1 digested with the same enzyme. The resulting plasmids pHW14–17 were introduced into gonococcal strains by transformation and selection on GC agar plates containing erythromycin. Plasmids were sequenced at GATC Biotech (Konstanz, Germany) using custom primers. PulG and XcpT expression were verified by immunoblotting using antibodies specific for PulG or XcpT.

Gel electrophoresis and Western blot analysis.
Protein samples containing SDS and β-mercaptoethanol were boiled for 5 min. All samples were separated by SDS-PAGE on 12 % or 15 % gels (Laemmli, 1970). Proteins were transferred to Immobilon-P Transfer Membranes (Millipore) using a Trans-Blot Semi-Dry transfer cell (Bio-Rad). Membranes were blocked in Tris-buffered saline (TBS) with 5 % non-fat dry milk. Immunoblotting was performed with polyclonal rabbit antibodies against PilA_Ft (Forslund et al., 2006), PilV_GC (Winther-Larsen et al., 2001), PulG, XcpT and Tfp from the gonococcal strain N400 (lot 904) (Winther-Larsen et al., 2007). P. aeruginosa PAK PilA-specific serum was a gift from E. Gotschlich (Rockefeller University). K. oxytoca PulG-specific serum was a gift from A. Pugsley (Pasteur Institute, Paris) and P. aeruginosa XcpT-specific serum was a gift from A. Filloux (IBSM-CNRS, Marseille Cedex). For the alkaline-phosphatase-conjugated secondary antibody system (Roche), the visualization was accomplished by incubating the filters with 0.1 % (w/v) NBT (nitro blue tetrazolium; Sigma) and 0.05 % (w/v) BCIP (5-bromo-4-chloro-3-indolyl phosphate; Sigma).

Immunofluorescence, immunogold and transmission electron microscopy.
Immunofluorescence microscopy was performed as described previously (Winther-Larsen et al., 2005). The pili were labelled using gonococcal PilE_GC specific serum (lot 904). Samples for transmission electron microscopy were prepared as published (Winther-Larsen et al., 2007) and viewed in a Hitachi (Tokyo) HU-11E-1 electron microscope. Immunogold labelling was carried out essentially as described previously (Winther-Larsen et al., 2007) using rabbit antiserum specific for PilE_GC (lot 904). Images shown correspond to fields that were representative of the overall observation. For statistical analysis the percentage of piliation was calculated from at least five sets of experiments in which the number of pili was counted per 600 gonococcal cells.

Tfp purification, transformation and twitching motility assay.
Transformation assays were carried out as described previously (Aas et al., 2002). Pili were purified by the ammonium sulphate procedure of Wolfgang et al. (1998a), with modifications (Winther-Larsen et al., 2007). Twitching motility assays were carried out in phenol-red-free DMEM (Gibco), supplemented with 2 mM L-glutamine, 8 mM sodium pyruvate, 5 mM ascorbic acid, 30 mM HEPES and 1 mg BSA ml^–1 at 37 °C on glass coverslips (Maier et al., 2004).

The repertoire of Tfp-related pilin genes in Francisella genomes
The genome sequence of the F. tularensis subsp. tularensis virulent strain SCHU S4 contains multiple ORFs similar to genes encoding components involved in Tfp biogenesis and Tfp structures in other bacterial species (Larsson et al., 2005). These ORFs include six genes whose predicted products have features typical for Tfp prepilins. Genome comparisons between different strains and subspecies of Francisella reveal several differences in the putative Tfp pilins between subsp. tularensis and subsp. holarctica (Fig. 1a). One locus containing three tandemly arrayed putative prepilin genes (pilA_Ft, pilE_Ft and pilV_Ft) is rearranged in some avirulent subsp. holarctica strains, including the live vaccine strain (Forslund et al., 2006; Svensson et al., 2005). These rearrangements are associated with an in-frame deletion spanning the pilA_Ft gene and the N-terminal-encoding part of pilE_Ft (Svensson et al., 2005). Furthermore, the residual pilE_Ft and pilV_Ft genes are predicted to be pseudogenes in subsp. holarctica due to non-sense mutations (Fig. 1a). In addition, internal deletions and a mutation within the stop codon change the reading frame of FTT0861_Ft (Larsson et al., 2005) (Fig. 1a), thereby extending the ORF in subsp. holarctica. The two remaining prepilin-like ORFs, FTT0230_Ft and FTT1314_Ft, are identical between subspp. tularensis and holarctica.

