A real-time semi-quantitative RT–PCR assay demonstrates that the pilE
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《核酸研究医学期刊》
Department of Microbiology-Immunology, Northwestern University Medical School 303 East Chicago Avenue, Chicago, IL 60611, USA
*To whom correspondence should be addressed. Tel: +1 312 503 9788; Fax: +1 312 503 1339; Email: h-seifert@northwestern.edu
ABSTRACT
A semi-quantitative real-time RT–PCR assay was designed to measure gonococcal pilin antigenicvariation (SQ-PCR Av assay). This assay employs 17 hybridization probe sets that quantitate subpopulations of pilin transcripts carrying different silent pilin copy sequences and one set that detects total pilE transcript levels. Mixtures of a DNA standard carrying the silent copy being detected and a clone encoding the starting pilE sequence, which is the majority pilE template, provided amplification curves that closely matched the experimental data and allowed an analysis of the contribution of different silent pilin copies to variation. The SQ-PCR Av assay was verified using DNA sequence analysis to demonstrate that this methodology allowed an accurate analysis of pilin variation. Both assays showed that with a specific starting pilE sequence, only a subset of the silent pilin copies recombine into pilE at a detectable level, and that this limited subset was reproducibly detected in replicate cultures. When an isogenic pilE sequence variant was examined using both assays, a new subset of silent copy sequences were detected recombining into pilE and the overall frequency of variation was increased. Thus, the parental pilE sequence influences the frequency of variation and the repertoire of pilin variants produced.
INTRODUCTION
Neisseria gonorrhoeae (also called Gc or the gonococcus) is a Gram-negative diplococcal bacterium and is the causative agent of the human sexually transmitted disease gonorrhea. No vaccines for gonorrhea exist in part because N.gonorrhoeae alters some of its surface elements, allowing it to evade the human immune system (1,2). Gonococcal attachment to human epithelial cells is mediated primarily by pili, surface structures composed of multiple units of the protein pilin (3,4). Pili mediate adherence to a number of different eukaryotic cell types, affect interactions with epithelia, endothelia and phagocytes (5), and are also required for full DNA transformation competence (6–8).
Pilin is encoded by the pilE gene present at a single expression locus in the strain FA1090. In addition to pilE, FA1090 also contains a total of 18 silent pilin gene copies present in 5 pilS loci and 1 silent pilin gene copy present in the pilE locus (Figure 1A) (9). These silent copies and pilE share a central semivariable (SV) region and 3' hypervariable (HV) region, but lack the 5' constant region and promoter found only at pilE. The 3' HV region can be further subdivided into a HV loop (HVL) and a HV tail (HVT). The HVL is flanked by two conserved regions, cys1 and cys2 (Figure 1B) (10).
Figure 1 Cartoons showing the pilin loci and the basis for the SQ-PCR assay. (A) Pilin loci of gonococcal strain FA1090. The N.gonorrhoeae strain FA1090 chromosome contains one pilin expression locus, pilE, and five silent pilin loci, pilS. The pilE locus contains the pilin expression gene pilE (light blue rectangle) and the upstream silent pilin copy (white rectangle). The wavy line indicates the pilE transcript. Each of the five pilS loci contain between one and six silent pilin gene copies (colored rectangles). The silent copies are shown using an abbreviated nomenclature. Silent pilin locus 1 (pilS1) gene copy 2 is shown as 1c2, pilS3 copy 3 is 3c3, etc. (B) The SQ-PCR Av assay to measure recombination at pilE. Five general portions of the pilE gene are shown: the 5' constant region, a central SV region, the conserved cys1 and cys2 regions that flank the HVL, which is followed by the hypervariable tail (HVT) (16). The wavy line indicates the transcript encoded by the pilE gene and the rectangles indicate coding regions of DNA. Primers are shown as arrows, and the sequence targeted by the pairs of hybridization probes is starred. The 5' primers are used with the pilE constant region hybridization probes, LC-HybCONS 1 and 2, and detect total pilE transcript levels. The 3' primers are used with the 17 pairs of pilS HV hybridization probes and detect individual pilS to pilE recombination at the HVL of pilE.
Pilin antigenic variation (Av) is defined as the high frequency change of amino acid residues in the pilin protein (11,12). During pilin Av, a portion of pilS replaces a portion of pilE, often producing a new pilin that is efficiently expressed on the cell surface. This RecA-dependent recombination of a pilS sequence into pilE is unidirectional because the donor pilS sequence remains unchanged while the recipient sequence is lost from pilE (13,14). Several conserved DNA segments that have been reported to be important for pilin Av include the cys2 sequence (15), as well as the correct spacing between cys1 and cys2 (16), and the 3'-untranscribed sequence of the Sma/Cla repeat (17). Av at pilE also requires RecA, and the RecF-like pathway of homologous recombination (18–20), but not the RecBCD pathway (18). The Rep helicase (21) and the RecA modulator RecX (22) are involved in pilin Av, but the replication restart protein PriA is not involved (23).
Many assays have been developed to analyze pilS to pilE recombination, but each has its shortcomings (17,24). The most common assay used to measure pilin Av uses the frequency of colonies appearing with a non-piliated colony morphology from a piliated population to estimate the frequency of pilin Av (18,19,22). However, this method underestimates the frequency of Av because not all changes in pilE lead to changes in colony morphology. To analyze pilin variation independent of colony morphology changes, Wainwright et al. (17) developed an RT–PCR-based assay to measure the transfer of silent copy sequences into pilE in a population of bacteria. Briefly, the assay measured the relative quantity of total and recombinant pilE by measuring the presence of new HVL sequences from either pilS1 copy 4 or pilS6 copy 1. Subsequently, Serkin et al. (24) improved upon the existing qualitative assay by mixing target and competitor RNA prior to reverse transcription such that competitor template served as an internal control for RT and PCR efficiency. This quantitative, competitive RT–PCR assay was capable of detecting recombination from multiple sources, yet was incomplete in that not all silent pilin copies were detected or could be individually assayed. Thus, in order to detect the contribution of all silent pilin copies individually, a real-time semi-quantitative RT–PCR (SQ-PCR Av) assay was designed to study pilS to pilE recombination.
This SQ-PCR Av assay was used to demonstrate that with a defined starting pilE sequence only a subset of the silent pilin copies are prevalent donors for pilin variation even between replicate cultures. Furthermore, with a different parental pilE sequence, a different representation of silent copy sequences was detected recombining into pilE, and the overall frequency also changed. Thus, the repertoire of silent copies that donate their sequences to create pilin variants depends on the parental sequence. These data demonstrated that a real-time RT–PCR assay can be used to investigate the mechanisms leading to antigenic variation of pilin in N.gonorrhoeae and shows that the starting pilE sequence has a major influence on the variants produced.
MATERIALS AND METHODS
Generation of SQ-PCR Av standards
The hypervariable loop (HVL) regions of all pilS copies were previously cloned into the pGEM-3 vector (24). These plasmids served as standards for the SQ-PCR Av assay. Clone E1r was a gift from Terri Hamrick and was used as a standard for assaying total pilE transcript levels (9). Clone pGADT7 + GcRecA#9 was used as a standard for the recA control (25). All plasmid standards were linearized by digestion with HincII (New England Biolabs). The linearized plasmid standards and Low DNA Mass Ladder (Invitrogen), which has a known concentration, were then run in a 0.8% agarose gel stained with ethidium bromide. Densitometry was performed using ImageQuant (Molecular Dynamics) to quantify the linear plasmid standards. Furthermore, the plasmid standards were normalized using SYBR Green I DNA dye and the LightCycler instrument. Molecules of the gene of interest were calculated as described previously (26), and serial dilutions representing 108–101 molecules were generated. Pilin competing-template mixed standards were generated by mixing the silent copy standard of interest with the parental copy standard at the total pilE transcript level. Non-pilin competing-template mixed standards were produced with opa PCR product, recA cDNA and Escherichia coli chromosome, each at the total pilE transcript level.
Bacterial strains and growth conditions
Gonococcal strain FA1090 recA6 pilin variants 1-81-S2 (27) and 1-81-S2(3c3) (this study) were used in all of the experiments. FA1090 recA6 contains an isopropyl-?-D-thiogalactopyranoside (IPTG)-regulatable gonococcal recA allele that produces a phenotypically RecA-deficient strain without IPTG in the growth medium and a RecA-proficient strain when grown in the presence of 1 mM IPTG that is indistinguishable from wild-type recA strain (28). Gonococci were grown on Gc Medium Base (Difco) with Kellogg supplements (GCB) (29) and 1 mM IPTG (Diagnostic Chemicals Ltd) at 37°C in 5% CO2 for 22, 26 or 30 h.
