A droplet digital PCR assay to measure pilin antigenic variation frequency in Neisseria gonorrhoeae

Abstract

ABSTRACT The strict human pathogen Neisseria 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 locus pilS3-copy 1 (S3C1) or a sequence conserved in all pilE variants (CYS2). The appropriate frequency of pilin antigenic variation is measured by the proportion of pilE amplicons 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 bacterium Neisseria gonorrhoeae. The rise of antibiotic-resistant gonorrhea is an urgent global threat to public health. Pilus antigenic variation is a gene conversion process that allows N. gonorrhoeae to evade host immune surveillance, and a mechanistic understanding of this process is crucial to understanding N. gonorrhoeae pathogenesis. This report shows that we can adopt a digital PCR methodology to quickly and accurately measure pilin antigenic variation.

Keywords

• Neisseria gonorrhoeae
• pilus
• antigenic variation
• gene conversion
• digital PCR