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and
H. Steven Seifert*
Northwestern University, Feinberg School of Medicine, Department of Microbiology-Immunology, Chicago, Illinois
Received 6 December 2006/ Accepted 29 January 2007
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Tfp expression is essential for N. gonorrhoeae pathogenesis, and only piliated bacteria are recovered from gonorrhea patients (36). Tfp are required for a variety of functions related to pathogenesis, including twitching motility, autoagglutination, and adherence to host epithelium (24, 40, 41). Tfp are also involved in high levels of natural DNA transformation in N. gonorrhoeae (32).
In Neisseria, the major Tfp subunit, pilin, is encoded by pilE. After being expressed in the cytosol, premature pilin subunits are transported across the inner membrane and are then cleaved by the prepilin peptidase PilD (15). Fully functional Tfp are dynamic structures that lengthen and retract via pilin polymerization and depolymerization, respectively. Two inner membrane-associated ATPases, PilF and PilT, are thought to antagonistically promote this extension and retraction, with PilF being involved in pilus elongation (15) and PilT being necessary for pilus retraction (26, 40). The portal though which the pilus extends or retracts across the outer membrane (OM) is PilQ (42).
PilQ belongs to a superfamily of OM proteins called secretins that function as large, surface-exposed, homooligomeric rings involved in secretion of intracellular macromolecules across the cellular envelope into the extracellular milieu (3, 38). In addition to Tfp expression, secretins are also required for type II and type III secretion, as reviewed by Bayan et al. (1), and in the secretion of certain filamentous phages (23). It has been reported that in multiple systems in a diverse group of organisms secretin multimers are remarkably resistant to denaturation in the presence of heat and sodium dodecyl sulfate (SDS) (18, 28).
Sequence analyses of secretins from several organisms have revealed that the C-terminal domain is highly conserved across genera, and regions in this domain are predicted to form ß-barrel structures, which are hallmarks of OM-spanning regions. Some data suggest that the C-terminal domain is directly involved in multimer assembly (3, 4). Conversely, the N-terminal domain is much less conserved among genera and is proposed to be periplasmic (2). The divergent nature of the N-terminal sequence and the suggested periplasmic localization indicate that the N terminus may be involved in specific interactions required for particular secretin functions.
In Neisseria, PilQ is the sole secretin and has been estimated to constitute approximately 10% of the OM mass in N. gonorrhoeae (28). Electron microscopy (EM) data suggest that PilQ in N. meningitidis exists as a dodecameric doughnut-shaped ring with symmetry suggestive of a tetramer of trimers in the OM (8-10). Far-Western blot data coupled with these EM data strongly suggest that PilQ directly interacts with Tfp and that this interaction stimulates a structural change in PilQ (9). In other organisms, EM analyses of secretins involved in filamentous phage secretion (29) and type II secretion in Klebsiella (5) revealed a similar general surface-exposed, multimeric ring structure in the OM. The variations among the secretins studied in these examples include variations in the nature and symmetry of a plug domain that is proposed to occlude the secretin pore, as well as the symmetry of the secretins (1).
In Neisseria, pilQ null mutants are nonpiliated and not competent for DNA transformation (13); however, the distinct phenotypes of two independently isolated mutants with spontaneous point mutations in pilQ in N. gonorrhoeae have suggested that PilQ can be involved in physiological events in addition to secretion of the pilus. The pilQ1 mutation, which results in an F563L substitution, allows N. gonorrhoeae to grow with hemoglobin as the sole source of iron when normal transport pathways are blocked, and the mutant is hypersensitive to the toxic effects of free heme (6). The pilQ1 strain is also more sensitive to certain antimicrobial compounds, suggesting that this mutation allows increased transport of large molecules through the PilQ complex. The pilQ1 mutant is able to express pili and undergoes transformation, albeit slightly less efficiently than the parent strain. Conversely, the pilQ2 mutation results in a E666K substitution, which appears to be a loss-of-function mutation (44). The pilQ2 mutant has a nonpiliated (P–) colony morphology and displays no pili, yet it still undergoes DNA transformation at a reduced frequency. These findings suggest that PilQ may participate directly in pilus-dependent processes and demonstrate that mutants with missense mutations can provide insight into PilQ function.
