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An Alternative DNA Structure Is Necessary for Pilin Antigenic Variation in Neisseria gonorrhoeae

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Science  07 Aug 2009:
Vol. 325, Issue 5941, pp. 764-767
DOI: 10.1126/science.1175653

Taking Shape

DNA recombination mechanisms enable certain pathogens to modify the proteins on their outer surfaces by rearranging their genes and so avoid repeated detection by the immune system. Cahoon and Seifert (p. 764) have found that antigenic variation of a single genetic locus in the human pathogen Neisseria gonorrhoeae is triggered by a specific cis-acting DNA element. This 16–base pair DNA sequence formed an unusual DNA structure in vitro; a guanine quartet (G4), which has been implicated in only a few other biological processes. The G4 forming sequence is required for processing the gene conversion reaction leading to antigenic variation. These findings have implications both for understanding mechanisms of DNA recombination and its role in microbial pathogenesis.

Abstract

Pathogens can use DNA recombination to promote antigenic variation (Av) of surface structures to avoid immune detection. We identified a cis-acting DNA sequence near the antigenically variable pilin locus of the human pathogen, Neisseria gonorrhoeae. This 16–base pair guanine (G)–rich sequence was required for pilin Av and formed a guanine quartet (G4) structure in vitro. Individual mutations that disrupted the structure also blocked pilin Av and prevented nicks required for recombination from occurring within the G4 region. A compound that binds and stabilizes G4 structures also inhibited pilin Av and prevented nicks from occurring on the G-rich strand. This site constitutes a recombination initiation sequence/structure that directs gene conversion to a specific chromosomal locus.

DNA recombination is a process that is shared by all DNA-carrying organisms and used for a variety of cellular processes, including DNA repair, genetic exchange, and meiotic chromosome segregation (1). Additionally, recombination mediates many high-frequency gene-diversification systems, including yeast mating-type switches, immunoglobin diversity, and pathogenesis-associated antigenic variation (Av) (24). Most recombination reactions occur at a low frequency, but several diversity-generating systems can enact programmed recombination reactions between specific loci at relatively high frequencies (2, 3, 5, 6).

Neisseria gonorrhoeae is the sole causative agent of gonorrhea and has evolved three high-frequency, diversity-generation systems to avoid immune surveillance (7). This antigenic variability of gonococcal populations is one reason that natural immunity to re-infection has never been demonstrated and has prevented the development of an effective vaccine. One of these Av systems is mediated by high-frequency gene-conversion events between one of many silent pilin loci and the single expressed pilin locus, pilE. This produces variant pilin proteins (2) that form antigenically divergent pili, which are the hairlike appendages expressed by many bacteria.

This system uses normal homologous recombination factors to mediate specialized gene-conversion reactions (812), but no DNA element required for pilin Av has been described. Previous work showed that some transposon insertions in the intergenic region upstream of pilE block pilin Av without altering pilin expression (11, 13). To define the DNA element being disrupted by these transposons, the pilE upstream region was randomly mutagenized, and the mutations were introduced into the gonococcal chromosome by DNA transformation and linkage to a transposon insertion that does not affect pilin Av (fig. S1A) (13, 14). Two independent screens were performed. In both screens, mutants unable to undergo pilin Av were selected by screening for a stable, piliated colony morphology (Avd phenotype, fig. S1B), whereas in the second screen, both Av deficient (Avd) and Av transformants were analyzed (table S1) (15). Coupled with site directed mutagenesis, DNA sequence analysis of the transformants revealed that alteration of any one of 12 guanine-cytosine (GC) base pairs within a 16–base pair (bp) region each inhibits pilin Av, whereas mutation of the other base pairs within or surrounding this region did not (Fig. 1, A and B, fig. S2, and table S2). The 12 GC base pairs defining this DNA element are conserved in 14 sequenced gonococcal isolates and three Neisseria meningitis genomes upstream of pilE, but they are not found in commensal Neisseria (table S3). The ability of the single-nucleotide mutations to replicate the genetic behavior of the pilin Av–disrupting transposon insertions was determined by introducing one of the GC base pair mutations into a recG/ruvB double mutant. Previously, the RecG and RuvABC Holliday junction processing pathways were both found to be required for pilin Av (10). RecA expression in a recG/ruvB double mutant is synthetically lethal but can be overcome by a pilin Av–disrupting transposon insertion (10). Introduction of one of the GC base pair mutations also rescued the RecA-dependent synthetically lethal phenotype, showing that these mutants have the identical phenotypes as the neighboring transposon insertions that act upstream of homologous recombination factors (fig. S3).

