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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.
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A droplet digital PCR assay to measure pilin antigenic variation frequency in Neisseria gonorrhoeae
ABSTRACT The strict human pathogenNeisseria gonorrhoeae(gonococcus [Gc]) infects an estimated 82 million individuals globally and is a World Health Organization-designated bacterial pathogen of public health importance due to escalating antimicrobial resistance. Gc vaccines have been hindered by Gc’s ability to evade immune surveillance in part by varying its major surface antigens like the type IV pilus. We developed a quick and precise method for measuring pilin antigenic variation (Av) frequency using droplet digital PCR (ddPCR) technology. Two fluorescent probes were designed to detect either the hypervariable tail region of silent pilin locuspilS3-copy 1 (S3C1) or a sequence conserved in allpilEvariants (CYS2). The appropriate frequency of pilin antigenic variation is measured by the proportion ofpilEamplicons carrying the recombinant S3C1 copy relative to the total pilE amplicons measured by CYS2. The ddPCR assay is specific for RecA-dependent pilin antigenic variation. The reduced frequency of pilin Av in strains lacking RecA-modulating recombination protein RecX and the DNA helicase RecQ confirms the ability of the assay to measure changes in pilin Av frequency. We used the ddPCR assay to determine that pilin Av frequency is altered by the colony densities on a solid medium. The ddPCR assay is an accurate, efficient way to measure Gc pilin Av frequency. IMPORTANCEGonorrhea is a sexually transmitted infectious disease of the human genital and nasopharyngeal mucosa caused by the host-restricted bacteriumNeisseria gonorrhoeae. The rise of antibiotic-resistant gonorrhea is an urgent global threat to public health. Pilus antigenic variation is a gene conversion process that allowsN. gonorrhoeaeto evade host immune surveillance, and a mechanistic understanding of this process is crucial to understandingN. gonorrhoeaepathogenesis. This report shows that we can adopt a digital PCR methodology to quickly and accurately measure pilin antigenic variation.
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- Award ID(s):
- 2239567
- PAR ID:
- 10662157
- Editor(s):
- D'Orazio, Sarah_E F
- Publisher / Repository:
- mSphere
- Date Published:
- Journal Name:
- mSphere
- Volume:
- 10
- Issue:
- 5
- ISSN:
- 2379-5042
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript
- Journal Article:
- https://doi.org/10.1128/msphere.00094-25