(31K): Fig. 1. Comparison of gene clusters encoding putative Tfp pilins in F. tularensis subspecies tularensis, holarctica and novicida. (a) All F. tularensis subspecies have direct repeats (DRs) within the N-terminal part of the pilAFt and pilEFt genes (solid arrows). In some subsp. holarctica strains, the DRs have mediated deletion of the pilAFt gene. pilEFt and pilVFt are not functional in subsp. holarctica due to non-sense mutations (arrowheads), and the FTT0861Ft pilin gene has an altered and extended reading frame due to a deletion event (Δ signs) causing mutations within the stop codon. (b) Alignment of the amino acid sequences of F. tularensis subsp. tularensis PilAFt and subsp. novicida PilAFn. The N-terminal 36 amino acids are identical while there are significant differences in the C-terminal part of these proteins. The solid arrow indicates the cleavage site of the prepilin.

Within the genome of subsp. novicida strain U112 (Rohmer et al., 2007), most of the Tfp-related genes are found to be similar to the Tfp genes in the highly virulent subsp. tularensis strain SCHU S4. However, one prominent difference is in the F. tularensis equivalent pilA, pilE and pilV gene cluster. Here, the C-terminal half of the first gene, pilA_Fn (FTN_0415), is significantly different, while the N-terminal part remains homologous to SCHU S4 pilA_Ft (Fig. 1b). In addition, some sequence differences are also seen in the intergenic regions of the pilAEV_Fn cluster (Forsberg & Guina, 2007).

Expression of Francisella Tfp-like pilins in N. gonorrhoeae
Owing to the conservation and promiscuity of the biogenesis machineries, expression of Tfp pilins in heterologous species has been successfully used to analyse pilus structure–function relationships (Elleman et al., 1986; Hoyne et al., 1992; Sauvonnet et al., 2000; Winther-Larsen et al., 2007). Here, we examined the propensities for the putative Francisella pilins to form Tfp in the heterologous Tfp expression system of N. gonorrhoeae, conditionally lacking the endogenous pilin subunit PilE (also denoted here as PilE_GC). To avoid potential problems related to heterologous expression levels, the Francisella pilin ORFs from subsp. tularensis strain SCHU S4 and pilA_Fn from subsp. novicida strain U112 were fused so as to create a glycine–phenylalanine junction to PilE_GC. Thus, the resulting translational fusion proteins carried the first seven residues of PilE_GC, but when processed by the PilD prepilin peptidase, the mature proteins are predicted to be entirely Francisella-derived. Despite repeated efforts, a translational fusion encoding the subsp. tularensis pilin FTT1314_Ft could not be obtained in N. gonorrhoeae due to accumulation of spontaneous mutations following transformation into N. gonorrhoeae. Immunoblot analysis verified that the remaining pilins, except for FTT0861_Ft, were expressed in the N. gonorrhoeae background (Fig. 2). Direct sequencing from genomic DNA, however, verified that the FTT0861_Ft clone was correct. All other pilins migrated as expected based on their calculated molecular masses, and the relative intensities of the signals varied between the antibody probes. The anti-PilE_GC antibodies were raised against the pilin subunits isolated from N. gonorrhoeae strain N400, and in a previous study it was suggested that the ability of heterologous Tfp antibodies to detect pilin subunits in immunoblotting involves exposure of a highly conserved epitope within or near the mature N-terminus following PilD cleavage (Patel et al., 1991). The PilA_Ft antibody was raised against the C-terminal part of the PilA_Ft protein (Forslund et al., 2006). Hence, this antibody is specific for the PilA_Ft pilin and would not recognize other pilins.