The goal of this study was to generate a library of pilQ missense mutants of N. gonorrhoeae to further understand the role of secretins in general and of PilQ in particular in terms of formation of stable multimers, pilus secretion, and pilus-dependent functions. To accomplish this goal, a method for performing error-prone mutagenesis of the pilQ chromosomal locus was devised. Upon mutagenesis of the middle one-third of pilQ, mutants with a P– colony morphology were isolated in order to identify mutations involved in pilus function. For each P– mutant isolated, Western blot analysis was performed to ascertain the effect of the mutation on PilQ monomer expression and heat- and SDS-resistant complex formation. Mutants expressing a nontruncated PilQ monomer were sequenced and analyzed to determine the effects of the mutations on pilus expression and pilus-dependent events. These missense mutants varied greatly in their ability to form stable PilQ complexes and to perform pilus-related functions. These mutants were grouped into five phenotypic classes based on PilQ functionality in terms of the ability to secrete pili, the ability to transform DNA, and the ability to adhere to host epithelial cells. Class 1 mutants were completely nonfunctional, and this class was a null class; class 2 mutants were minimally functional; class 3 mutants were moderately functional; class 4 mutants were differentially functional; and the single class 5 mutant was highly functional.
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MATERIALS AND METHODS |
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All bacterial strains were grown on GCB plates with Kellogg's supplements I and II (19) at 37°C in a 5% CO2 humidified atmosphere. Erythromycin was used in GCB at a concentration of 0.5 µg/ml, chloramphenicol was used in GCB at a concentration of 1 µg/ml, and nalidixic acid was used in transformation assays at a concentration of 1 µg/ml.
Construction of pMB9. A 2,557-bp DNA fragment from FA1090, starting 55 bp downstream of the pilQ coding start site and ending 438 bp downstream of the pilQ stop codon, was amplified using primers PilQSacIITop (5'-TTTCAGACGGCATCCGCGGGAAACATTACAGAC-3') and PilQDS2Bot (5'-GGCGGAAATCCGAACACGTCC-3'), which replaced both a G at position 70 and an A at position 72 with C to generate a SacII site between positions 68 and 73 relative to the start site of pilQ (Fig. 2A). This fragment was cloned into the pSMART ampicillin-resistant vector using the CloneSmart Blunt cloning system (Lucigen). The erythromycin resistance (Ermr) gene from pJD1145 (25) was amplified using primers ErmUpTopBamHI (5'-GGATCCGCCGTCTGAAGTTTGACAGCTTATCATCGCGTGC-3') and ErmBot (5'-CACAAAAAATAGGTACACGAAAAC-3'), resulting in a BamHI restriction site immediately upstream of the start of the Ermr gene, and the fragment was inserted into the naturally occurring NdeI restriction site 17 bp downstream of the pilQ stop codon by blunt end cloning (Fig. 2A).
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The mutagenized PCR products were gel purified and cloned back into the pMB9 vector from which pilQ region 2 had previously been removed by digestion with BsmI and XhoI (Fig. 2A). The new plasmid pool, with various region 2 pilQ mutations, was transformed into N. gonorrhoeae strain RM11.2, the parental strain, which does not permit plasmid replication. Since the plasmid is unable to replicate in Neisseria and there is no homology between N. gonorrhoeae and the plasmid vector, selection for transformants on erythromycin ensured that the Ermr cassette had recombined onto the bacterial chromosome along with some part of the mutant pilQ locus and the pilQ downstream region.
Sequence analysis. DNA sequencing was performed commercially (SeqWright, Houston, TX). The primers used for sequencing the pilQ region 2 mutants were designed to anneal to DNA flanking the outer edges of region 2, so any mutations at the extreme 5' or 3' end of the region could be detected (Fig. 2). Primer PilQ757Top (5'-CAGCTGATTATCACAACAACCGGC-3') anneals to DNA 757 to 780 bp downstream of the pilQ start site, and primer PilQ1566Bot (5'-CCGGCAGGTTGATTTTGGTTTGG-3') anneals to DNA 1,566 to 1,588 bp downstream of the pilQ start site. DNA sequence analysis of pilE was performed as previously described (34).