RNA extraction and reverse transcription
Gonoccoci were swabbed from solid media into ice-cold phosphate-buffered saline (PBS), and Trizol extraction of total RNA was performed according to the supplier's instructions (Invitrogen). DNase treatment was performed twice (24), followed by RNA purification using the RNeasy RNA cleanup protocol (Qiagen) as described previously (24). RNA was run in a 0.8% agarose gel stained with ethidium bromide to partially quantitate and assess the stability of the RNA. 28S ribosomal RNA bands were compared and normalized by performing densitometry with ImageQuant software (Molecular Dynamics). After RNA levels from the experimental samples were normalized, cDNA synthesis was performed as described by Serkin and Seifert (24) using the primer LC-CYS2rev for the 3' HV region of pilE, LC-CONSrev for the 5' constant region of pilE or LC-recArev for recA (Supplementary Material). All reverse transcription reactions were performed with a no reverse transcriptase control in order to control for contaminating DNA in the SQ-PCR Av assay. The reverse transcription reaction was purified using the Qiaquick PCR Purification Kit (Qiagen).
Oligonucleotides
All oligonucleotides used are listed in Supplementary Table 1. The forward primer LC-CYS1for and reverse primer LC-CYS2rev hybridize to two previously characterized pilin regions, cys1 and cys2 (30), conserved in all N.gonorrhoeae pilE and pilS copies (9). LC-CYS1for and LC-CYS2rev were used in conjunction with the silent pilin copy probes (Supplementary Material). A second primer set consisting of forward primer LC-CONSfor and LC-CONSrev anneals to the 5' constant region of the N.gonorrhoeae pilE gene and was used in conjunction with the pilE 5' constant region hybridization probes to detect total pilE transcript levels. The third primer set consists of forward primer LC-recAfor and reverse primer LC-recArev and anneals to the N.gonorrhoeae recA gene. The recA primers were used in conjunction with the recA hybridization probes to detect recA transcript levels, which served as a control for RNA normalization. All primers were reverse phase high-performance liquid chromatography purified (IT Biochem). Primer and probe melting temperatures were calculated using the freeware program TM Utility v.1.3 (Idaho Technology, http://www.idahotech.com/downloads_up/pTmutility_form.htm).
Hybridization probes were designed to specifically target 17 of the 18 silent pilin gene copies (9). An alignment of all pilS copies using the software program AlignX (InforMax, Inc.) allowed unique sequences within the HVL to be identified as potential hybridization probe targets for each silent pilin copy (Supplementary Figure 1). Because the HVL sequences share sequence similarity (Supplementary Material), probe target sequences for each silent copy were compared against all the other FA1090 silent pilin gene copies (using the BLAST program) to ensure that the hybridization probe sequences specifically hybridize only to the targeted silent copy. The HVL sequence of one silent pilin copy, pilS1 copy 5, is identical to the HVL sequence of pilS2 copy 5. Thus, no pilS1 copy 5 hybridization probes were designed; instead, the pair of pilS2 copy 5 hybridization probes detects both pilS1 copy 5 and pilS2 copy 5. The distance between the donor and acceptor probes in all probe sets was 1 base (31). All donor probes were labeled with fluorescein at the 3' end, while all acceptor probes were labeled at the 5' end with LightCycler-Red 640-N-hydroxysuccinimide ester. Acceptor probes also had phosphate groups attached to the 3' end to prevent probe extension (IT Biochem).
Each of the pairs of hybridization probes used the same primer pair for amplification (Figure 1B). Clones carrying the HVL sequence of each silent copy bordered by the conserved cys1 and cys2 priming sites were isolated for use as standards to quantitate the proportion of each silent HVL sequence that had transferred into the pilE of an experimental sample. The purified plasmid standards were gel quantitated as described above, and 10-fold dilutions of these standards were used in each SQ-PCR Av experiment.
The annealing temperature of the hybridization probe pairs were adjusted to yield maximal detection of the targeted silent pilin gene copy sequence while concurrently minimizing undesired hybridization to sequences from the other silent pilin gene copies. Pairs of hybridization probes were deemed optimized when they met two conditions: (i) that all other untargeted silent pilin gene copies were detected at levels <1% of the targeted copy (data not shown) and (ii) that the detection limit was 105 copies of linear plasmid mixed standard or less.
Analysis of Southern colony variants and sequencing for the verification of the SQ-PCR Av data
FA1090 1-81-S1 recA6 was grown at 37°C in CO2 on GCB agar +/– IPTG for 22 h and was swabbed into ice-cold PBS and plated onto GCB agar. After 22 h, 1055 individual colonies of Gc were selected and frozen in 15 μl of GCBG (GCB with 20% glycerol). The frozen stocks of the individual colonies of Gc were passaged onto GCB agar. Individual colonies from each revived culture were lysed (1% Triton X-100, 2 mM Tris, pH 8, 2 mM EDTA) and the pilE gene was PCR amplified using PILRBS (17) and SP3A (32) in a PTC-100 thermocycler (MJ Research). The pilE PCR products were electrophoresed on a 0.8% TBE agarose gel, denatured, neutralized and blotted to nylon as described previously (33). The blots were probed with a 3' end labeled digoxigenin-11-ddUTP (Boehringer Mannheim) oligonucleotide specific to the parental FA1090 1-81-S2 recA6 HVL sequence (Supplementary Material, LC-Hyb2c1 1). The blots were probed at 67°C for 2 h and were washed with 0.1x SSC, 0.1% SDS twice at 67°C for 20 min and twice with 0.5x SSC, 0.1% SDS at room temperature. The blots were exposed to Kodak BioMax XAR film. A positive control, pilE amplified PCR product of FA1090 1-81-S2 recA6, and a negative control, pilE amplified PCR product of RM11.2 (6c1 in the HVL) were included in each Southern blot. About 3% of the isolates did not hybridize to the 2c1 parental probe in the HVL. These isolates were sequenced to determine the sequence changes in the pilE HVL. Furthermore, 5% of randomly selected 2c1 positive clones were sequenced and found to carry the 2c1 HVL sequence, showing that we were not missing any HVL variants. For sequencing, the PCR products were treated with shrimp alkaline phosphatase and exonuclease 1 to remove primers and dNTPs. PCR-generated pilin templates (16–33 ng) were used in each sequencing reaction with the PILRBS primer (17) and were analyzed using a Beckman Coulter CEQ 2000XL DNA system as specified by the manufacturer (Beckman).
Sequencing assay of antigenic variation for validation of the SQ-PCR Av assay
A sequencing assay was performed to verify the results of the SQ-PCR Av assay when using the strain FA1090 1-81-S2(3c3) recA6. This assay was performed as described previously (23). Briefly, FA1090 1-81-S2 recA6 and variant 1-81-S2 3c3 recA6 were grown on plates containing IPTG and incubated at 37°C in 5% CO2 for 20 h. Five starter colonies from each variant were then passaged onto separate GCB plates. A total of 45–50 colonies arising from each starting colony were passaged two more times to ensure clonal populations, and the pilE gene of each colony was sequenced as described above.
Real-time RT–PCR and data analysis
Real-time RT–PCR was performed on the LightCycler instrument (Roche Diagnostics). All assays used hybridization probes containing 2 μl of LightCycler DNA Master Hybridization Probes mix (Roche Diagnostics), 3.2 μl of MgCl2 (for a final concentration of 5 mM), 2 μl of each primer (0.5 mM), 2 μl of donor hybridization probe (0.2 mM), 2 μl of acceptor hybridization probe (0.2 mM), 2 μl of template and PCR-grade sterile water for a final volume of 20 μl. PCR amplification for the hybridization probes assays began with initial denaturation (30 s at 95°C) followed by 40 cycles of denaturation (2 s at 95°C), annealing (56–66°C for silent copy probes, 53°C for pilE and 60°C for recA) and extension (5 s at 72°C). For the optimized annealing temperatures for each pair of hybridization probes see Supplementary Material. Fluorescence in channels F1 and F2 was acquired at the end of every extension step, and the ratio of F2/F1 was analyzed.
Data were exported from LightCycler software and imported into Excel (Microsoft). Standard curves, P-values and Pearson Correlation Coefficients ‘R’ were generated using the Fit linear function. Pie charts were generated in Microcal Origin (Microcal Software Inc.).
