Focusing homologous recombination: pilin antigenic variation in the pathogenic Neisseria

Abstract

Some pathogenic microbes utilize homologous recombination to generate antigenic variability in targets of immune surveillance. These specialized systems rely on the cellular recombination machinery to catalyse dedicated, high-frequency reactions that provide extensive diversity in the genes encoding surface antigens. A description of the specific mechanisms that allow unusually high rates of recombination without deleterious effects on the genome in the well-characterized pilin antigenic variation systems of Neisseria gonorrhoeae and Neisseria meningitidis is presented. We will also draw parallels to selected bacterial and eukaryotic antigenic variation systems, and suggest the most pressing unanswered questions related to understanding these important processes.

Figure 1. The pilin expression locus guanine quartet (pilE G4)

A. The pilE G4 motif and surrounding DNA sequence. Mutation of GC bps (boxed in black) completely blocks pilin antigenic variation while mutation of G3 (boxed in grey) has some residual activity. The DNA element underlined in black forms a guanine quartet (G4 or G-quadruplex) motif. When G3 is mutated a second G4 motif can form (underlined in grey) using the G0 base instead (See Figure 1C)). B. Cartoon of a guanine quartet structure. An all parallel G4 structure composed of the pilE G4 sequence is shown where guanines are numbered as indicated in Figure 1A. C. Kinetic pilus-dependent colony phase variation assay: This assay measures the average number of visible pilus-dependent colony morphology changes occurring over time and is a surrogate measure of antigenic variation. Mutation of G0 has no effect on phase variation while mutation of G3 shows some residual activity. Mutation of both G0 and G3 shows a loss of phase variation comparable to the G4 mutant strain (G4mt, G12→A). Error bars represent the standard error of the mean of 10 colonies. Asterisk indicates P<0.05 as measured by two-tailed Student’s T test.

Figure 2. A working model for N. gonorrhoeae pilin antigenic variation

Shown is the pilin expression locus (pilE) and upstream region. We propose that initiation of pilin antigenic variation begins with the formation of the pilE G4 structure which may be mediated by structure-specific binding proteins. Once a single stranded nick occurs either by a G4 specific nuclease or by a replication stall, (the latter is shown), RecQ and/or Rep unwind the pilE G4 structure and RecJ degrades the single stranded DNA substrate. Then homologous pairing occurs between pilE and a pilS donor. Next, RecOR assists RecA-mediated strand exchange that is modulated by RecX and/or RdgC. Then the Holliday junction DNA recombination intermediate is processed by RecG and RuvAB which is then resolved by RuvC.