SDS-PAGE and Western blotting. Whole-cell lysates were prepared as described previously (44). Samples were run on SDS-polyacrylamide gels (10%), separated by electrophoresis, and electrotransferred to a polyvinylidene difluoride membrane in 10 mM CAPS (N-cyclohexyl-3-aminopropanesulfonic acid) buffer with 10% methanol at pH 11 for 1.5 h. Upon transfer, the membrane was blocked with 5% dry milk in Tris-buffered saline with 0.5% Tween for 1 h and then incubated with a 1:100,000 dilution of a polyclonal anti-PilQ antibody (graciously provided by C. E. Wilde, Indiana University School of Medicine) for 1 h at room temperature. The membrane was washed three times with Tris-buffered saline with 0.5% Tween and then incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Chemicon International, Temecula, CA) for 1 h and washed three times. Blots were developed with the ECL-Plus Western detection reagent (Amersham Biosciences) used according to the manufacturer's instructions.
Transformation assays. Transformation assays were performed as previously described, with slight modifications (44). Strains were grown for 18 h on GCB plates and resuspended in liquid GCB to an optical density at 600 nm of approximately 0.2. Then 20 µl of a bacterial resuspension was added to 200 µl of liquid GCB containing supplements, 5 mM MgSO4, 1 mM IPTG, and 10 ng of plasmid pSY6, which carries a gyrB mutation (33) that confers nalidixic acid resistance (Nalr) in N. gonorrhoeae. Transformation mixtures were incubated at 37°C for 15 min. The reaction mixtures were then treated with 1 U RQ1 DNase (Promega), incubated at 37°C for 5 min, and added to 2 ml of liquid GCB plus supplements. After 5 h of incubation at 37°C in the presence of 5% CO2, the reaction mixtures were 10-fold serially diluted and spotted onto GCB plates in the presence and absence of 1 µg/ml nalidixic acid. The efficiencies reported below were determined by dividing the number of CFU/ml recovered on nalidixic acid-containing by the total number of CFU/ml recovered on nonselective plates.
Adherence assays. ME180 human endocervical epithelial cells (ATCC HTB 33) were maintained in RPMI 1640 medium without L-glutamine (Mediatech, Inc) supplemented with 5% fetal bovine serum (RPMI-FBS) containing penicillin (100 U/ml), streptomycin (100 mg/ml), and amphotericin B (2.5 µg/ml) at 37°C in the presence of 5% CO2. All adherence assays were performed in the absence of antibiotics and amphotericin B.
Standard CFU adherence assays were performed as described previously, with slight modifications (21). Briefly, 1 ml of a culture containing 5 x 105 ME180 cells/ml was added to each well of a 24-well culture dish (Corning) and incubated for 24 h prior to the assay. N. gonorrhoeae was cultured on GCB plates for 18 h and was gathered with Dacron swabs, suspended in RPMI-FBS, and diluted to obtain a concentration of approximately 1 x 107 CFU/ml. One milliliter of a bacterial suspension was incubated in each well for 1 h. Monolayers were washed three times with phosphate-buffered saline (PBS) and incubated with 1% saponin for 10 min. Two milliliters of RPMI-FBS was then added to each well, the monolayers were disrupted by pipetting, and 10-fold serial dilutions were prepared and plated on GCB plates. The results were determined by dividing the number of CFU/ml recovered by total number of CFU/ml initially placed in each well.
Immunofluorescence microscopy for detection of surface-exposed PilQ. All N. gonorrhoeae strains were labeled with the fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) (Molecular Probes-Invitrogen), which enters a bacterial cell and irreversibly binds to proteins associated with the membrane. After 18 h of growth on solid medium, approximately 109 N. gonorrhoeae cells were swabbed into 1 ml of 5 mM MgSO4 in PBS (MPBS), centrifuged at 3,000 x g for 5 min, and resuspended in 1 ml of a solution containing 0.2 mg/ml CFDA-SE in MPBS prepared according to the manufacturer's instructions. Bacteria were incubated in the presence of CFDA-SE for 20 min at 37°C, washed by centrifugation at 3,000 x g for 5 min, and resuspended in 1 ml MPBS. Resuspended bacteria were placed on top of a glass coverslip and incubated at room temperature for 45 min. The coverslip was washed once with MPBS, and the bacteria were fixed by adding 1 ml of 4% paraformaldehyde in PBS for 15 min and then washed three times with PBS and blocked with 10% goat serum in PBS for 45 min at room temperature.