RESULTS
Development of a SQ-PCR Av assay to detect sequence changes in the pilE HVL
Many assays have been developed to measure the frequency of pilin Av, but each has limitations (17,20,24). Each previously developed Av assay underestimates the frequency of variation and records only a small proportion of the possible recombination events comprising pilin Av. The specificity supplied by the hybridization step of real-time RT–PCR was used to attempt to detect recombination of all 18 silent gene copies into the pilE gene within a large population of bacteria.
A total of 18 pairs of hybridization probes were designed, one probe set reacted specifically with the constant region only found in pilE and the other 17 probe sets were designed to measure the level of the 18 silent copy HVL sequences (two HVL sequences are identical) in a population of gonococci. To test whether the hybridization probes could detect recombinant pilE sequences, N.gonorrhoeae strain FA1090 1-81-S2 recA6 was grown on solid media with or without 1 mM IPTG to control pilin Av. Total RNA was extracted and then reverse transcribed to pilin cDNA for real-time RT–PCR analysis. Pilin cDNA was normalized using the conserved region real-time RT–PCR primers and probes and adjusted to a concentration of 1 x 108 molecules per reaction. Once pilin cDNA was normalized, real-time RT–PCR was performed to quantify the parental 2c1 HVL sequence (Figure 2). Fluorescent acquisition curves of the cDNA from the experimental 22 h pilin cDNA sample of Gc was similar in slope and character to the purified standards during the early amplification cycles, although in the later cycles the slopes diverged between the pilin cDNA and the standards. During the earlier cycles, the experimental cDNA sample was estimated to contain 9.7 x 107 molecules carrying the parental 2c1 sequence. This measurement suggested that 97% of the pilE genes in this population retained the parental 2c1 sequence, while 3% had recombined with silent copies to produce variant pilE genes with a changed HVL. To investigate this further, real-time RT–PCR was used to measure the appearance of new HVL sequences into pilE from the other silent copies. However, fluorescent acquisition curves using the HVL probes all demonstrated a severely depressed slope that could not be compared with or quantified using the purified standard curves (Figure 3A). Some of the silent copy probes produced a signal above background (Figure 3A), while other silent copies were undetectable using these probes (data not shown).
Figure 2 Real-time RT–PCR measurement of total pilE transcript levels. The fluorescence acquisition curves are plots of the fluorescence detected versus cycle number. The most concentrated standard (108 molecules, squares) begins to rise above background at approximately cycle number 13. The closed symbols represent dilutions of the purified standard (squares, 108 molecules; circles, 107 molecules; triangles, 106 molecules; and diamonds, 105 molecules). The cross symbol indicates the no template control, which contained the master mixture and water instead of template. The star indicates the experimental sample of cDNA of Gc grown for 22 h on plates with IPTG to induce RecA expression and antigenic variation and is superimposed on the 108 standard curve.
Figure 3 RT–PCR analysis of 3c1 cloned standards and mixed standards for 3c1. (A) Representative fluorescence acquisition curves for 3c1 hybridization probes with dilutions of 3c1 standards (closed squares, 108 molecules; circles, 107 molecules; triangles, 106 molecules; and diamonds, 105). This is compared with pilin cDNA detected with 3c1 hybridization probes (star). The opened star is the no RT control for contaminating DNA. The cross symbol is the no template control (background level). (B) Fluorescence acquisition curves for 3c1 hybridization probes with dilutions of 3c1 cloned standards (closed squares, 108 molecules; circles, 107 molecules; triangles, 106 molecules; and diamonds, 105) and dilutions of 3c1 standards mixed with the 108 parental copy standard (2c1) (open squares, 108 3c1 molecules; circles, 107 3c1 molecules; triangles, 106 3c1 molecules; and diamonds, 105 3c1 molecules). Total pilin cDNA detected with 3c1 hybridization probes is indicated by the star.
It was unclear whether the frequency of recombination of each silent copy was too low to be quantified by this assay, or whether the complex mixture of cDNAs present in the experimental pilin cDNA presents a problem for the real-time RT–PCR technology. It was probably that there was an excess of either (1) any non-pilin competing template or (2) competing pilin sequences (predominately the parental copy) that interfered with amplification or detection of recombinant pilE sequences within the experimental sample. To test whether any non-pilin competing template interfered with detection, amplification curves of different dilutions of the pilE standard in question were mixed with recA cDNA, opa PCR product, or E.coli chromosomal DNA. These real-time RT–PCR amplification curves were similar in slope to the purified standards (data not shown), showing that an excess of non-pilin competing templates does not interfere with the assay. To determine whether the excess of pilE molecules carrying the parental HVL sequence interfered with detection, an excess of purified DNA molecules carrying the parental 2c1 HVL sequence was mixed with different amounts of purified template carrying the HVL sequence being detected. The mixed samples produced depressed fluorescence acquisition curves during real-time RT–PCR, which replicated those recorded using pilin cDNA (Figure 3B). These data suggest that the excess of parental 2c1 sequences in the pilin cDNA sample was preventing the lower concentration of recombinant molecules from being quantified using the hybridization probes. Since the mixed standards produced fluoresence acquisition curves with different slopes, depending on the amount of the specific standard present in the mixture, we asked whether these mixed standards could be used to estimate the amount of recombinant HVL sequence from each silent copy in a complex mixture of pilE sequences. All 16 HVL probes produced depressed amplification curves when the target HVL sequence was limiting within an excess of parental pilE templates, and for each probe set, the florescence acquisition curves became increasingly depressed as the level of the probe reactive sequence was decreased relative to the parental molecules (data not shown). With each probe set, 105 molecules of the minority sequence were detectable above background within the excess of 108 molecules of the 2c1 pilE template (data not shown).
These mixed standards were used to approximate the amount of recombinant pilE in a population, and the participation of each silent copy to the variation in the HVL region. Comparing each HVL probe set against a mixed standard carrying various amounts of each HVL standard mixed with 2c1 template, six additional silent copy sequences produced detectable amplification curves using pilE cDNA, while probes for the remaining 10 silent copy sequences detected nothing above background (data not shown). Pilin cDNA from a culture grown without IPTG, which does not undergo pilin Av, produced undetectable levels of amplification with all of the silent copy probes, showing that the amplification detected was RecA-dependent, as is pilin Av (data not shown). The contribution of all six detectable silent copy sequences to pilin variation was estimated by comparing the amplification of each HVL subpopulation to the mixed standard (Figure 3B). Surprisingly, the seven detectable HVL sequences were detected at differing frequencies in pilE. The HVL sequence originating from pilS2 copy 1 (3c1) was estimated to recombine at a frequency of 1.25% of the total pilE molecules. The other detectable HVL sequences were estimated to occur at a frequency of 0.60% for 1c1, 0.40% for 6c1 and 0.25% for 1c3, 2c6 and 3c3. The sum of the recombinant pilE molecules along with the parental pilE molecules together corresponded to the 108 molecules of pilE present in each sample. While these results strongly suggested that this assay was accurately describing the recombinant population of pilE variants, the validity of these findings was confirmed using a different approach.
Independent validation of the repertoire of silent pilin sequences producing pilin Av
To confirm the results of the SQ-PCR Av assay, individual colonies from the same population examined using the SQ-PCR Av assay were isolated by growing out individual clones from the population on solid medium without IPTG. Clones that had undergone pilin variation in the HVL region were identified by the absence of hybridization signal to an oligonucleotide specific for the parental 2c1 HVL sequence (Supplementary Table 2, LC-Hyb2c1 1). This hybridization screen confirmed that 97% of the 1055 colonies assayed retained 2c1 in the HVL (data not shown). The pilE gene was amplified from the 32 colonies that did not hybridize with the 2c1 probe and the pilE gene sequenced to determine the silent copies that had donated sequences to alter the HVL region. All of the 2c1 hybridization negative clones were found to express a new pilin variant with different silent copy sequences carried in the HVL region (Figure 4). Six non-parental silent gene HVL sequences were found in pilE using the sequencing assay, and this was a similar repertoire of donor silent copies as that determined by the SQ-PCR Av assay (Figure 4). The non-parental silent gene sequences found in pilE HVL often extended into the SV region (data not shown). None of the HVL sequences that were undetectable in the SQ-PCR assay was found in the sequenced variants (data not shown). While the exact frequency of each silent copy recombining into pilE was not identical in the two assays, they were strikingly similar—considering the semi-quantitative estimate of frequency used in the SQ-PCR Av assay and the limited number of colonies assayed by sequencing (Figure 4). From these data, we concluded that the SQ-PCR Av assay for pilin variation can accurately estimate the frequency of variation and reliably detect the silent copies that participate in producing pilin variation. These results show that within this one bacterial culture, only a subset of silent copies contributed to the 3% of variant pilE HVL sequences.
