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

Abstract

The 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.

Fig. 1. Effect of HJ processing mutants on pilin Av, DNA repair and DNA transformation

Parental strain is FA1090 expressing the 1-81-S2 pilin variant isolated from a human volunteer infection (Seifert et al., 1994) carrying the recA6 allele. This strain is phenotypically RecA- without ITPG in the growth medium and RecA+ Gc when grown in the presence of IPTG (Seifert, 1997). All strains are isogenic except for the indicated loss-of-function mutation and where appropriate the wild type gene expressed from an irrelevant locus. A. Pilin variation was measured using the pilus-dependent colony morphology change (PDCMC) assay as previously described (Sechman et al., 2005). Approximately 10 P+ colonies were assayed and the number of nonpiliated outgrowths recorded for each time indicated. B. UV survival was measured by exposing dilutions of the indicated strains to the indicated UV dose. Each value is reported relative tot the unirradiated control. The survival curves for the parental strain and the complemented ruvB:B and ruvC:C cultures are indistinguishable. C. DNA transformation competence was tested using the pSY6 plasmid which carries a gyrB allele that confers resistance to nalidixic acid when recombined inot the chromosome (Stein et al., 1991). Error bars represent the standard error of the mean of at least three experiments. * Indicates statistical difference from recA6 at p≤0.05. # Indicates statistical difference from ruvB and ruvC at p≤0.05.

Fig 2. Effect of mutations in HJ processing on RecA-dependent growth and pilin variation

Strains are the same as in Fig. 1 and were grown for 24 hrs. A. Colonies of HJ processing mutants without IPTG (RecA-). B. Colonies of HJ processing mutants with IPTG (RecA+) C. Quantification of HJ processing mutant growth in colonies with (+IPTG) or without (-IPTG), relative to the parental strain.

Fig. 3. Analysis of survivors of RecA induction

A. Percent of P^- colony variants after IPTG induction. Strains are the same as in Fig. 1 and were grown for 24 hrs in the presence of IPTG, collected and grown on solid medium without IPTG for 24 hrs before scoring for pilus-dependent colony morphology (PDCMC) under a stereomicroscope. B. Percent of P^- colony variants with a pilE deletion after IPTG induction. Random colonies from panel A which showed a nonpiliated colony morphology were lysed and the presence of the pilE locus determined by PCR using the oligonucleotides PILSART and SP3A (Wright et al., 1994). * indicates statistically different than parental by student t-test with a p<0.5

Fig. 4. Deletion of pilE in recG ruvA and recG ruvB mutant strains alleviates the RecA-dependent growth defect

All variants are FA1090 and contain the recA6 allele. P+- indicates a variant with intact pilE, piliated colony morphology. P- represents a variant with an intact pilE, nonpiliated colony morphology. ΔP represents a variant with a deleted pilE, nonpiliated colony morphology. RecA- indicates that the strain was grown in the absence if IPTG, and RecA+ indicates that the strain was grown in the presence of IPTG. Growth of each strain was measured by determining cfu/colony after 24 hrs. Error bars represent the standard error of the mean of three experiments. * Indicates a statistically significant difference from recA6 P+. # indicates a statistically significant difference from recGruvA and recGruvB P+ RecA- and P-RecA+ at p≤0.05

Fig 5. A cis-acting transposon mutation rescues the HJ processing deficiency growth phenotype

A. Map of the pilE upstream region showing the location of a transposon insertion that is deficient for pilin Av and produces a piliated nonvarying colonial morphology (P⁺_nv) (pilE::Tn#1) or is WT for pilin Av (pilE::Tn #9). The distance between the sites of transposon insertions is 50 bp. The upstream silent pilin locus (USS), repeat sequence (RS1 & RS2), promoter (P) and transcriptional start (arrow) are shown. B. The pilE::Tn5#9 and pilE::Tn5#1 transposon derived mutants differentially affect the RecA-dependent growth of the HJ processing mutants.

Fig 6. A proposed pathway for recombination leading to pilin Av

A. Initiation event in region, possibly a SS nick, defined by the pilE::Tn5#1 transposon insertion B. Creation of a gapped substrate by the action of the exonuclease RecQ and/or the nucleases RecJ and Rep. C. Formation of one or two HJs by RecAX and RecOR. D. Branch migration by RuvAB and RecG followed by resolution by RuvC and possibly another undefined endonuclease for RecG.