Surface-exposed PilQ was visualized using PilQ antiserum whose cross-reactive antibodies had been removed by whole-cell adsorption with a pilQ null mutant. To adsorb the antiserum, approximately 1011 RM11.2 pilQ::cat cells grown overnight on solid medium were swabbed into 3 ml liquid GCB, and 30 µl of antiserum was added and incubated at 4°C on a moving rotor for 18 h. Following incubation, the adsorbed antiserum was clarified by centrifugation and sterilized by passage through a 0.2-µm syringe filter. One hundred microliters of the cleaned antiserum was added to fixed bacteria on the coverslip and incubated at room temperature in a humidified chamber for 1 h. After incubation, the coverslip was washed three times with PBS, and 100 µl of Alexa Fluor 647 goat anti-rabbit antiserum (Invitrogen) diluted 1:200 in PBS was added and incubated at room temperature in a humid chamber for 1 h. After incubation the coverslip was washed three times with PBS and once in distilled deionized water. The coverslip was covered with 10 µl Fluoromount (Southern Biotechnology, Birmingham, AL) containing 2.5 mg/ml propyl gallate (ICN Biomedicals, Inc., Costa Mesa, CA) to prevent photobleaching and mounted on a slide. Samples were examined with a Leica DMIRE2 microscope. CFDA-SE-labeled N. gonorrhoeae was visualized using a Leica green fluorescent protein filter, PilQ was indirectly visualized using a CY5 filter, and the images were overlaid. Images were analyzed using the Openlab software (Agilent Technologies, Palo Alto, CA).
Immunoelectron microscopy for detection of pilus expression. Immunogold transmission EM (TEM) with an antipilin antibody was performed as described previously (6). Values were obtained by dividing the number of pilus bundles observed by the total number of bacterial cells observed. For each mutant, at least two grids were observed and at least 200 cells were analyzed. To be classified as a pilus bundle, a structure had to display filamentous striations and be labeled with gold particles (Fig. 3). No unbundled pili were detected.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Plasmid pMB9 carrying the entire N. gonorrhoeae pilQ gene minus the first 54 bp of the coding sequence was constructed (Fig. 2A). Region 2 of pilQ, which is flanked by naturally occurring XhoI and BsmI restriction sites, encodes amino acid residues 258 through 504 and was mutated via error-prone PCR in the presence of 5 mM MgCl2. A pool of mutated region 2 plasmids was isolated, and the pool of mutated inserts was cloned between the XhoI and BsmI sites of pMB9 to reconstitute pilQ with mutations throughout region 2. The pool of mutated pMB9 plasmids was transformed into N. gonorrhoeae strain FA1090 pilus variant RM11.2 recA6, a highly piliated strain (21), with selection for the Ermr cassette inserted into the downstream flanking sequences of pilQ.
Following mutagenesis, approximately 2,000 Ermr mutant colonies were isolated and screened to identify those with a nonpiliated (P–) colony morphology, as described previously (21). A total of 204 P– Ermr colonies were identified, and chromosomal DNA was isolated from each colony and used to retransform the RM11.2 parent strain to confirm that Ermr was linked to the P– colony morphology (transformational backcross). Approximately 97% of the backcrossed mutants (a total of 197 mutants) exhibited erythromycin resistance and the P– colony morphology, indicating that the mutation or mutations causing the P– colony morphology were linked to the pilQ locus. PilQ Western blot analysis of these 197 mutants revealed that 138 mutants had no detectable full-length PilQ monomer (data not shown). DNA sequence analysis of pilQ of 20 randomly selected mutants with no detectable monomer showed that 19 of these mutants had nonsense mutations and one had three substitutions (E409K, V327I, and R475H). Mutants that lacked a stable PilQ monomer were not tested further.