(42K): Fig. 2. Expression of putative Francisella pilin genes in N. gonorrhoeae. Total bacterial lysates were separated by SDS-PAGE under denaturing conditions and proteins were identified by immunoblotting using anti-PilAFt (upper panel) or anti-PilEGC (lower panel) antibodies. Strains used (lanes from left): MW24 (pilEGC); GE2 (iga : : pilEGC), KS59 (iga : : pilAFt), KS60 (iga : : pilEFt), KS61 (iga : : pilVFt), KS62 (iga : : FTT0861Ft), KS63 (iga : : FTT0230Ft), KS64 (iga : : pilAFn).

PilA from F. tularensis subsp. tularensis and subsp. novicida supports formation of Tfp-like structures in N. gonorrhoeae
As a first approach, transmission electron microscopy (TEM) was used to examine if the putative Francisella pilins could form pilus-like filaments in N. gonorrhoeae. Structures resembling Tfp were only observed for the PilA_Fn-expressing strain (KS64) (Fig. 3) and at levels well below those seen for endogenous Tfp. In addition, these structures appeared as single filaments compared to the pilus bundles usually observed for wild-type gonococcal cells. Next, surface exposure of the pilin-like proteins was examined by immunofluorescence microscopy (IFM) using antibodies shown to be reactive in immunoblotting. Although no immunolabelling was detected using rabbit antibodies raised against PilA_Ft, antibodies raised against wild-type pili from N. gonorrhoeae strain N400 revealed the presence of Tfp-like appendages in strains expressing either PilA_Fn (KS64) or PilA_Ft (KS59) (Fig. 4a). No immunoreactive filaments were detected in the strains expressing PilE_Ft (KS60), PilV_Ft (KS61), FTT0861_Ft (KS62) or FTT0230_Ft (KS63) (Table 2 and IFM not shown). The immunoreactive filaments seen using the N400 Tfp antiserum in cells expressing PilA_Fn and PilA_Ft differed from those seen in the wild-type N. gonorrhoeae background. First, the numbers of fluorescent appendages were considerably fewer in the Francisella PilA-expressing backgrounds (Table 2). Second, the appendages were longer than the short pilus bundles observed in the wild-type background. To determine if the filaments seen for Francisella PilA by IFM were directly related to those detected by TEM, immunogold-labelling TEM using the N400 PilE_GC antiserum was carried out. As the appendages were specifically decorated with gold particles (Fig. 4b), we propose that the structures observed by TEM are the same as those detected by IFM.

(181K): Fig. 3. Piliation of wild-type and mutant gonococcal strains expressing pilA from Francisella subsp. tularensis and subsp. novicida, analysed by negative-stained TEM. Strains: GE2 (wt, iga : : pilEGC); KS59 (iga : : pilAFt) and KS64 (iga : : pilAFn).

(71K): Fig. 4. IFM of N. gonorrhoeae expressing various pilin genes from F. tularensis. Gonococcal cells (green) and Tfp-like structures (red) were detected using indirect IFM. Anti-PilEGC specific antibodies (lot 904) were used to detect the Tfp-like structures. Strains: GE2 (iga : : pilEGC), MW24 (pilEGC), KS59 (iga : : pilAFt) and KS64 (iga : : pilAFn). Note that the appearance of Tfp seen here is quite similar to that seen using the same type of technique for E. coli cells expressing the PulG pseudopilus as bundles (Vignon et al., 2003). (b) Piliation of gonococcal strain KS64 (iga : : pilAFn) detected by immunogold labelling and TEM using anti-PilEGC (lot 904) antibodies (10 nm gold particles).

