Analyzing Neisseria gonorrhoeae Pilin Antigenic Variation Using 454 Sequencing Technology

Abstract

Many pathogens use homologous recombination to vary surface antigens in order to avoid immune surveillance. Neisseria gonorrhoeae, the bacterium responsible for the sexually transmitted infection gonorrhea, achieves this in part by changing the sequence of the major subunit of the type IV pilus in a process termed pilin antigenic variation (Av). The N. gonorrhoeae chromosome contains one expression locus (pilE) and many promoterless, partial-coding silent copies (pilS) that act as reservoirs for variant pilin information. Pilin Av occurs by high-frequency gene conversion reactions, which transfer pilS sequences into the pilE locus. We have developed a 454 sequencing-based assay to analyze the frequency and characteristics of pilin Av that allows a more robust analysis of pilin Av than previous assays. We used this assay to analyze mutations and conditions previously shown to affect pilin Av, confirming many but not all of the previously reported phenotypes. We show that mutations or conditions that cause growth defects can result in Av phenotypes when analyzed by phase variation-based assays. Adapting the 454 sequencing to analyze pilin Av demonstrates the utility of this technology to analyze any diversity generation system that uses recombination to develop biological diversity. IMPORTANCEMeasuring and analyzing complex recombination-based systems constitute a major barrier to understanding the mechanisms used to generate diversity. We have analyzed the contributions of many gonococcal mutations or conditions to the process of pilin antigenic variation.

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

“…There were four common silent copy choices among all the strains tested. The tail sequence of silent copies 2c1 and 6c1 (2c1:6c1 HV T ) was one of the common events, similar to previously published results Rotman et al, 2016). We also observed a mosaic sequence containing both 1-81-S2 and the 2c1:6c1 tail sequence that was not observed in the negative control strains.…”

“…Mutation of the G4 sRNA promoter elements had previously been shown to lower or completely abrogate pilin Av, measured by the surrogate PDCMC assay . In addition, a previously published 454 sequencing assay confirmed that a mutation in the −35 region of the sRNA promoter reduced pilin Av frequencies (Rotman et al, 2016). We have confirmed and extended these results to show that the sRNA transcription initiation is rate-limiting for pilin Av.…”

“…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). These pilin Av frequency values ascertained by PacBio sequencing are the first to use an unregulated Gc strain and confirmed the effect of the gar promoter mutations to previous results using different sequencing technologies and an IPTG-regulated strain Rotman et al, 2016). These results confirm that that the gar transcription initiation is rate-limiting for pilin Av.…”

“…Pilin Av requires many conserved recombination and repair factors that normally function in single-strand gap and double-strand break repair including RecA, RecOR and RecG (Mehr and Seifert, 1997;Mehr and Seifert, 1998;Sechman et al, 2005). When other recombination and repair factors, RecQ, RecJ and Rep, are mutated, pilin Av is lowered, but not totally abrogated, indicating that these factors are involved in the gene conversion process, but may have redundant roles (Mehr and Seifert, 1998;Skaar et al, 2002;Kline and Seifert, 2005;Rotman et al, 2016;Xu and Seifert, 2018).…”

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

“…Pilin Av requires many conserved common recombination and repair factors that process gap repair and double strand breaks (21). Inactivation of some required factors, such as RecA (22), RecO, RecR and RecG, completely abrogate pilin Av (23)(24)(25), while mutation of other factors, such as RecQ, Rep, and RecJ reduce pilin Av frequencies (24,(26)(27)(28)(29).…”