DNA sequence analysis of pilQ was performed for all 59 mutants with a full-length PilQ monomer to identify the amino acid substitutions that resulted in the P– colony morphology. Of these 59 pilQ mutants, 2 had four amino acid substitutions, 8 had three substitutions, 13 had two substitutions, and 36 had single substitutions. Since identification of individual residues involved in PilQ structure and function was the aim of this study, the 36 mutants with single residue changes were the focus of this work. Some of these mutants had redundant amino acid substitutions, so there was a total of 19 unique amino acid substitutions (Fig. 2B), and further analysis was performed with these mutants. Collectively, these 19 pilQ mutants had substitutions in 15 residues. The most N-terminal substitution was Q307K, and the most C-terminal substitution was K490E. At 12 residues there was a single substitution (Q307K, V332D, I369L, L375P, L402S, L436P, L444P, D448V, V477D, E478K, S484P, and K490E), at two residues there were two different substitutions (D438Vand D438G and I472N and I472T), and at one residue there were three substitutions (D464G, D464Y, and D464N) (Fig. 2B and Table 1).
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PilQ is surface exposed in missense mutants. Since all of the pilQ missense mutants expressed the full-length monomer subunit and some of them expressed detectable levels of an SDS-resistant multimer, immunofluorescence microscopy was performed with all 19 mutants to determine whether the mutant PilQ was surface exposed. All pilQ missense mutants had detectable levels of surface-exposed PilQ, while the pilQ::cat null mutant showed no PilQ reactivity (Fig. 4). It is noteworthy that for the parent strain, as well as for all 19 missense mutants, some cells had no PilQ reactivity, some cells were totally covered with PilQ reactivity, and some cells had punctate areas of PilQ reactivity (Fig. 4). These results demonstrate that the diminished pilus expression and altered pilus-related events observed for the missense mutants were not due to interference with the ability of PilQ to localize to the OM or to be surface exposed.
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pilQ missense mutants with no detectable pili have growth defects in a pilT background. N. gonorrhoeae pilQ null mutants with a regulatable pilT background have severe growth defects when PilT is depleted compared to the growth of a pilT or pilQ single mutant (42). To determine whether the pilQ missense mutations used in this study had a similar effect in a pilT mutant background, all 19 pilQ missense mutations were transformed into a strain with an IPTG-inducible pilT gene (40). All of the pilQ mutants with no detectable pili as determined by TEM had a growth defect when PilT was not expressed, and this defect was rescued in the presence of IPTG (Table 1 and data not shown). Conversely, all of the strains with detectable levels of pili, even very low levels, grew like the parental strain when PilT was depleted (Table 1). This finding shows that pilQ missense mutants with no detectable pilus secretion react to PilT depletion like the pilQ null mutant reacts. Furthermore, we concluded that with this collection of pilQ missense mutants, any detectable pilus secretion is sufficient to rescue pilQ pilT double mutants from such growth defects (42).
pilQ missense mutants have a wide range of transformation efficiencies. The DNA transformation efficiency was determined for all 19 missense mutants, along with the wild-type parent strain and the pilQ::cat null mutant. As seen previously with N. gonorrhoeae (44), the difference between the level of acquisition of a Nalr marker by a wild-type strain and the level of acquisition of a Nalr marker by a pilQ null mutant was several orders of magnitude (Fig. 5). For four mutants (Q307K, I369L, L375P, and D438V) the transformation levels approached that of the parental strain, and all but one of these four mutants (D438V) secreted levels of pili that were near wild-type levels (Fig. 5 and Table 1). For six mutants (V332D, L402S, D438G, E487K, S484P, and K490E) the transformation levels were diminished yet detectable; three of these mutants expressed reduced levels of pili (D438G, E478K, and K490E), while the other three displayed no detectable pili (V332D, L402S, and S484P) (Fig. 5 and Table 1). For the remaining nine mutants (L436P, L444P, D448V, D464G, D464Y, D464N, I472N, I472T, and V477D) the transformation efficiencies were below the detection limit, similar to the transformation efficiency of the pilQ null mutant. None of these nine mutants expressed pili (Fig. 5 and Table 1). It is notable that all but one amino acid substitution that abolished transformation were in a single region spanning residues 444 to 477 (Fig. 5B). While all pilQ mutants with detectable pili were able to undergo transformation, not all transformable pilQ mutants expressed pili, an observation made previously (22, 44). This observation supports the current hypothesis that transformation is not dependent upon the presence of fully formed pili as much as it is dependent upon the presence of functional pilus assembly apparatuses and suggests that the role of PilQ in the formation of the pilus assembly apparatus is less stringent for transformation than for expression of the pilus.