Figure 4 Analysis of the silent copies recombining into the HVL of 2c1. (A) Detection of donor silent pilin copies using the SQ-PCR Av assay for N.gonorrhoeae strain FA1090 1-81-S2 recA6 grown for 22 h on solid media in the presence of IPTG. (B) Detection of donor silent pilin copies using the pilE sequencing assay for N. gonorrhoeae strain FA1090 1-81-S2 recA6 grown for 22 h on solid media in the presence of IPTG. The parental copy sequence, 2c1, was retained in 97% of the pilE templates in both assays, and the proportion of silent copy donors to the 3% of variant pilE HVL sequences is shown.
Having verified the findings of SQ-PCR Av assay, the reproducibility of the non-uniform donation of silent copy sequences was assayed in repeat experiments using the same starting pilin variant within replicate bacterial cultures. Two cultures of N.gonorrhoeae strain FA1090 1-81-S2 recA6 were grown with or without IPTG for 22 h in two separate experiments, and two cultures were grown on separate days and each sampled at 22, 26 and 30 h. In each case, growth without IPTG resulted in no detectable recombination in pilE. Similar copies were detected in all four IPTG grown cultures at 22 h (Figure 5). The most prevalent gene copy detected was the parental copy, 2c1. Other detectable gene copies included 1c1, 2c6, 3c1, 3c2, 3c3 and 6c1. Essentially the same subset of silent copies recombined into the pilE HVL in replicate cultures, although the exact frequency for each silent copy sequence did vary from culture to culture, and in some cultures a new silent copy was detected (Figure 5). Taken together, these results show that the spectrum of pilE HVL variants produced during pilin Av is not random and that some silent copies are over represented, while others are under represented during pilin variation.
Figure 5 Similar silent copies are detected in separate experiments over time. Detection of newly recombined silent pilin copies using the SQ-PCR Av assay for N.gonorrhoeae strain FA1090 1-81-S2 recA6 with 2c1 as the parental pilE sequence grown for 22 (A–D), 26 (C and D) and 30 (D) h on solid media in the presence of IPTG in four separate experiments. The parental copy, 2c1, was retained in 95% (A and B) and 94% (C and D) of the pilE cDNA templates. The graphs represent 5–6% of the pilE transcripts that have changed from parental. The legend depicts the silent HVL sequences detected in pilE.
The starting pilin sequence influences the frequency of pilin variation and the spectrum of silent copies acting as donors
There were two main explanations for why a reproducible, nonrandom recombination of the silent copy HVL sequences into pilE was observed. Either the starting pilE sequence dictates the repertoire of the donor silent copies used as recombination donors, or the nonrandom use of silent copy donors reflects a mechanistic limit on recombination that is independent of the expressed gene. In order to differentiate between these possibilities, a FA1090 1-81-S2 recA6 pilin variant containing the 3c3 sequence within the entire pilE locus was recovered from the sequencing assay and used as the parental variant in the SQ-PCR Av assay. The 3c3 variant is 80% identical to the 2c1 variant which is a lower percent identity than a majority of the other silent copy sequences (data not shown).
FA1090 1-81-S2(3c3) recA6 was analyzed using the SQ-PCR Av assay as described above using the mixed standards to estimate the overall frequency of pilin variation and to measure the recombination potential of each silent copy. The 3c3 variant showed a much higher Av frequency than the 2c1 variant, with 12% HVL variation after 22 h of IPTG induction as compared with 3–5% variation with the 2c1 variant (Figures 5 and 6). Analysis of the contribution of each silent copy to variants derived from the 3c3 variant demonstrated a different repertoire of silent copies than were found during the 2c1 variation assays. Not only was the repertoire of silent copies arising during Av different between the 2c1 and 3c3 variants, but a broader representation of silent copy donors was found with the 3c3 parental sequence than the 2c1 variant, and the distribution of silent copy donors was more diverse (Figures 5 and 6).
Figure 6 A different representation of silent copies is detected with a different parental pilE sequence. Detection of newly recombined silent pilin copies using the SQ-PCR Av assay using three replicate cultures (panels A–C) of strain FA1090 1-81-S2(3c3) S2 recA6 grown for 22 h on solid media in the presence of IPTG. The parental silent copy was calculated to be 88% of the total number of copies (data not shown). The graphs represent the remaining 12% of the pilE transcripts that have changed from parental. The legend depicts the silent HVL sequences detected in pilE.
To confirm the results of the SQ-PCR Av assay with the 3c3 variant, the frequency and spectrum of variants produced in the 3c3 variant were analyzed by sequencing the pilE gene of random colonies isolated after IPTG induction of RecA. A level of 9% variation in the HVL was detected by the sequencing assay as opposed to 12% variation measured by the SQ-PCR Av assay. Considering the limitation of both assays, these values are in reasonable agreement. New silent sequences were found in both the SV and HVT regions, verifying that the SQ-PCR Av assay only records a subset of the total pilE variation. The greater repertoire of silent copy donors was also observed in the sequencing assay (data not shown). We conclude that the parental pilE sequence influences both the frequency of variation and the diversity of variants produced.
DISCUSSION
A SQ-PCR Av assay was developed to detect all pilS to pilE recombination at the HVL region in a population of Gc. The complex nature of the pilin cDNA isolated from Gc prevented efficient real-time RT–PCR analysis even though the primers and probes worked as expected on purified standards. This inefficient real-time RT–PCR was not owing to the use of hybridization probes since we obtained similar results using Taqman probes (data not shown). It has not been investigated whether other detection technologies could provide increased specificity to allow a more quantitative assay using purified standards. It is most likely that even though most of the HVL sequences are divergent (Supplementary Figure 1), when the parental sequence is in excess it out competes with the real target sequence during the hybridization step and therefore prevents efficient detection.
By using mixed standards, the complex mixture of pilin cDNA templates was mimicked and the frequency with which each detectable silent copy contributed to pilin Av was determined. The accuracy of these estimates relies on comparing the amplification of mixed standards with pilin cDNA samples and can be estimated to be within a 3-fold of the real level and often as close as 2-fold. While frequencies produced are not precise, they allow a good estimate of the relative use of all detected silent copies as donors for pilin Av. The silent copies that were undetected may also recombine into pilE and produce variants, just at a level undetectable by either assay. The actual frequency with which these undetected silent copies participate in variation at pilE is unknown, but is at least 5-fold lower than the least frequent detected copy, and therefore the variants carrying the undetected copies are truly under represented in this bacterial population. However, even if these under represented copies participate in pilin Av rarely, they could still contribute to bacterial colonization if they are not recognized by an adaptive immune response.
It is not clear why the starting pilE sequence influences the frequency at which silent copies recombine into pilE. There was no pattern for the donor silent copies recombined more or less frequently in relationship to their position (upstream or downstream) to pilE or their position in a silent locus. As a homologous recombination mediated process (34), pilin Av may respond to the ability of the pilE recipient and the donor silent copy sequence to allow heteroduplex formation due to sequence similarity. The silent copies that recombined into pilE with the 2c1 starting pilE variant did show a higher degree of sequence similarity to the 2c1 variant (Clustal W, AlignX, Vector NTI) compared with the silent copies that recombined less frequently. However, these sequence similarity predictions were not supported with the donor silent copy spectrum observed with the 3c3 starting pilE variant. Therefore, no rules based on the two examples examined here can be established.
The use of this SQ-PCR Av assay has resulted in several important conclusions about the mechanism of pilin Av. First, the recombination potential of different silent pilin gene copies with pilE is not uniform. This observation means that within a limited population of gonococci, immune responses are likely to be raised only to a subset of potential pilin variants. Initially, this would allow the remainder of the pilin variants to contribute to escape of the immune response and be saved for future immune evasion. However, the silent copies that recombine at a very low frequency may become targeted over time as the immune response recognizes and eliminates the more dominant variants. Second, we have clearly demonstrated that the parental pilE sequence can influence the frequency and repertoire of pilin variants arising in a population. This observation suggests that particular pilin variants may act in a unique manner during a gonococcal infection, which could explain the various clinical manifestations of the disease in different individuals.
SUPPLEMENTARY MATERIAL
Supplementary Material is available at NAR Online.
ACKNOWLEDGEMENTS
The authors thank Alison Criss and Kimberly Kline for comments on this work and the manuscript. This work was supported by NIH grants R01/R37 AI33443 and R01 AI44239. Funding to pay the Open Access publication charges for this article was provided by the NIH.