Table 2. Francisella PilA pilin supports genetic transformation but not twitching motility in N. gonorrhoeae

In an attempt to confirm the composition of the structures seen for the PilA_Ft and PilA_Fn backgrounds, we carried out standard pilus purification schemes (Winther-Larsen et al., 2007; Wolfgang et al., 1998a). However, no pili were recovered using these protocols. It has previously been demonstrated that levels of detectable P. aeruginosa PilA_PAK-derived pili expressed in N. gonorrhoeae can be dramatically increased in a mutant background lacking the PilT retraction ATPase (Winther-Larsen et al., 2007). However, the use of this background in these instances had no influence on the levels of appendages detected by IFM, TEM or purification attempts (Supplementary Figs S1 and S2 and data not shown, respectively).

Evidence for Francisella PilA protein glycosylation in N. gonorrhoeae
Although epitope sharing between unrelated Tfp pilins has been documented in Western-type immunoblotting experiments using heterologous anti-Tfp antisera, it seemed curious that antibodies to strain N400 Tfp would react with the PilA_Fn- and PilA_Ft-associated surface structures in N. gonorrhoeae. A recent study documented that immunization of rabbits with endogenous N400 Tfp results in antibodies that recognize epitopes associated with the galactose-2,4-diacetamido-2,4,6-trideoxyhexose (Gal-DATDH) disaccharide linked at serine 63 of PilE_GC (Vik et al., 2009). There is also indirect evidence that PilA_Ft is post-translationally modified in its endogenous host (Forslund et al., 2006). To determine if PilA_Ft and PilA_Fn might be O-glycosylated in N. gonorrhoeae, we examined the relative motilities of the proteins in defined N. gonorrhoeae protein glycosylation (pgl) mutants and variants (Aas et al., 2007a). Null mutations in core genes essential for protein glycosylation (pglC, pglD, pglF and pglO) in N. gonorrhoeae were associated with an increase in PilA_Fn migration, while in a pglA mutant background in which only the DATDH monosaccharide is linked to proteins, the migration was intermediate between that seen in the wild-type and null mutants (Fig. 5a). Furthermore, dramatically retarded migration was observed in a pglE_ON variant background in which a Gal-Gal-DATDH trisaccharide is linked to proteins. Virtually identical results were seen for the behaviour of PilA_Ft, with the exception of the pglE_ON variant background, in which four distinct antigenic forms migrating more slowly than the form seen in the glycosylation null mutants were detected. Taken together, these findings suggest that Francisella PilA proteins undergo glycosylation in N. gonorrhoeae. We then tested the potential influence of protein glycosylation on the ability of the antibodies to react with the PilA-associated appendages by IFM. For both the Francisella PilA_Fn and PilA_Ft proteins, the glycosylation null mutation pglC abrogated the labelling of the structures (Fig. 5b). However, the absence of PglC did not affect surface organelle expression, as Tfp-like structures were still visible by TEM in the PilA_Fn-expressing pglC mutant strain (Fig. 5c).

(44K): Fig. 5. Influence of the N. gonorrhoeae glycosylation mutants on PilAFt and PilAFn. (a) Influence on relative migration of PilAFt and PilAFn. Immunoblot of whole-cell lysates using anti-PilAPAK antibodies. The strains used were as follows. Upper panel: first lane MW24 (pilEGC), and then iga : : pilAFn (KS64), iga : : pilEGC (GE2), pglC (KS183), pglD (KS185), pglA (KS181), pglI (KS191), pglEON (KS187), pglF (KS189), pglO (KS193). Lower panel: first lane MW24 (pilEGC), and then iga : : pilAFt (KS59), iga : : pilEGC (GE2), pglC (KS182), pglD (KS184), pglA (KS180), pglI (KS190), pglEON (KS186), pglF (KS188), pglO (KS192). (b) Influence on the cross-reactivity of Francisella PilA pilins with anti-PilEGC antibodies (lot 904). Gonococcal cells (green) and Tfp-like structures (red) were detected using indirect IFM. Strains: iga : : pilAFt (KS59), iga : : pilAFt, pglC (KS182), iga : : pilAFn (KS64), iga : : pilAFn, pglC (KS183). Note that the appearance of Tfp seen here is quite similar to that seen using the same type of technique for E. coli cells expressing the PulG pseudopilus (Vignon et al., 2003). (c) Piliation analysis by negative-stained TEM of strain iga : : pilAFn, pglC (KS183). Scale bars, 200 nm.