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DISCUSSION |
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There are nine class 1 mutants, which have no detectable pili, exhibit no detectable transformation, exhibit no pilus-mediated adherence, and thus are classified as pilQ null mutants (Fig. 2B and Table 1). The substitutions in this class are L436P, L444P, D448V, D464G, D464Y, D464N, I472N, I472T, and V477D. None of these substitutions is conservative. There are two leucine-to-proline substitutions, which is noteworthy because the introduction of proline residues is known to disrupt ß-strands, which are important for proper functioning of OM-spanning proteins (7). It is also noteworthy that three mutants with substitution of the aspartic acid at residue 464 were isolated and all have similar phenotypes. Additionally, two mutants with substitution of the isoleucine at residue 472 were isolated and have similar phenotypes (Table 1). No class 1 mutant consistently produced SDS-stable PilQ multimers, even though the mutants displayed surface-exposed PilQ (Fig. 1 and 4). All of the class 1 substitutions are clustered in the C-terminal domain of region 2, which is hypothesized to be involved in PilQ multimer formation (3, 4); hence, the most probable explanation for the class 1 mutant phenotype is that PilQ is transported to the outer membrane but do not form stable dodecamers and thus do not form a functional pilus apparatus.
There are three class 2 mutants, which do not secrete pili, exhibit low yet detectable levels of transformation, adhere like the pilQ null mutant, and are classified as minimally functional mutants. The class 2 substitutions span nearly the length of region 2 (V332D, L402S, and S484P) (Fig. 2B and Table 1). Like the substitutions in the class 1 mutants, none of these substitutions are conservative, and the PilQ protein is surface exposed (Fig. 4). One mutant (V332D) has a high level of a detectable SDS-stable multimer, one mutant (L402S) has a greatly diminished level of an SDS-stable multimer compared to the parent strain, and one mutant (S484P) has no detectable SDS-stable multimer (Fig. 1). By isolating transformable mutants with no detectable pili yet finding no mutants with pili that do not transform, we confirmed previous work that suggested that the pilus assembly apparatus can still mediate transformation even without extended pili (22, 44). We predict that the class 2 mutant proteins form a minimally functional secretin structure and that these mutant proteins either allow transformation of a small subset of cells or form a partially functional assembly apparatus in all cells. Regardless of which possibility is correct, the class 2 mutant PilQ proteins do not complete a functional pilus assembly apparatus and thus do not secrete pili; thus, the proteins do not facilitate adherence.
There are four class 3 mutants, which display detectable yet reduced levels of pili, have intermediate levels of transformation, exhibit little or no adherence, and therefore are classified as moderately functional mutants. The amino acid substitutions in the class 3 mutants are D438V, D438G, E478K, and K490E (Fig. 2B and Table 1). The D438V substitution results in a higher transformation efficiency and statistically higher level of adherence than D438G, although both transformation and adherence are still significantly reduced compared to the transformation and adherence of the parent strain. Only one class 3 mutant (D438V) produces consistently detectable levels of an SDS-resistant multimer, although the level is lower than that of the parent strain (Fig. 1).
It is likely that the class 3 mutants form an assembly apparatus that is functional to the point of allowing transformation and some level of pilus expression, yet not sufficient for proper adherence. The decreased adherence may be the result of a structural defect in the pili secreted by the mutant PilQ; alternatively, the reduced number pili secreted by these pilQ mutants may be insufficient for pilus-mediated adherence. The fact that the moderately functional class 3 substitutions are clustered in the same C-terminal domain of region 2 as the substitutions of the class 1 null mutants suggests that either the residues affected in class 3 mutants are not as critical as those in class 1 mutants for the formation of a functional apparatus or that the amino acid substitutions in class 3 mutants retain partial functionality compared to the class 1 amino acid substitutions.
