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A real-time semi-quantitative RT-PCR assay demonstrates that the pilE sequence dictates the frequency and characteristics of pilin antigenic variation in Neisseria gonorrhoeae.

ABSTRACT: A semi-quantitative real-time RT-PCR assay was designed to measure gonococcal pilin antigenic variation (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.

SUBMITTER: Rohrer MS

PROVIDER: S-EPMC1148172 | biostudies-literature | 2005

REPOSITORIES: biostudies-literature

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A real-time semi-quantitative RT-PCR assay demonstrates that the pilE sequence dictates the frequency and characteristics of pilin antigenic variation in Neisseria gonorrhoeae.

Rohrer Melissa S MS Lazio Matthew P MP Seifert H Steven HS

Nucleic acids research 20050609 10

A semi-quantitative real-time RT-PCR assay was designed to measure gonococcal pilin antigenic variation (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 ...[more]

PMID: 15947134

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Project description: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.

Dataset Information

A real-time semi-quantitative RT-PCR assay demonstrates that the pilE sequence dictates the frequency and characteristics of pilin antigenic variation in Neisseria gonorrhoeae.

Publications

A real-time semi-quantitative RT-PCR assay demonstrates that the pilE sequence dictates the frequency and characteristics of pilin antigenic variation in Neisseria gonorrhoeae.

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