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*To whom correspondence should be addressed. Tel: +1 312 503 9788; Fax: +1 312 503 1339; Email: h-seifert@northwestern.edu
ABSTRACT
A semi-quantitative real-time RT–PCR assay was designed to measure gonococcal pilin antigenicvariation (SQ-PCR Av assay). This assay employs 17 hybridization probe sets that quantitate subpopulations of pilin transcripts carrying different silent pilin copy sequences and one set that detects total pilE transcript levels. Mixtures of a DNA standard carrying the silent copy being detected and a clone encoding the starting pilE sequence, which is the majority pilE template, provided amplification curves that closely matched the experimental data and allowed an analysis of the contribution of different silent pilin copies to variation. The SQ-PCR Av assay was verified using DNA sequence analysis to demonstrate that this methodology allowed an accurate analysis of pilin variation. Both assays showed that with a specific starting pilE sequence, only a subset of the silent pilin copies recombine into pilE at a detectable level, and that this limited subset was reproducibly detected in replicate cultures. When an isogenic pilE sequence variant was examined using both assays, a new subset of silent copy sequences were detected recombining into pilE and the overall frequency of variation was increased. Thus, the parental pilE sequence influences the frequency of variation and the repertoire of pilin variants produced.
INTRODUCTION
Neisseria gonorrhoeae (also called Gc or the gonococcus) is a Gram-negative diplococcal bacterium and is the causative agent of the human sexually transmitted disease gonorrhea. No vaccines for gonorrhea exist in part because N.gonorrhoeae alters some of its surface elements, allowing it to evade the human immune system (1,2). Gonococcal attachment to human epithelial cells is mediated primarily by pili, surface structures composed of multiple units of the protein pilin (3,4). Pili mediate adherence to a number of different eukaryotic cell types, affect interactions with epithelia, endothelia and phagocytes (5), and are also required for full DNA transformation competence (6–8).
Pilin is encoded by the pilE gene present at a single expression locus in the strain FA1090. In addition to pilE, FA1090 also contains a total of 18 silent pilin gene copies present in 5 pilS loci and 1 silent pilin gene copy present in the pilE locus (Figure 1A) (9). These silent copies and pilE share a central semivariable (SV) region and 3' hypervariable (HV) region, but lack the 5' constant region and promoter found only at pilE. The 3' HV region can be further subdivided into a HV loop (HVL) and a HV tail (HVT). The HVL is flanked by two conserved regions, cys1 and cys2 (Figure 1B) (10).
Figure 1 Cartoons showing the pilin loci and the basis for the SQ-PCR assay. (A) Pilin loci of gonococcal strain FA1090. The N.gonorrhoeae strain FA1090 chromosome contains one pilin expression locus, pilE, and five silent pilin loci, pilS. The pilE locus contains the pilin expression gene pilE (light blue rectangle) and the upstream silent pilin copy (white rectangle). The wavy line indicates the pilE transcript. Each of the five pilS loci contain between one and six silent pilin gene copies (colored rectangles). The silent copies are shown using an abbreviated nomenclature. Silent pilin locus 1 (pilS1) gene copy 2 is shown as 1c2, pilS3 copy 3 is 3c3, etc. (B) The SQ-PCR Av assay to measure recombination at pilE. Five general portions of the pilE gene are shown: the 5' constant region, a central SV region, the conserved cys1 and cys2 regions that flank the HVL, which is followed by the hypervariable tail (HVT) (16). The wavy line indicates the transcript encoded by the pilE gene and the rectangles indicate coding regions of DNA. Primers are shown as arrows, and the sequence targeted by the pairs of hybridization probes is starred. The 5' primers are used with the pilE constant region hybridization probes, LC-HybCONS 1 and 2, and detect total pilE transcript levels. The 3' primers are used with the 17 pairs of pilS HV hybridization probes and detect individual pilS to pilE recombination at the HVL of pilE.
Pilin antigenic variation (Av) is defined as the high frequency change of amino acid residues in the pilin protein (11,12). During pilin Av, a portion of pilS replaces a portion of pilE, often producing a new pilin that is efficiently expressed on the cell surface. This RecA-dependent recombination of a pilS sequence into pilE is unidirectional because the donor pilS sequence remains unchanged while the recipient sequence is lost from pilE (13,14). Several conserved DNA segments that have been reported to be important for pilin Av include the cys2 sequence (15), as well as the correct spacing between cys1 and cys2 (16), and the 3'-untranscribed sequence of the Sma/Cla repeat (17). Av at pilE also requires RecA, and the RecF-like pathway of homologous recombination (18–20), but not the RecBCD pathway (18). The Rep helicase (21) and the RecA modulator RecX (22) are involved in pilin Av, but the replication restart protein PriA is not involved (23).
Many assays have been developed to analyze pilS to pilE recombination, but each has its shortcomings (17,24). The most common assay used to measure pilin Av uses the frequency of colonies appearing with a non-piliated colony morphology from a piliated population to estimate the frequency of pilin Av (18,19,22). However, this method underestimates the frequency of Av because not all changes in pilE lead to changes in colony morphology. To analyze pilin variation independent of colony morphology changes, Wainwright et al. (17) developed an RT–PCR-based assay to measure the transfer of silent copy sequences into pilE in a population of bacteria. Briefly, the assay measured the relative quantity of total and recombinant pilE by measuring the presence of new HVL sequences from either pilS1 copy 4 or pilS6 copy 1. Subsequently, Serkin et al. (24) improved upon the existing qualitative assay by mixing target and competitor RNA prior to reverse transcription such that competitor template served as an internal control for RT and PCR efficiency. This quantitative, competitive RT–PCR assay was capable of detecting recombination from multiple sources, yet was incomplete in that not all silent pilin copies were detected or could be individually assayed. Thus, in order to detect the contribution of all silent pilin copies individually, a real-time semi-quantitative RT–PCR (SQ-PCR Av) assay was designed to study pilS to pilE recombination.
This SQ-PCR Av assay was used to demonstrate that with a defined starting pilE sequence only a subset of the silent pilin copies are prevalent donors for pilin variation even between replicate cultures. Furthermore, with a different parental pilE sequence, a different representation of silent copy sequences was detected recombining into pilE, and the overall frequency also changed. Thus, the repertoire of silent copies that donate their sequences to create pilin variants depends on the parental sequence. These data demonstrated that a real-time RT–PCR assay can be used to investigate the mechanisms leading to antigenic variation of pilin in N.gonorrhoeae and shows that the starting pilE sequence has a major influence on the variants produced.
MATERIALS AND METHODS
Generation of SQ-PCR Av standards
The hypervariable loop (HVL) regions of all pilS copies were previously cloned into the pGEM-3 vector (24). These plasmids served as standards for the SQ-PCR Av assay. Clone E1r was a gift from Terri Hamrick and was used as a standard for assaying total pilE transcript levels (9). Clone pGADT7 + GcRecA#9 was used as a standard for the recA control (25). All plasmid standards were linearized by digestion with HincII (New England Biolabs). The linearized plasmid standards and Low DNA Mass Ladder (Invitrogen), which has a known concentration, were then run in a 0.8% agarose gel stained with ethidium bromide. Densitometry was performed using ImageQuant (Molecular Dynamics) to quantify the linear plasmid standards. Furthermore, the plasmid standards were normalized using SYBR Green I DNA dye and the LightCycler instrument. Molecules of the gene of interest were calculated as described previously (26), and serial dilutions representing 108–101 molecules were generated. Pilin competing-template mixed standards were generated by mixing the silent copy standard of interest with the parental copy standard at the total pilE transcript level. Non-pilin competing-template mixed standards were produced with opa PCR product, recA cDNA and Escherichia coli chromosome, each at the total pilE transcript level.
Bacterial strains and growth conditions
Gonococcal strain FA1090 recA6 pilin variants 1-81-S2 (27) and 1-81-S2(3c3) (this study) were used in all of the experiments. FA1090 recA6 contains an isopropyl-?-D-thiogalactopyranoside (IPTG)-regulatable gonococcal recA allele that produces a phenotypically RecA-deficient strain without IPTG in the growth medium and a RecA-proficient strain when grown in the presence of 1 mM IPTG that is indistinguishable from wild-type recA strain (28). Gonococci were grown on Gc Medium Base (Difco) with Kellogg supplements (GCB) (29) and 1 mM IPTG (Diagnostic Chemicals Ltd) at 37°C in 5% CO2 for 22, 26 or 30 h.