There are two class 4 mutants, which display high levels of pili and high, albeit less than parental, levels of transformation, yet adhere to ME180 cells with an efficiency that is similar to that of the pilQ null mutant. The two mutants have similar high levels of an SDS-resistant multimer (Fig. 1). Because of these opposite effects on transformation and piliation relative to adherence efficiency, these mutants are classified as differentially functional mutants. The substitutions are I369L and L375P (Fig. 2B and Table 1). Although the two mutations result in similar phenotypes, one substitution is highly conserved (I369L), while the other is nonconservative (L375P). The I369L mutant displays secreted pili at a level that is approximately twofold higher than the level observed for the L375 mutant (41 and 17%, respectively); however, both mutants are more similar to the parent strain (35%) than to the mutant with the next highest level of pilus secretion (D438V [2.6%]). These data confirm previous studies demonstrating that Neisseria pilus expression is necessary, but not sufficient, for adherence (27, 39). However, this is the first time that PilQ has been directly implicated in adherence.
There are two hypotheses that explain why these differentially functional mutants express high levels of pili but exhibit low levels of adherence. The first hypothesis is that the mutant PilQ secretes pili that are not functional for mediating adherence, which suggests that the isoleucine at residue 369 and the leucine at residue 375 are somehow involved in the secretion of adherence-facilitating pili. The second hypothesis is that there is no defect in the pili secreted by the mutant PilQ; instead, PilQ plays a direct role in adherence, and the mutations disrupt this role. Either way, these phenotypes suggest that the two residues substituted in class 4 mutants are involved in interactions between PilQ and another component required for adherence to host epithelium, but we cannot differentiate between the two hypotheses at this time.
Class 5 consists of a single mutant with the substitution Q307K (Fig. 2B and Table 1). This mutant is classified as a functional mutant and is similar to the parental strain in terms of piliation and adherence. Although this mutant transforms better than any of the pilQ mutants examined in this study, the transformation efficiency is still significantly lower than that of the parent strain. By isolating mutants that display high levels of pili yet still have a P– colony morphology, we demonstrated that factors in addition to parental levels of pilus expression are involved in colony morphology.
It is likely that this pilQ mutant forms a pilus assembly apparatus that is not as stable as the parent apparatus, yet is functionally adequate to secrete pili, allowing parental levels of adherence and near-parental levels of transformation. The data also suggest that the glutamine at residue 307, the most N-terminal residue substituted in this study, is involved in some aspect of Neisseria physiology that affects colony morphology, since the Q307K mutant still has a nonpiliated colony morphology compared to the parental strain. As stated above, the differences in piliation, pilus function, and colony morphology are not the result of pilin antigenic variation, as the pilE sequence of all missense mutants was shown to be identical to that of the parent strain.
In sum, we identified 15 PilQ residues in N. gonorrhoeae that, when replaced, alter colony morphology. The replacements have diverse effects on PilQ multimer stability, secretion of pili, DNA transformation, and adherence to host epithelium. Since PilQ is surface exposed in all of our missense mutants, the altered function is not a result of defective transport to the OM. The fact that mutants with no stable PilQ multimer still had surface-exposed PilQ indicates that formation of SDS-stable multimers is not required for PilQ mobilization to the OM. Furthermore, we demonstrated that the oligomer status of PilQ as determined by SDS-PAGE does not necessarily predict the biological activity of a given mutant, a concept that has been suggested previously for N. gonorrhoeae (22, 44), although not based on a collection of pilQ mutants of this size.
The observations made using a collection of pilQ missense mutants in this study clearly indicate that gonococcal PilQ may be directly involved in pilus-dependent functions and is not simply a passive pore in the OM. Whether this is because the pilus that is expressed from the mutant secretin is altered via direct interactions between the mature pilus and PilQ, which has been suggested previously (9), or because PilQ directly interacts with other components involved in transformation and adherence has not been determined yet. Based on the results of this study, additional mutagenesis of the other two regions of pilQ and additional analysis of some of the region 2 mutants obtained are warranted in order to further understand the role of PilQ in the biology of the Tfp and possibly the pathogenesis of N. gonorrhoeae.
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ACKNOWLEDGMENTS |
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This work was supported by NIH grants R01 AI055977 to H.S.S. and F32 AI065091 to R.A.H.
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FOOTNOTES |
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Published ahead of print on 2 February 2007.
Present address: University of Michigan, Department of Molecular, Cellular, and Developmental Biology, 830 North University, Ann Arbor, MI 48109.
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REFERENCES |
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