Fig. 1

Identification of a DNA sequence in the intergenic region upstream of pilE required for gonococcal pilin Av. (A) Mutational spectrum of genetic screens. Cartoon of the mutagenized region from the transposon insertion (red triangle) that has no effect on pilin Av (13) to 165 bp downstream showing the location of the previously identified Av-disrupting transposons (blue triangle) (11, 13). The x axis indicates the base number, whereas the y axis indicates the number of mutations isolated per base. Screen 1 (bottom axis) represents 103 Avd mutants (only mutants with mutations <9 are shown) (table S1A). Screen 2 (top axis) represents 204 Av transformants and 106 Avd mutants (only mutants with mutations <17 are shown) (table S1B). The region linked to the Avd phenotype is shown with a purple box. Bars indicate mutations that allow Av (black), single mutations that cause an Avd phenotype (red), and Avd mutants that have mutations within the boxed region (blue) but have mutations elsewhere (green). (B) Bases required for pilin Av. Mutation of individual purple-boxed base pairs results in an Avd phenotype. Solid purple boxes indicate a complete block of pilin Av, whereas the G-3 mutant shows residual activity (fig. S2). The DNA element on the bottom strand forms a G4 motif (black underlined bases 1 to 16); when G-3 is mutated, the alternative G4 using G-0 (gray box) is shown underlined in gray.

The G-rich sequence defined by the genetic screens conforms to a guanine-quartet (G4) motif (Fig. 1B) (16, 17). G4-forming sequences have been implicated in many biological processes (1821), but their existence within a cell has yet to be proven. To test whether the pilE-linked G4 motif forms a G4 structure, we synthesized a series of oligonucleotides and evaluated their ability to form a G4 structure. CD spectrum analysis showed that the DNA sequence required for pilin Av adopts a parallel G4 structure and that mutation of two thymines in the loop regions has no effect on the structure (Fig. 2, A and B). A methylase-protection assay revealed two alternative G4 structures (Fig. 2C). Mutation of the G-3 residue yielded a mutant with residual pilin Av, suggesting that this alternate G4 structure can form when the G-3 site is mutated (Fig. 1B and fig. S2). Replication-stop assays with three different DNA polymerases confirmed that the G4 structure forms and can halt replication (fig. S4). Individual mutations that block pilin Av were also found to block structure formation in all three assays (Fig. 2, A and C, and fig. S4).

Fig. 2

Analysis of G4 structure formation in vitro. (A) Circular dichroism (CD) spectrometry. The hairpin oligonucleotide forms a double-stranded DNA molecule producing a B-form DNA CD spectrum (26). The AvG4 oligonucleotide has the pilE G4 sequence that allows pilin Av; Av-1 and Av-2 oligonucleotides have A-to-T (gray) mutations that do not alter Av, and all produce parallel G4 structure CD spectrums (26). Avd-1 and Avd-2 oligonucleotides contain G-to-A (gray) transversions that block pilin Av and show altered CD spectrums. (B) Parallel G4 structure. Three G-quartets (squares) in a parallel G4 configuration. G, guanine; T, thymine. (C) Dimethyl sulfate (DMS) methylase-protection assay. Formed G4 guanines are protected from DMS attack (27). Representative electropherograms of a FAM-labeled oligonucleotide are shown. The oligonucleotide contains I, the wild-type G4 sequence (AvG4 underlined), or II and III, two mutations that block pilin Av (Avd-1 and Avd-2, underlined) (three independent repeats). The x axis shows a 52-base region, and the y axis shows relative fluorescence (RF). Blue and red traces indicate G4-forming and nonforming conditions, respectively. Under G4-forming conditions, AvG4 forms two alternative G4 structures (5′GGGTGGGTTGGGTGGGG or 5′GGGTGGGTTGGGTGGGG; guanines forming the structure are underlined; asterisks), whereas mutant oligonucleotides do not form either structure.

There are 46 predicted G4-forming sequences in the gonococcal chromosome (16, 17), and none of these sequences are identical to the pilE-linked G4, nor are they located near any gene involved in pilin Av. Mutation of two irrelevant G4-forming sequences in the genome had no effect on pilus phase variation, thus ruling out a nonspecific role for other G4 sequences in pilin Av (fig. S5). When a copy of the pilE-linked G4 sequence was introduced upstream of the pilS7 silent pilin locus at a position similar to the pilE-linked copy in a strain where the pilE-linked copy was mutated (fig. S6), there were no observed changes at pilS7 or pilE, even though 4 to 31 pilE variants carrying pilS7 sequences would normally be found in a pool of 374 progeny (table S4) (2). Moreover, when the pilE-linked G4 was replaced with four other G4-forming sequences or an inverted copy, pilin Av was lost (Fig. 3C). Thus, the specific G4-forming sequence, its position, and its orientation are all essential to allow pilin Av.