PilA from F. tularensis subsp. tularensis and subsp. novicida supports competence for natural genetic transformation in N. gonorrhoeae
It has previously been demonstrated that the expression of P. aeruginosa pilin PilA_PAK can partially complement a defect in competence for natural transformation in a gonococcal strain lacking its endogenous pilin (Aas et al., 2002). Using the same strategy, we examined whether Francisella pilins could function in competence for transformation in an otherwise unpiliated N. gonorrhoeae background. Only the strain expressing PilA_Fn from subsp. novicida (KS72) supported transformation levels just above that seen for the unpiliated control strain (Table 2). It should be noted that compared to growth on plates, strains expressing PilV_Ft (KS69) and pilin FTT0230_Ft (KS71) grew poorly in liquid culture, a property which may have a negative impact on the transformation phenotype for these strains. In a study by Aas et al. (2002), two pilin-like proteins, ComP_GC and PilV_GC, were shown to influence the transformation levels, but not Tfp expression, in N. gonorrhoeae. Whereas ComP_GC is essential for natural transformation, and its elevated expression leads to enhanced transformation frequencies, PilV_GC inhibits transformability by antagonizing ComP function. As such, pilV_GC mutants phenocopy ComP-overexpressing strains (comP_oe). When tested in N. gonorrhoeae comP_oe or pilV_GC backgrounds, the expression of PilA_Fn led to an over 500-fold increase in transformation frequency (Table 2). PilA_Ft expression also led to an increase in transformability, but at more modest levels (20–40-fold). None of the other four Francisella pilins altered transformability in either of the two backgrounds. As noted previously, the expression of both PilV_Ft (KS75, KS83) and FTT0230_Ft (KS77, KS85) was associated with poor growth in liquid culture, which could potentially also affect the results here.

The three sets of transformation experiments described above were all conducted in a genetic background where the expression of the endogenous gonococcal pilin was under the control of the lac promoter, although non-induced. Studies have shown that very low levels of PilE_GC are sufficient for high-level transformability (Aas et al., 2007b; Long et al., 2001). Therefore, it was possible that physiological alterations associated with high-level expression of Francisella pilins might perturb levels of LacI repressor, allowing residual expression of PilE_GC. To address this concern, the PilA_Fn- and PilA_Ft-expressing pilV_GC strains were re-engineered so that the inducible pilE_GC allele was disrupted by a chloramphenicol-resistance gene cassette (Hegge et al., 2004). The transformation frequency was essentially unaltered in this background compared to the pilV_GC inducible pilE_GC parent strains (Table 2) proving that residual PilE_GC expression could not account for the enhanced transformation frequencies.

As dynamic polymers, Tfp undergo rounds of extension and retraction events that are commonly a prerequisite for Tfp-associated genetic transformation (Wolfgang et al., 1998a). In N. gonorrhoeae, the ability to undergo twitching motility on surfaces is directly correlated with pilus retraction activity (Merz et al., 2000). However, none of the Francisella pilins supported twitching motility when assessed by microscopic examination for movement at the periphery of colonies or by the slide culture method (Table 2).

