Transposon Mutagenesis Identifies Sites Upstream of the Neisseria gonorrhoeae pilE Gene That Modulate Pilin Antigenic Variation

Abstract

Gene conversion mediates the variation of virulence-associated surface structures on pathogenic microorganisms, which prevents host humoral immune responses from being effective. One of the best-studied gene conversion systems is antigenic variation (Av) of the pilin subunit of the Neisseria gonorrhoeae type IV pilus. To identify cis-acting DNA sequences that facilitate Av, the 700-bp region upstream of the pilin gene pilE was targeted for transposon mutagenesis. Four classes of transposon-associated mutations were isolated, distinguishable by their pilus-associated phenotypes: (i) insertions that did not alter Av or piliation, (ii) insertions that blocked Av, (iii) insertions that interfered with Av, and (iv) insertions that interfered with pilus expression and Av. Mutagenesis of the pilE promoter did not affect the frequency of Av, directly demonstrating that pilin Av is independent of pilE transcription. Two stretches of sequence upstream of pilE were devoid of transposon insertions, and some deletions in these regions were not recoverable, suggesting that they are essential for gonococcal viability. Insertions that blocked pilin Av were located downstream of the RS1 repeat sequence, and deletion of the region surrounding these insertions completely abrogated pilin Av, confirming that specific sequences 5 to pilE are essential for the recombination events underlying pilin Av.

Pilin Antigenic Variation Occurs Independently of the RecBCD Pathway in Neisseria gonorrhoeae

“…By utilizing the previously described sequencing assay for pilin Av (6,18), we conclusively demonstrated that recB and recD mutants show the same rate of pilin Av regardless of the length of RecA induction (Fig. 2 to 5).…”

“…To assess the pilin Av rates of recB and recD mutants in MS11 and FA1090, a previously described sequencing assay was performed in which RecA expression was induced in N. gonorrhoeae for a given period of time and the pilE gene was sequenced from each of the progeny colonies (6,18). Subsequent to RecA induction, seven P ϩ colonies were picked at random and grown in the absence of IPTG to lock in the pilE sequence.…”

“…Another report, using the reverse transcription-PCR and coconversion assays, suggested that pilin Av is diminished in recB mutants in both MS11 and FA1090 (16). Thus, the roles of the individual RecBCD pathway genes and the entire pathway in pilin Av are controversial.As DNA sequence analysis of many samples has become faster, more accurate, and more cost-effective, an assay in which direct sequencing of the pilE genes of multiple random piliated (P ϩ ) N. gonorrhoeae colonies has been developed and shown to have a high level of sensitivity and statistical power (6,18). In this assay, a previously described isopropyl-␤-Dthiogalactopyranoside (IPTG)-inducible recA strain (38) is grown for a certain time on IPTG, allowing pilin Av to occur.…”

“…The pilin Av sequencing assay was performed as described previously (6,18). Briefly, all six strains were grown on solid GCB medium with 1 mM IPTG, allowing the expression of RecA for the times described in Results below (Table 1).…”

“…The pilin Av rate was determined by dividing the pilin Av frequency by the number of generations each sample was grown in the presence of IPTG as determined by a CFU/colony assay at the time of harvest. Determination of statistical significance using the Wilcoxon rank sum test was performed as described previously (18). A pilin Av event was defined as the number of pilS copies donated in a single pilE variant.…”