RNA extraction and reverse transcription
Gonoccoci were swabbed from solid media into ice-cold phosphate-buffered saline (PBS), and Trizol extraction of total RNA was performed according to the supplier's instructions (Invitrogen). DNase treatment was performed twice (24), followed by RNA purification using the RNeasy RNA cleanup protocol (Qiagen) as described previously (24). RNA was run in a 0.8% agarose gel stained with ethidium bromide to partially quantitate and assess the stability of the RNA. 28S ribosomal RNA bands were compared and normalized by performing densitometry with ImageQuant software (Molecular Dynamics). After RNA levels from the experimental samples were normalized, cDNA synthesis was performed as described by Serkin and Seifert (24) using the primer LC-CYS2rev for the 3' HV region of pilE, LC-CONSrev for the 5' constant region of pilE or LC-recArev for recA (Supplementary Material). All reverse transcription reactions were performed with a no reverse transcriptase control in order to control for contaminating DNA in the SQ-PCR Av assay. The reverse transcription reaction was purified using the Qiaquick PCR Purification Kit (Qiagen).
Oligonucleotides
All oligonucleotides used are listed in Supplementary Table 1. The forward primer LC-CYS1for and reverse primer LC-CYS2rev hybridize to two previously characterized pilin regions, cys1 and cys2 (30), conserved in all N.gonorrhoeae pilE and pilS copies (9). LC-CYS1for and LC-CYS2rev were used in conjunction with the silent pilin copy probes (Supplementary Material). A second primer set consisting of forward primer LC-CONSfor and LC-CONSrev anneals to the 5' constant region of the N.gonorrhoeae pilE gene and was used in conjunction with the pilE 5' constant region hybridization probes to detect total pilE transcript levels. The third primer set consists of forward primer LC-recAfor and reverse primer LC-recArev and anneals to the N.gonorrhoeae recA gene. The recA primers were used in conjunction with the recA hybridization probes to detect recA transcript levels, which served as a control for RNA normalization. All primers were reverse phase high-performance liquid chromatography purified (IT Biochem). Primer and probe melting temperatures were calculated using the freeware program TM Utility v.1.3 (Idaho Technology, http://www.idahotech.com/downloads_up/pTmutility_form.htm).
Hybridization probes were designed to specifically target 17 of the 18 silent pilin gene copies (9). An alignment of all pilS copies using the software program AlignX (InforMax, Inc.) allowed unique sequences within the HVL to be identified as potential hybridization probe targets for each silent pilin copy (Supplementary Figure 1). Because the HVL sequences share sequence similarity (Supplementary Material), probe target sequences for each silent copy were compared against all the other FA1090 silent pilin gene copies (using the BLAST program) to ensure that the hybridization probe sequences specifically hybridize only to the targeted silent copy. The HVL sequence of one silent pilin copy, pilS1 copy 5, is identical to the HVL sequence of pilS2 copy 5. Thus, no pilS1 copy 5 hybridization probes were designed; instead, the pair of pilS2 copy 5 hybridization probes detects both pilS1 copy 5 and pilS2 copy 5. The distance between the donor and acceptor probes in all probe sets was 1 base (31). All donor probes were labeled with fluorescein at the 3' end, while all acceptor probes were labeled at the 5' end with LightCycler-Red 640-N-hydroxysuccinimide ester. Acceptor probes also had phosphate groups attached to the 3' end to prevent probe extension (IT Biochem).
Each of the pairs of hybridization probes used the same primer pair for amplification (Figure 1B). Clones carrying the HVL sequence of each silent copy bordered by the conserved cys1 and cys2 priming sites were isolated for use as standards to quantitate the proportion of each silent HVL sequence that had transferred into the pilE of an experimental sample. The purified plasmid standards were gel quantitated as described above, and 10-fold dilutions of these standards were used in each SQ-PCR Av experiment.
The annealing temperature of the hybridization probe pairs were adjusted to yield maximal detection of the targeted silent pilin gene copy sequence while concurrently minimizing undesired hybridization to sequences from the other silent pilin gene copies. Pairs of hybridization probes were deemed optimized when they met two conditions: (i) that all other untargeted silent pilin gene copies were detected at levels <1% of the targeted copy (data not shown) and (ii) that the detection limit was 105 copies of linear plasmid mixed standard or less.
Analysis of Southern colony variants and sequencing for the verification of the SQ-PCR Av data
FA1090 1-81-S1 recA6 was grown at 37°C in CO2 on GCB agar +/– IPTG for 22 h and was swabbed into ice-cold PBS and plated onto GCB agar. After 22 h, 1055 individual colonies of Gc were selected and frozen in 15 μl of GCBG (GCB with 20% glycerol). The frozen stocks of the individual colonies of Gc were passaged onto GCB agar. Individual colonies from each revived culture were lysed (1% Triton X-100, 2 mM Tris, pH 8, 2 mM EDTA) and the pilE gene was PCR amplified using PILRBS (17) and SP3A (32) in a PTC-100 thermocycler (MJ Research). The pilE PCR products were electrophoresed on a 0.8% TBE agarose gel, denatured, neutralized and blotted to nylon as described previously (33). The blots were probed with a 3' end labeled digoxigenin-11-ddUTP (Boehringer Mannheim) oligonucleotide specific to the parental FA1090 1-81-S2 recA6 HVL sequence (Supplementary Material, LC-Hyb2c1 1). The blots were probed at 67°C for 2 h and were washed with 0.1x SSC, 0.1% SDS twice at 67°C for 20 min and twice with 0.5x SSC, 0.1% SDS at room temperature. The blots were exposed to Kodak BioMax XAR film. A positive control, pilE amplified PCR product of FA1090 1-81-S2 recA6, and a negative control, pilE amplified PCR product of RM11.2 (6c1 in the HVL) were included in each Southern blot. About 3% of the isolates did not hybridize to the 2c1 parental probe in the HVL. These isolates were sequenced to determine the sequence changes in the pilE HVL. Furthermore, 5% of randomly selected 2c1 positive clones were sequenced and found to carry the 2c1 HVL sequence, showing that we were not missing any HVL variants. For sequencing, the PCR products were treated with shrimp alkaline phosphatase and exonuclease 1 to remove primers and dNTPs. PCR-generated pilin templates (16–33 ng) were used in each sequencing reaction with the PILRBS primer (17) and were analyzed using a Beckman Coulter CEQ 2000XL DNA system as specified by the manufacturer (Beckman).
Sequencing assay of antigenic variation for validation of the SQ-PCR Av assay
A sequencing assay was performed to verify the results of the SQ-PCR Av assay when using the strain FA1090 1-81-S2(3c3) recA6. This assay was performed as described previously (23). Briefly, FA1090 1-81-S2 recA6 and variant 1-81-S2 3c3 recA6 were grown on plates containing IPTG and incubated at 37°C in 5% CO2 for 20 h. Five starter colonies from each variant were then passaged onto separate GCB plates. A total of 45–50 colonies arising from each starting colony were passaged two more times to ensure clonal populations, and the pilE gene of each colony was sequenced as described above.
Real-time RT–PCR and data analysis
Real-time RT–PCR was performed on the LightCycler instrument (Roche Diagnostics). All assays used hybridization probes containing 2 μl of LightCycler DNA Master Hybridization Probes mix (Roche Diagnostics), 3.2 μl of MgCl2 (for a final concentration of 5 mM), 2 μl of each primer (0.5 mM), 2 μl of donor hybridization probe (0.2 mM), 2 μl of acceptor hybridization probe (0.2 mM), 2 μl of template and PCR-grade sterile water for a final volume of 20 μl. PCR amplification for the hybridization probes assays began with initial denaturation (30 s at 95°C) followed by 40 cycles of denaturation (2 s at 95°C), annealing (56–66°C for silent copy probes, 53°C for pilE and 60°C for recA) and extension (5 s at 72°C). For the optimized annealing temperatures for each pair of hybridization probes see Supplementary Material. Fluorescence in channels F1 and F2 was acquired at the end of every extension step, and the ratio of F2/F1 was analyzed.
Data were exported from LightCycler software and imported into Excel (Microsoft). Standard curves, P-values and Pearson Correlation Coefficients ‘R’ were generated using the Fit linear function. Pie charts were generated in Microcal Origin (Microcal Software Inc.).
