Loss of both Holliday junction processing pathways is synthetically lethal in the presence of gonococcal pilin antigenic variation

Abstract

SummaryThe obligate human pathogen Neisseria gonorrhoeae (Gc) has co-opted conserved recombination pathways to achieve immune evasion by way of antigenic variation (Av). We show that both the RuvABC and RecG Holliday junction (HJ) processing pathways are required for recombinational repair, each can act during genetic transfer, and both are required for pilin Av. Analysis of double mutants shows that either the RecG or RuvAB HJ processing pathway must be functional for normal growth of Gc when RecA is expressed. HJ processing-deficient survivors of RecA expression are enriched for non-piliated bacteria that carry large deletions of the pilE gene. Mutations that prevent pilin variation such as recO, recQ, and a cis-acting pilE transposon insertion all rescue the RecA-dependent growth inhibition of a HJ processing-deficient strain. These results show that pilin Av produces a recombination intermediate that must be processed by either one of the HJ pathways to retain viability, but requires both HJ processing pathways to yield pilin variants. The need for diversity generation through frequent recombination reactions creates a situation where the HJ processing machinery is essential for growth and presents a possible target for novel antimicrobials against gonorrhoea.

Neisseria gonorrhoeae MutS Affects Pilin Antigenic Variation through Mismatch Correction and Not by pilE Guanine Quartet Binding

“…6). This result is consistent with previous reports using a phase-variable pilC strain (49). Both the ⌬mutS and the mutS-F32A mutations decreased the viability of the IPTG-induced ruvB recG mutant an additional order of magnitude relative to the MMC-proficient parent (Fig.…”

“…Holliday junctions are central intermediates that arise during homologous recombination, and RuvAB and RecG are helicases that catalyze the branch migration of the Holliday junction intermediates to extend or remove heteroduplexes (48). Initiation of pilin AV in a ruvB recG double mutant results in a synthetic lethality due to unresolved recombination intermediates at the pilE locus (49). Cells that cannot undergo pilin AV (e.g., due to a mutation of recA, deletion of the pilE gene, or disruption to the upstream G4) are rescued from the synthetic lethality (13,49).…”

“…Initiation of pilin AV in a ruvB recG double mutant results in a synthetic lethality due to unresolved recombination intermediates at the pilE locus (49). Cells that cannot undergo pilin AV (e.g., due to a mutation of recA, deletion of the pilE gene, or disruption to the upstream G4) are rescued from the synthetic lethality (13,49). Since MutS acts to prevent recombination at the pilE locus, we reasoned that the absence of mutS would exacerbate the ruvB recG synthetic lethality.…”

“…One means to escape the ruvB recG synthetic lethality is the chance deletion of the pilE locus or the partial duplication of the pilE gene (49). In the parental locked pilC strain, only 2.7% of The legend depicts the silent copies by position (e.g., "2c1" represents "pilS2 copy 1").…”

Type IV Pilin Proteins: Versatile Molecular Modules

“…Homologous recombination was previously shown to mediate pilin antigenic variation (231,278,353,357), but no specific genetic element capable of triggering the recombination event had been identified. A cis-acting 16-base guanine-rich sequence-designated the G4-RIS or "recombination initiation sequence"-located upstream of pilE on the lagging strand, was shown to form a guanine quartet (G4) which facilitates nick formation within the G4 DNA (74).…”