A Double-Strand Break Does Not Promote Neisseria gonorrhoeae Pilin Antigenic Variation

Abstract

The major subunit of the type IV pilus (T4p) of Neisseria gonorrhoeae undergoes antigenic variation (AV) dependent on a guanine quadruplex (G4) DNA structure located upstream of the pilin gene. Since the presence of G4 DNA induces genome instability in both eukaryotic and prokaryotic chromosomes, we tested whether a double-strand break (DSB) at the site of the pilE G4 sequence could substitute for G4-directed pilin AV. The G4 motif was replaced by an I-SceI cut site, and the cut site was also introduced to locations near the origin of replication and the terminus. Expression of the I-SceI endonuclease from an irrelevant chromosomal site confirmed that the endonuclease functions to induce double-strand breaks at all three locations. No antigenic variants were detected when the G4 was replaced with the I-SceI cut site, but there was a growth defect from having a DSB in the chromosome, and suppressor mutations that were mainly deletions of the cut site and/or the entire pilE gene accumulated. Thus, the pilE G4 does not act to promote pilin AV by generating a DSB but requires either a different type of break, a nick, or more complex interactions with other factors to stimulate this programmed recombination system. IMPORTANCE Neisseria gonorrhoeae, the causative agent of gonorrhea, possesses a DNA recombination system to change one of its surface-exposed antigens. This recombination system, known as antigenic variation, uses an alternate DNA structure to initiate variation. The guanine quadruplex DNA structure is known to cause nicks or breaks in DNA; however, much remains unknown about how this structure functions in cells. We show that inducing a break by different means does not allow antigenic variation, indicating that the DNA structure may have a more complicated role.

Genomic Stress Responses Drive Lymphocyte Evolvability: An Ancient and Ubiquitous Mechanism

“…[ 99,100 ] Similarities between antigenic variation in unicellular parasites and antigen receptor gene variation in lymphocytes are also manifested in the role of G4 heteroduplex motifs, four‐stranded structures, which act as breakpoint sites for gene conversion in N. gonorrhoeae and T. brucei and which are also known to operate as targeting sites for AID during CSR. [ 101–104 ] Furthermore, allelic exclusion mechanisms are in place in lymphocytes and in T. brucei to ensure clonality of antigen receptors and variant surface glycoproteins (VSG), accordingly. [ 105–107 ] Thus, relying on conserved molecules and homologous repair pathways, gene conversion in lymphocytes and gene conversion in unicellular parasites are fundamentally equivalent.…”

Cas9-mediated endogenous plasmid loss in Borrelia burgdorferi

“…Repair of a site-specific DSB in Neisseria gonorrhoeae, when there are no homologous sequences to provide a template for recombinational repair, occurs at such low frequencies that less than one cell in ten thousands survives this type of genome lesion [46]. In contrast, the presence of short (5 to 23 base pairs) homologous sequences flanking an endonuclease-induced DSB led to RecA-mediated repair in a small fraction of cells [46]. Since most B. burgdorferi plasmids are not needed for growth in axenic culture, induction of DNA lesions in B. burgdorferi plasmids should cause plasmid loss if DNA repair is inefficient.…”

Cas9-mediated endogenous plasmid loss in Borrelia burgdorferi

“…Indeed, in the absence of a recombinational donor sequence, exogenously induced double-stranded DNA breaks (DSBs) in the chromosome can be lethal in several bacteria, including Escherichia coli [40, 41], streptococci [42], Clostridium cellulolyticum [43], and the spirochete Leptospira biflexa [44]. Repair of a site-specific DSB in Neisseria gonorrhoeae , when there are no homologous sequences to provide a template for recombinational repair, occurs at such low frequencies that less than one cell in ten thousands survives this type of genome lesion [45]. In contrast, the presence of short (5 to 23 base pairs) homologous sequences flanking an endonuclease-induced DSB led to RecA-mediated repair in a small fraction of cells [45].…”

“…Repair of a site-specific DSB in Neisseria gonorrhoeae , when there are no homologous sequences to provide a template for recombinational repair, occurs at such low frequencies that less than one cell in ten thousands survives this type of genome lesion [45]. In contrast, the presence of short (5 to 23 base pairs) homologous sequences flanking an endonuclease-induced DSB led to RecA-mediated repair in a small fraction of cells [45]. Since most B. burgdorferi plasmids are not needed for growth in axenic culture, induction of DNA lesions in B. burgdorferi plasmids should cause plasmid loss if DNA repair is inefficient.…”