Antigenic Variation in Neisseria gonorrhoeae Occurs Independently of RecQ-Mediated Unwinding of the pilE G Quadruplex

Abstract

The obligate human pathogen Neisseria gonorrhoeae alters its cell surface antigens to evade the immune system in a process known as antigenic variation (AV). During pilin AV, portions of the expressed pilin gene (pilE) are replaced with segments of silent pilin genes (pilS) through homologous recombination. The pilE-pilS exchange is initiated by formation of a parallel guanine quadruplex (G4) structure near the pilE gene, which recruits the homologous recombination machinery. The RecQ helicase, which has been proposed to aid AV by unwinding the pilE G4 structure, is an important component of this machinery. However, RecQ also promotes homologous recombination through G4-independent duplex DNA unwinding, leaving the relative importance of its G4 unwinding activity unclear. Previous investigations revealed a guanine-specific pocket (GSP) on the surface of RecQ that is required for G4, but not duplex, DNA unwinding. To determine whether RecQ-mediated G4 resolution is required for AV, N. gonorrhoeae strains that encode a RecQ GSP variant that cannot unwind G4 DNA were created. In contrast to the hypothesis that G4 unwinding by RecQ is important for AV, the RecQ GSP variant N. gonorrhoeae strains had normal AV levels. Analysis of a purified RecQ GSP variant confirmed that it retained duplex DNA unwinding activity but had lost its ability to unwind antiparallel G4 DNA. Interestingly, neither the GSP-deficient RecQ variant nor the wild-type RecQ could unwind the parallel pilE G4 nor the prototypical c-myc G4. Based on these results, we conclude that N. gonorrhoeae AV occurs independently of RecQ-mediated pilE G4 resolution.IMPORTANCE The pathogenic bacteria Neisseria gonorrhoeae avoids clearance by the immune system through antigenic variation (AV), the process by which immunogenic surface features of the bacteria are exchanged for novel variants. RecQ helicase is critical in AV and its role has been proposed to stem from its ability to unwind a DNA secondary structure known as a guanine quadruplex (G4) that is central to AV. In this work, we demonstrate that the role of RecQ in AV is independent of its ability to resolve G4s and that RecQ is incapable of unwinding the G4 in question. We propose a new model of RecQ's role in AV where the G4 might recruit or orient RecQ to facilitate homologous recombination.

Keywords: DNA helicase; G quadruplex; Neisseria gonorrhoeae; antigen variation.

FIG 1

Comparison of the structures of antiparallel and parallel G4s and bacterial RecQ helicases. (A) Model of a parallel G4, such as the c-myc and pilE G4s used in this study. Each blue structure represents four guanine bases in a quartet structure. G4-forming sequences are shown under each model. (B) Model of an antiparallel G4 typified by the telomeric G4. (C) Comparison of the domain architecture of RecQ helicases from bacterial species. The location of the GSP is denoted by the blue lines. (D) Sequence alignment of GSP between bacterial RecQ helicases. Residues that directly interact with a guanine base within the GSP are boxed.

FIG 2

Role of GSP in N. gonorrhoeae antigenic variation. Pilin-dependent colony morphology changes in N. gonorrhoeae variants are depicted. Each point represents the average colony phase variation score of three biological replicates, with 10 colonies assessed for each replicate. Error bars represent the standard errors of the mean (n = 10), and an asterisk indicates a P value of <0.05, as determined by a two-tailed Student t test relative to the FA0190 strain.

FIG 3

smFRET studies of NgRecQ helicase activity. (A) Scheme depicting the smFRET strategy used to monitor DNA unwinding by NgRecQ. (B) NgRecQ-mediated unwinding of duplex DNA. Histograms of the smFRET signals for the DNA alone (top), or 12-min after the addition of ATP and NgRecQ or NgRecQ Asp307Ala are shown. (C and D) Same as in panel B but for the antiparallel telo-G4 and parallel pilE G4 substrates, respectively.

FIG 4

Model of the role of RecQ in N. gonorrhoeae AV. After formation of the pilE G4, RecQ (domains colored as in Fig. 1) binds to the G4. (Left) The RecQ HRDC domains bind to the G4 or nearby DNA, orienting RecQ to unwind the pilE gene. RecJ degrades the unwound ssDNA behind RecQ. RecQ cannot unwind the pilE G4, so it remains folded and recruits RecA for strand exchange by homologous recombination. (Right) Alternatively, HRDC domains could destabilize the G4, allowing for unwinding by another G4 resolving helicase. Once the G4 obstruction is removed, RecQ unwinds the pilE gene to facilitate RecA loading and homologous recombination.