Expression of the T2SS pseudopilin XcpT from P. aeruginosa or PulG from K. oxytoca does not support genetic transformation in N. gonorrhoeae
Both the transformation and microscopy data noted above support the hypothesis that PilA_Ft and PilA_Fn are Tfp pilins. It is important to note, however, that T2SS proteins of the PulG family can, when overexpressed, result in the formation of pseudopili with an appearance similar to Tfp (Durand et al., 2003; Vignon et al., 2003). Few studies, if any, have explored whether the expression of T2SS pseudopilins can support Tfp-associated functions. To examine this possibility in more detail, the T2SS proteins PulG and XcpT from K. oxytoca and P. aeruginosa, respectively, were cloned and expressed in N. gonorrhoeae. Like the Francisella pilins, both were fused at the prepilin peptidase processing site in addition to fusions generated at the putative ATG translational start codons. The expression of both PulG and XcpT in N. gonorrhoeae was verified using antibodies specific for each protein (Supplementary Fig. S3). The transformation assay revealed that neither PulG nor XcpT supported genetic transformation in N. gonorrhoeae in either a wild-type or pilV_GC mutant background (Table 2). Moreover, no pilus-like filamentous structures could be detected by TEM (data not shown).

Expression of Francisella PilA-associated appendages depends on Tfp biogenesis factors
The expression of Tfp depends on several canonical Tfp assembly factors (Carbonnelle et al., 2006; Martin et al., 1995; Nudleman et al., 2006) that are unique to Tfp systems as they are lacking in T2SSs. We expressed both PilA_Ft and PilA_Fn in a selection of mutant backgrounds lacking such components, including PilF_GC, PilM_GC or PilN_GC, to investigate whether the Francisella-associated filament requires the endogenous N. gonorrhoeae Tfp assembly machinery. PilM_GC and PilN_GC are Tfp assembly factors that act in unknown capacities (Carbonnelle et al., 2006), while PilF_GC (PilB in P. aeruginosa) is believed to be the subunit polymerization ATPase (O'Toole & Kolter, 1998). Pilus expression was abolished in all three biogenesis-defective mutant backgrounds (Supplementary Fig. S4).

As previously shown, homologues of Tfp structural and biogenesis components influence the pathogenesis of F. tularensis subsp. holarctica and subsp. novicida infections (Forslund et al., 2006; Zogaj et al., 2008). There are clear polymorphisms and gross differences in the content of genes encoding these components that correlate with the relative virulence of Francisella species (Forslund et al., 2006). However, there remains a large gap in our knowledge of how these components function in shaping pathogenic potential. With the aim of beginning to resolve these issues, we conducted trans-species complementation studies and found that one particular class of pilin-like proteins in subspp. tularensis and novicida can complement Tfp-related features and functions in a strain of N. gonorrhoeae lacking its endogenous pilin. Despite their orthologous nature, however, PilA_Ft and PilA_Fn behaved differently in this background. While expression of both pilins individually was associated with the expression of Tfp-like appendages detectable by IFM, the levels of the appendages were lower in the PilA_Ft-expressing strain. Consistent with these differences, TEM detected Tfp-like organelles only in the PilA_Fn-expressing strain. These findings were paralleled by dramatic differences in the abilities of these two pilin-like proteins to support competence for natural genetic transformation. In backgrounds optimized to enhance DNA binding and uptake, transformability in the PilA_Fn-expressing strain was increased nearly 1000-fold relative to the negative control background and 50-fold relative to the isogenic PilA_Ft-expressing strain. Interestingly, competence for transformation has been reported for subsp. novicida (Gallagher et al., 2008; Tyeryar & Lawton, 1970). The significance of this results in relation to our data is difficult to interpret since the components required for DNA uptake in subsp. novicida have yet to be characterized. It is important to note here that all available data back the notion that transformability in N. gonorrhoeae absolutely requires an intact Tfp extension/retraction system including a major pilin subunit (Wolfgang et al., 1998a). We hypothesize accordingly that the appendages seen by IFM and TEM are in fact filamentous polymers made up of PilA_Fn and are structural equivalents of bona fide Tfp. This proposal is supported by the fact that antibodies raised against endogenous Tfp (composed of PilE_GC) that detect PilA_Fn protein in immunoblotting also react with the PilA_Fn-associated structures as seen by IFM and TEM. This proposal is further backed by the observations that these structures are not seen in cells individually lacking PilM_GC, PilN_GC and PilF_GC, which are associated specifically with the Tfp assembly system, as they have no counterparts in related T2SSs.