RESULTS
Development of a SQ-PCR Av assay to detect sequence changes in the pilE HVL
Many assays have been developed to measure the frequency of pilin Av, but each has limitations (17,20,24). Each previously developed Av assay underestimates the frequency of variation and records only a small proportion of the possible recombination events comprising pilin Av. The specificity supplied by the hybridization step of real-time RT–PCR was used to attempt to detect recombination of all 18 silent gene copies into the pilE gene within a large population of bacteria.
A total of 18 pairs of hybridization probes were designed, one probe set reacted specifically with the constant region only found in pilE and the other 17 probe sets were designed to measure the level of the 18 silent copy HVL sequences (two HVL sequences are identical) in a population of gonococci. To test whether the hybridization probes could detect recombinant pilE sequences, N.gonorrhoeae strain FA1090 1-81-S2 recA6 was grown on solid media with or without 1 mM IPTG to control pilin Av. Total RNA was extracted and then reverse transcribed to pilin cDNA for real-time RT–PCR analysis. Pilin cDNA was normalized using the conserved region real-time RT–PCR primers and probes and adjusted to a concentration of 1 x 108 molecules per reaction. Once pilin cDNA was normalized, real-time RT–PCR was performed to quantify the parental 2c1 HVL sequence (Figure 2). Fluorescent acquisition curves of the cDNA from the experimental 22 h pilin cDNA sample of Gc was similar in slope and character to the purified standards during the early amplification cycles, although in the later cycles the slopes diverged between the pilin cDNA and the standards. During the earlier cycles, the experimental cDNA sample was estimated to contain 9.7 x 107 molecules carrying the parental 2c1 sequence. This measurement suggested that 97% of the pilE genes in this population retained the parental 2c1 sequence, while 3% had recombined with silent copies to produce variant pilE genes with a changed HVL. To investigate this further, real-time RT–PCR was used to measure the appearance of new HVL sequences into pilE from the other silent copies. However, fluorescent acquisition curves using the HVL probes all demonstrated a severely depressed slope that could not be compared with or quantified using the purified standard curves (Figure 3A). Some of the silent copy probes produced a signal above background (Figure 3A), while other silent copies were undetectable using these probes (data not shown).
Figure 2 Real-time RT–PCR measurement of total pilE transcript levels. The fluorescence acquisition curves are plots of the fluorescence detected versus cycle number. The most concentrated standard (108 molecules, squares) begins to rise above background at approximately cycle number 13. The closed symbols represent dilutions of the purified standard (squares, 108 molecules; circles, 107 molecules; triangles, 106 molecules; and diamonds, 105 molecules). The cross symbol indicates the no template control, which contained the master mixture and water instead of template. The star indicates the experimental sample of cDNA of Gc grown for 22 h on plates with IPTG to induce RecA expression and antigenic variation and is superimposed on the 108 standard curve.
Figure 3 RT–PCR analysis of 3c1 cloned standards and mixed standards for 3c1. (A) Representative fluorescence acquisition curves for 3c1 hybridization probes with dilutions of 3c1 standards (closed squares, 108 molecules; circles, 107 molecules; triangles, 106 molecules; and diamonds, 105). This is compared with pilin cDNA detected with 3c1 hybridization probes (star). The opened star is the no RT control for contaminating DNA. The cross symbol is the no template control (background level). (B) Fluorescence acquisition curves for 3c1 hybridization probes with dilutions of 3c1 cloned standards (closed squares, 108 molecules; circles, 107 molecules; triangles, 106 molecules; and diamonds, 105) and dilutions of 3c1 standards mixed with the 108 parental copy standard (2c1) (open squares, 108 3c1 molecules; circles, 107 3c1 molecules; triangles, 106 3c1 molecules; and diamonds, 105 3c1 molecules). Total pilin cDNA detected with 3c1 hybridization probes is indicated by the star.
It was unclear whether the frequency of recombination of each silent copy was too low to be quantified by this assay, or whether the complex mixture of cDNAs present in the experimental pilin cDNA presents a problem for the real-time RT–PCR technology. It was probably that there was an excess of either (1) any non-pilin competing template or (2) competing pilin sequences (predominately the parental copy) that interfered with amplification or detection of recombinant pilE sequences within the experimental sample. To test whether any non-pilin competing template interfered with detection, amplification curves of different dilutions of the pilE standard in question were mixed with recA cDNA, opa PCR product, or E.coli chromosomal DNA. These real-time RT–PCR amplification curves were similar in slope to the purified standards (data not shown), showing that an excess of non-pilin competing templates does not interfere with the assay. To determine whether the excess of pilE molecules carrying the parental HVL sequence interfered with detection, an excess of purified DNA molecules carrying the parental 2c1 HVL sequence was mixed with different amounts of purified template carrying the HVL sequence being detected. The mixed samples produced depressed fluorescence acquisition curves during real-time RT–PCR, which replicated those recorded using pilin cDNA (Figure 3B). These data suggest that the excess of parental 2c1 sequences in the pilin cDNA sample was preventing the lower concentration of recombinant molecules from being quantified using the hybridization probes. Since the mixed standards produced fluoresence acquisition curves with different slopes, depending on the amount of the specific standard present in the mixture, we asked whether these mixed standards could be used to estimate the amount of recombinant HVL sequence from each silent copy in a complex mixture of pilE sequences. All 16 HVL probes produced depressed amplification curves when the target HVL sequence was limiting within an excess of parental pilE templates, and for each probe set, the florescence acquisition curves became increasingly depressed as the level of the probe reactive sequence was decreased relative to the parental molecules (data not shown). With each probe set, 105 molecules of the minority sequence were detectable above background within the excess of 108 molecules of the 2c1 pilE template (data not shown).
These mixed standards were used to approximate the amount of recombinant pilE in a population, and the participation of each silent copy to the variation in the HVL region. Comparing each HVL probe set against a mixed standard carrying various amounts of each HVL standard mixed with 2c1 template, six additional silent copy sequences produced detectable amplification curves using pilE cDNA, while probes for the remaining 10 silent copy sequences detected nothing above background (data not shown). Pilin cDNA from a culture grown without IPTG, which does not undergo pilin Av, produced undetectable levels of amplification with all of the silent copy probes, showing that the amplification detected was RecA-dependent, as is pilin Av (data not shown). The contribution of all six detectable silent copy sequences to pilin variation was estimated by comparing the amplification of each HVL subpopulation to the mixed standard (Figure 3B). Surprisingly, the seven detectable HVL sequences were detected at differing frequencies in pilE. The HVL sequence originating from pilS2 copy 1 (3c1) was estimated to recombine at a frequency of 1.25% of the total pilE molecules. The other detectable HVL sequences were estimated to occur at a frequency of 0.60% for 1c1, 0.40% for 6c1 and 0.25% for 1c3, 2c6 and 3c3. The sum of the recombinant pilE molecules along with the parental pilE molecules together corresponded to the 108 molecules of pilE present in each sample. While these results strongly suggested that this assay was accurately describing the recombinant population of pilE variants, the validity of these findings was confirmed using a different approach.
Independent validation of the repertoire of silent pilin sequences producing pilin Av
To confirm the results of the SQ-PCR Av assay, individual colonies from the same population examined using the SQ-PCR Av assay were isolated by growing out individual clones from the population on solid medium without IPTG. Clones that had undergone pilin variation in the HVL region were identified by the absence of hybridization signal to an oligonucleotide specific for the parental 2c1 HVL sequence (Supplementary Table 2, LC-Hyb2c1 1). This hybridization screen confirmed that 97% of the 1055 colonies assayed retained 2c1 in the HVL (data not shown). The pilE gene was amplified from the 32 colonies that did not hybridize with the 2c1 probe and the pilE gene sequenced to determine the silent copies that had donated sequences to alter the HVL region. All of the 2c1 hybridization negative clones were found to express a new pilin variant with different silent copy sequences carried in the HVL region (Figure 4). Six non-parental silent gene HVL sequences were found in pilE using the sequencing assay, and this was a similar repertoire of donor silent copies as that determined by the SQ-PCR Av assay (Figure 4). The non-parental silent gene sequences found in pilE HVL often extended into the SV region (data not shown). None of the HVL sequences that were undetectable in the SQ-PCR assay was found in the sequenced variants (data not shown). While the exact frequency of each silent copy recombining into pilE was not identical in the two assays, they were strikingly similar—considering the semi-quantitative estimate of frequency used in the SQ-PCR Av assay and the limited number of colonies assayed by sequencing (Figure 4). From these data, we concluded that the SQ-PCR Av assay for pilin variation can accurately estimate the frequency of variation and reliably detect the silent copies that participate in producing pilin variation. These results show that within this one bacterial culture, only a subset of silent copies contributed to the 3% of variant pilE HVL sequences.
