PacBio amplicon sequencing method to measure pilin antigenic variation frequencies ofNeisseria gonorrhoeae

Abstract

WORD LIMIT 200 words)Gene diversification is a common mechanism pathogens use to alter surface structures to aid in immune avoidance. Neisseria gonorrhoeae uses a gene conversion-based diversification system to alter the primary sequence of the gene encoding the major subunit of the pilus, pilE. Antigenic variation occurs when one of the non-expressed 19 silent copies donates part of its DNA sequence to pilE. We have developed a method using Pacific Biosciences amplicon sequencing and custom software to determine pilin antigenic variation frequencies. The program analyzes 37 variable regions across the strain FA1090 1-81-S2 pilE gene and can be modified to determine sequence variation from other starting pilE sequences or other diversity generation systems. Using this method, we measured pilin antigenic variation frequencies for various derivatives of strain FA1090 and showed we can also analyze pilin antigenic variation frequencies during macrophage infection.

Transcriptional initiation of a small RNA, not R‐loop stability, dictates the frequency of pilin antigenic variation in Neisseria gonorrhoeae

“…), it was possible that changes in R‐loop levels or R‐loop stability might still alter pilin Av frequencies. A PacBio sequencing assay was used to determine the pilin Av frequency of various strains (Ozer et al , ). Deep sequencing is the best method for determining Av frequency when the strains tested have different growth rates, a factor relevant to this work, as the rnhA + nicsP lac ::rnhA strain exhibits a slower growth in the absence of IPTG.…”

“…The garP −35 mutation was previously reported to lower pilin Av rates using the pilus-dependent colony morphology change (PDCMC) assay in an inducible recA background (recA6) . Pilin Av frequencies of garP −10 and garP −35 mutants without a regulatable recA (parental strain, FA1090) were determined by a novel method using PacBio sequencing (Ozer et al, 2019). All starting strains were confirmed to have the same, predominate 1-81-S2 pilE sequence by Sanger sequencing of each progenitor colony used to generate the progeny used for PacBio sequencing; however, since pilin Av was unregulated each founder colony would start with different amounts of variant pilE subpopulations that were below the level of detection.…”

“…Individual amplicon sequences were determined by repetitive PacBio SMRTbell sequencing to reduce sequencing errors. The resulting sequences were demultiplexed, filtered for quality and analyzed to determine the pilin Av frequency for each strain (Ozer et al, 2019). The parental strain, FA1090, had an average pilin Av frequency of 18.1%, the garP −35 strain had a frequency of 6.7% and the garP −10 mutant strain had a frequency of 0.1% (Table 1 and Supporting Table 1).…”

“…Deep sequencing is the best method for determining Av frequency when the strains tested have different growth rates, a factor relevant to this work, as the rnhA + nicsP lac ::rnhA strain exhibits a slower growth in the absence of IPTG. Additionally, the longer reads generated during PacBio sequencing allows for the pilS silent copy donors to be quantified and defines the types of recombinants produced (Supporting Figs 5 and 6) (Ozer et al, 2019).…”

“…While gar does form an R‐loop, modulating the R‐loop stability by regulating RNase HI expression in Gc does not alter pilE G4 or pilin Av frequencies. We detected pilin Av frequencies using long‐read Pacific Biosciences amplicon sequencing and a custom analysis software (Ozer et al ., ). These results show that the process of sRNA transcription is rate‐limiting for the G4 structure formation and pilin Av, while the R‐loop stability does not alter pilin Av.…”