A shortcoming in determining if the structures seen in gonococci are Tfp has been the inability to purify the appendages and characterize their composition. This same situation confounds previous studies examining the relationships between Tfp-related components and the expression of extracellular appendages in both F. tularensis subsp. holarctica and subsp. novicida. Zogaj et al. (2008) identified pilus-like appendages in subsp. novicida and also studied the presence or absence of these structures in a variety of mutant backgrounds. Based on indirect genetic data, the authors proposed that PilE4 (orthologous to what is termed FTT0861 in this work) is the major structural subunit of Tfp. Somewhat surprisingly, the appendages were seen independently of the expression of homologues of the PilQ secretin and PilC inner-membrane protein families (P. aeruginosa nomenclature) that are essential for Tfp expression in most species. Similar studies of the live vaccine strain, LVS (subsp. holarctica), have concluded that this strain also expresses Tfp-like structures (Chakraborty et al., 2008). It is difficult to assess the potential relevance/applicability of our results to the work with the LVS, as that strain carries a mutation that inactivates expression of PilA_Ft, the pilin-like protein we examined. In the case of subsp. novicida, however, our findings increase the possibility that PilA_Fn is the major structural component of Tfp. All studies so far have failed to purify and structurally characterize these Tfp-like structures. Therefore, it is not possible to establish which pilin is the major subunit of Tfp in the different subspecies and strains studied. However, our results provide evidence that pilin-like proteins from Francisella are capable of interacting with components of an established Tfp system. Additionally, comparative studies of these particular systems have a unique potential to increase our understanding of the basic genotype–phenotype relationships underlying Tfp-related biology. For example, F. tularensis subspecies appear to lack a homologue of the canonical PilM Tfp assembly factor present in virtually all other type IVa pilin Tfp-expressing species (Gil et al., 2004; Larsson et al., 2005). Moreover, the pilT genes (encoding the pilus retraction ATPase in Tfp systems) in subsp. holarctica strains were reported to carry identical nonsense mutations leading to a truncated protein which, based on all available data on this class of ATPases, would lead to disrupted function (Satyshur et al., 2007). Similarly, the pilE4 genes (orthologous to the gene encoding FTT0861) of subsp. holarctica strains carry identical frame-shift mutations leading to dramatic structural alterations in the C-terminal segment of the ORF. These latter findings are consistent with the idea that the functions served by the corresponding products are no longer associated with a fitness advantage to these strains and that the genes are undergoing decay. Thus, changes in Tfp-related genes and their products may be implicated in evolutionary events within Francisella species.

We are indebted to A. Pugsley (Pasteur Institute, Paris) and A. Filloux (IBSM-CNRS, Marseille Cedex) for the gift of plasmids and antibodies. This work was supported by funds from the Research Council of Norway Functional Genomics initiative (FUGE) directed through The Consortium of Advanced Microbial Sciences and Technologies (CAMST) (M. K. and H. C. W.-L.), the Swedish Research Council (Å. F.) and the Kempe Foundation (E. S.). We thank Åshild Vik, Jennifer Ritchie, Moa Lavander and Jeanette Bröms for critical reading of the manuscript.

Edited by: L. S. Frost

Footnotes

†Present address: Department of Food Safety and Infection Biology, Norwegian School of Veterinary Science, Post Box 8146, 0033 Oslo, Norway.

A supplementary table of primers and four supplementary figures are available with the online version of this paper.