Figure 4 Analysis of the silent copies recombining into the HVL of 2c1. (A) Detection of donor silent pilin copies using the SQ-PCR Av assay for N.gonorrhoeae strain FA1090 1-81-S2 recA6 grown for 22 h on solid media in the presence of IPTG. (B) Detection of donor silent pilin copies using the pilE sequencing assay for N. gonorrhoeae strain FA1090 1-81-S2 recA6 grown for 22 h on solid media in the presence of IPTG. The parental copy sequence, 2c1, was retained in 97% of the pilE templates in both assays, and the proportion of silent copy donors to the 3% of variant pilE HVL sequences is shown.
Having verified the findings of SQ-PCR Av assay, the reproducibility of the non-uniform donation of silent copy sequences was assayed in repeat experiments using the same starting pilin variant within replicate bacterial cultures. Two cultures of N.gonorrhoeae strain FA1090 1-81-S2 recA6 were grown with or without IPTG for 22 h in two separate experiments, and two cultures were grown on separate days and each sampled at 22, 26 and 30 h. In each case, growth without IPTG resulted in no detectable recombination in pilE. Similar copies were detected in all four IPTG grown cultures at 22 h (Figure 5). The most prevalent gene copy detected was the parental copy, 2c1. Other detectable gene copies included 1c1, 2c6, 3c1, 3c2, 3c3 and 6c1. Essentially the same subset of silent copies recombined into the pilE HVL in replicate cultures, although the exact frequency for each silent copy sequence did vary from culture to culture, and in some cultures a new silent copy was detected (Figure 5). Taken together, these results show that the spectrum of pilE HVL variants produced during pilin Av is not random and that some silent copies are over represented, while others are under represented during pilin variation.
Figure 5 Similar silent copies are detected in separate experiments over time. Detection of newly recombined silent pilin copies using the SQ-PCR Av assay for N.gonorrhoeae strain FA1090 1-81-S2 recA6 with 2c1 as the parental pilE sequence grown for 22 (A–D), 26 (C and D) and 30 (D) h on solid media in the presence of IPTG in four separate experiments. The parental copy, 2c1, was retained in 95% (A and B) and 94% (C and D) of the pilE cDNA templates. The graphs represent 5–6% of the pilE transcripts that have changed from parental. The legend depicts the silent HVL sequences detected in pilE.
The starting pilin sequence influences the frequency of pilin variation and the spectrum of silent copies acting as donors
There were two main explanations for why a reproducible, nonrandom recombination of the silent copy HVL sequences into pilE was observed. Either the starting pilE sequence dictates the repertoire of the donor silent copies used as recombination donors, or the nonrandom use of silent copy donors reflects a mechanistic limit on recombination that is independent of the expressed gene. In order to differentiate between these possibilities, a FA1090 1-81-S2 recA6 pilin variant containing the 3c3 sequence within the entire pilE locus was recovered from the sequencing assay and used as the parental variant in the SQ-PCR Av assay. The 3c3 variant is 80% identical to the 2c1 variant which is a lower percent identity than a majority of the other silent copy sequences (data not shown).
FA1090 1-81-S2(3c3) recA6 was analyzed using the SQ-PCR Av assay as described above using the mixed standards to estimate the overall frequency of pilin variation and to measure the recombination potential of each silent copy. The 3c3 variant showed a much higher Av frequency than the 2c1 variant, with 12% HVL variation after 22 h of IPTG induction as compared with 3–5% variation with the 2c1 variant (Figures 5 and 6). Analysis of the contribution of each silent copy to variants derived from the 3c3 variant demonstrated a different repertoire of silent copies than were found during the 2c1 variation assays. Not only was the repertoire of silent copies arising during Av different between the 2c1 and 3c3 variants, but a broader representation of silent copy donors was found with the 3c3 parental sequence than the 2c1 variant, and the distribution of silent copy donors was more diverse (Figures 5 and 6).
Figure 6 A different representation of silent copies is detected with a different parental pilE sequence. Detection of newly recombined silent pilin copies using the SQ-PCR Av assay using three replicate cultures (panels A–C) of strain FA1090 1-81-S2(3c3) S2 recA6 grown for 22 h on solid media in the presence of IPTG. The parental silent copy was calculated to be 88% of the total number of copies (data not shown). The graphs represent the remaining 12% of the pilE transcripts that have changed from parental. The legend depicts the silent HVL sequences detected in pilE.
To confirm the results of the SQ-PCR Av assay with the 3c3 variant, the frequency and spectrum of variants produced in the 3c3 variant were analyzed by sequencing the pilE gene of random colonies isolated after IPTG induction of RecA. A level of 9% variation in the HVL was detected by the sequencing assay as opposed to 12% variation measured by the SQ-PCR Av assay. Considering the limitation of both assays, these values are in reasonable agreement. New silent sequences were found in both the SV and HVT regions, verifying that the SQ-PCR Av assay only records a subset of the total pilE variation. The greater repertoire of silent copy donors was also observed in the sequencing assay (data not shown). We conclude that the parental pilE sequence influences both the frequency of variation and the diversity of variants produced.
DISCUSSION
A SQ-PCR Av assay was developed to detect all pilS to pilE recombination at the HVL region in a population of Gc. The complex nature of the pilin cDNA isolated from Gc prevented efficient real-time RT–PCR analysis even though the primers and probes worked as expected on purified standards. This inefficient real-time RT–PCR was not owing to the use of hybridization probes since we obtained similar results using Taqman probes (data not shown). It has not been investigated whether other detection technologies could provide increased specificity to allow a more quantitative assay using purified standards. It is most likely that even though most of the HVL sequences are divergent (Supplementary Figure 1), when the parental sequence is in excess it out competes with the real target sequence during the hybridization step and therefore prevents efficient detection.
By using mixed standards, the complex mixture of pilin cDNA templates was mimicked and the frequency with which each detectable silent copy contributed to pilin Av was determined. The accuracy of these estimates relies on comparing the amplification of mixed standards with pilin cDNA samples and can be estimated to be within a 3-fold of the real level and often as close as 2-fold. While frequencies produced are not precise, they allow a good estimate of the relative use of all detected silent copies as donors for pilin Av. The silent copies that were undetected may also recombine into pilE and produce variants, just at a level undetectable by either assay. The actual frequency with which these undetected silent copies participate in variation at pilE is unknown, but is at least 5-fold lower than the least frequent detected copy, and therefore the variants carrying the undetected copies are truly under represented in this bacterial population. However, even if these under represented copies participate in pilin Av rarely, they could still contribute to bacterial colonization if they are not recognized by an adaptive immune response.
It is not clear why the starting pilE sequence influences the frequency at which silent copies recombine into pilE. There was no pattern for the donor silent copies recombined more or less frequently in relationship to their position (upstream or downstream) to pilE or their position in a silent locus. As a homologous recombination mediated process (34), pilin Av may respond to the ability of the pilE recipient and the donor silent copy sequence to allow heteroduplex formation due to sequence similarity. The silent copies that recombined into pilE with the 2c1 starting pilE variant did show a higher degree of sequence similarity to the 2c1 variant (Clustal W, AlignX, Vector NTI) compared with the silent copies that recombined less frequently. However, these sequence similarity predictions were not supported with the donor silent copy spectrum observed with the 3c3 starting pilE variant. Therefore, no rules based on the two examples examined here can be established.
The use of this SQ-PCR Av assay has resulted in several important conclusions about the mechanism of pilin Av. First, the recombination potential of different silent pilin gene copies with pilE is not uniform. This observation means that within a limited population of gonococci, immune responses are likely to be raised only to a subset of potential pilin variants. Initially, this would allow the remainder of the pilin variants to contribute to escape of the immune response and be saved for future immune evasion. However, the silent copies that recombine at a very low frequency may become targeted over time as the immune response recognizes and eliminates the more dominant variants. Second, we have clearly demonstrated that the parental pilE sequence can influence the frequency and repertoire of pilin variants arising in a population. This observation suggests that particular pilin variants may act in a unique manner during a gonococcal infection, which could explain the various clinical manifestations of the disease in different individuals.
SUPPLEMENTARY MATERIAL
Supplementary Material is available at NAR Online.
ACKNOWLEDGEMENTS
The authors thank Alison Criss and Kimberly Kline for comments on this work and the manuscript. This work was supported by NIH grants R01/R37 AI33443 and R01 AI44239. Funding to pay the Open Access publication charges for this article was provided by the NIH.
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