Fig. 3

Stabilization, substitution, or inversion of the pilE G4 structure inhibits Av. (A) Kinetic pilus-dependent colony phase-variation assay. A representative assay (n = 3) of NMM-treated or untreated RecA+ and RecA– bacteria (RecA is required for pilin Av) with a mean phase-variation score (11) and SEM (error bars) of 10 colonies is shown. The mean phase variation of NMM-treated RecA+ is different from untreated RecA+ by Student’s t test, P < 0.05 (asterisk). (B) DNA sequencing assay for pilin Av frequency (2). 28 piliated progeny from seven founder colonies of untreated and NMM-treated bacteria were analyzed. Blue diamonds and pink x marks show the mean frequency of untreated and NMM-treated founders, respectively. Horizontal bars show the median of the means. The pilin Av frequency of NMM-treated bacteria is less than that for untreated bacteria by Wilcoxon rank-sum test, P < 0.05 (asterisk). (C) G4 substitution colony phase-variation assay. RecA+ and RecA– strains have the wild-type pilE G4 sequence. The pilE G4 inverted strain has the identical G4 sequence inverted in the same location with no other sequence changes. The pilE G4 Avd-1 mutant has a G-to-A transversion at G-5 that blocks pilin Av. The other mutants contain different G4-forming sequences: a parallel G4 from T. thermophila (28), an in vitro characterized antiparallel G4 (29), and two G4s (IGR1172 G4 and NGO816 G4) found in other locations in the gonococcal chromosome. A representative assay (n = 3) with mean phase variation score (11) and SEM (error bars) of 10 colonies is shown. All mutants are different than RecA+ by Student’s t test, P < 0.05 (asterisk).

To directly probe for a role of the G4 structure in live gonococci, we used N-methyl mesoporphyrin IX (NMM) (fig. S7, A and B), a compound that can enter live cells and specifically bind G4 DNA, but not duplex DNA (22, 23). Gonococci grown on a concentration of NMM that did not alter growth (fig. S7C) were assayed for pilin phase and Av. Bacterial growth on NMM showed significantly decreased pilus phase and Av (Fig. 3, A and B). Surprisingly, ~70% of piliated antigenic variants arising on NMM were produced by recombination with pilS3 copy 1, which normally contributes to ~20% of variants produced from the same parental variant (table S4) (2). These results are consistent with formation and further processing of the G4 structure being required for pilin Av.

Because homologous recombination is initiated by a nick or break, we used break-site mapping (BSM) (24) to assay for G4-dependent nicks or breaks in this chromosomal region. We detected nicks throughout the pilE upstream region that were at a higher concentration per base pair in the G4-forming sequence in antigenically variant bacteria (table S5) and in a higher density throughout the region as compared with the analogous region in pilS7 (table S6). Occasionally, we also detected double-strand breaks in the G4-forming sequence (Fig. 4A). We did not detect any nicks or breaks in the G-rich sequence in the Avd-1 G4 point mutant, suggesting that nicks are the result of the G4 structure (Fig. 4, A and B). NMM-treated bacteria had a similar pattern of nicks as untreated bacteria on the top strand, but we did not detect any nicks on the bottom strand (Fig. 4, F and G). To test whether specific proteins required for pilin Av process the pilE G4, we performed BSM of selected mutants. A recJ exonuclease mutant and recQ/rep helicase double mutant are both deficient in pilin Av. BSM of the recJ mutant showed an increase in nicks at the G4-forming sequence, but all G4 localized nicks were lost in a recJ/Avd-1 mutant (Fig. 4, D and E). A recQ/rep double mutant showed a slight increase in the number of nicks (Fig. 4C). Thus, an intact G4-forming sequence is required to produce nicks in the G4 DNA, and these nicks are processed by RecJ exonuclease and RecQ and Rep helicases. The involvement of RecQ in pilin Av (9, 11) is consistent with the established affinity of RecQ helicases for G4 structures (23, 25).

Fig. 4

The pilE G4 is processed during pilin Av. A 40-bp region upstream of pilE containing the pilE G4 (red) is shown. Arrows indicate the location of nicks detected by BSM (24) on the top or bottom DNA strand with the number detected in four replicate reactions from two separate cultures. Asterisks mark double-strand breaks. (A) Tn#9 is an Av strain that was used in the genetic screen (black arrows) with an isogenic G4 mutant strain, Avd-1 (red arrows). (B to G) Parental strain nicks (black arrows). (B) G4 mutant-strain Avd-1 nicks (red arrows). (C) recQ/rep double-mutant nicks (purple arrows). (D) recJ mutant-strain nicks (green arrows). (E) G4 Avd-1/ recJ double-mutant nicks (yellow arrows). (F) NMM-treated parental-strain nicks (blue arrows). (G) NMM-treated G4 mutant-strain Avd-1 nicks (orange arrows).

Our data suggest that formation of the pilE G4 structure is required for pilin Av. It is likely that the structure forms only when the DNA duplex is melted, possibly during DNA replication, because the G-rich sequence is on the lagging strand. As only the pilE G4-forming sequence can mediate pilin Av, we propose that this G4 structure is a specialized recombination initiation sequence/structure (G4-RIS). The recognition that some G4-forming sequences are important for transcription (G4-TSC) and telomere maintenance (G4-TEL) suggests divergent evolution of these sequences/structures for diverse molecular processes.

Supporting Online Material

www.sciencemag.org/cgi/content/full/325/5941/764/DC1

Materials and Methods

Figs. S1 to S7

Tables S1 to S8

References

References and Notes

  1. Materials and methods are available as supporting material on Science Online.
  2. We thank A. Chen and P. Schook for reading the manuscript and the Seifert laboratory members and W. Anderson for input. This work was supported by NIH grants R37AI033493, R01AI055977, and R01AI044239 to H.S.S. L.A.C. was partially supported by NIH grant T32GM08061.
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