The frequency and rate of pilin antigenic variation in Neisseria gonorrhoeae

Abstract

The pilin antigenic variation (Av) system of Neisseria gonorrhoeae (Gc) mediates unidirectional DNA recombination from silent gene copies into the pilin expression locus. A DNA sequencing assay was developed to accurately measure pilin Av in a population of Gc strain FA1090 arising from a defined pilin progenitor under non-selective culture conditions. This assay employs a piliated parental Gc variant with a recA allele whose promoter is replaced by lac-regulatory elements, allowing for controlled induction of pilin Av. From this assay, the frequency of pilin Av was measured as 0.13 recombination events per cell, with a corresponding rate of pilin Av of 4x10(-3) events per cell per generation. Most pilin variants retained the parental piliation phenotype, providing the first comprehensive analysis of piliated variants arising from a piliated progenitor. Sequence analysis of pilin variants revealed that a subset of possible recombination events predominated, which differed between piliated and non-piliated progeny. Pilin Av exhibits the highest reported frequency of any pathogenic gene conversion system and can account for the extensive pilin variation detected during human infection.

Figure 1. Recombination events in P+ variants of Gc strain FA1090

pilS copies are abbreviated as in Table 1. The positions in pilE of the semivariable (SV), hypervariable loop (HV_L) and tail (HV_T), and conserved cys1 and cys2 sequences are indicated. Sequences conserved among all pilS copies are gray and divergent regions are white; both are to scale. Recombination events involving the same donor pilS copy are grouped together and are presented in decreasing order of frequency of appearance in pilE. Hatched boxes indicate recombinant pilS sequence in 1-81-S2, and flanking sequence shared by 1-81-S2 and the donor pilS copy is indicated by flanking black lines. Six pilS copies (boxed) participated in more than one recombination event at pilE (labeled A, B, etc.). The alignment of each pilS copy with the 1-81-S2 sequence is shown above the corresponding recombination events; regions of each pilS copy that share identity with 1-81-S2 are indicated by black bars and divergent sequences by white boxes. In recombination events where two pilS copies could have served as donor (2c4/6c1 and 2c1/6c1), flanking sequence shared with both copies is indicated with a black line; sequence shared with only one copy is indicated by a dotted line. In pilS2c1/6c1, the dotted line represents additional homology between pilS2c1 and 1-81-S2 not present in pilS6c1; in pilS2c4, the dotted line represents additional homology between pilS6c1 and 1-81-S2 not present in pilS2c4. The number of times each recombination event was recovered is indicated at the right. The eight pilin variants recovered in independent cultures of Gc are indicated by arrowheads. A326G and A326G/A350G are sequences found in multiple pilS copies.

Figure 2. Recombination events in P- variants of Gc strain FA1090

Labeling of divergent and conserved regions, and alignments of pilS copies with pilE, is as in Fig. 1. C395A and A355G/G358A are sequences found in multiple pilS copies. Black arrowheads indicate the position of frameshifts that are predicted to cause premature truncation of pilin.

Figure 3. pilS copies in strain FA1090 that produce truncated pilin proteins and confer P- colony morphology

Alignments of pilS copies with 1-81-S2 pilE begin at nucleotide 159 of pilE, numbered from the first nucleotide of the ATG start codon. Sequence divergences from 1-81-S2 pilE are indicated; identical nucleotide to 1-81-S2, (-); nucleotide missing relative to other pilS copies or pilE, (*). Frameshifts relative to 1-81-S2 are circled. Each pilS sequence terminates with the predicted stop codon (boxed).