Antimutator Role of DNA Glycosylase MutY in Pathogenic Neisseria Species

Abstract

Genome alterations due to horizontal gene transfer and stress constantly generate strain on the gene pool of Neisseria meningitidis, the causative agent of meningococcal (MC) disease. The DNA glycosylase MutY of the base excision repair pathway is involved in the protection against oxidative stress. MC MutY expressed in Escherichia coli exhibited base excision activity towards DNA substrates containing A:7,8-dihydro-8-oxo-2-deoxyguanosine and A:C mismatches. Expression in E. coli fully suppressed the elevated spontaneous mutation rate found in the E. coli mutY mutant. An assessment of MutY activity in lysates of neisserial wild-type and mutY mutant strains showed that both MC and gonococcal (GC) MutY is expressed and active in vivo. Strikingly, MC and GC mutY mutants exhibited 60-to 140-fold and 20-fold increases in mutation rates, respectively, compared to the wild-type strains. Moreover, the differences in transitions and transversions in rpoB conferring rifampin resistance observed with the wild type and mutants demonstrated that the neisserial MutY enzyme works in preventing GC3AT transversions. These findings are important in the context of models linking mutator phenotypes of disease isolates to microbial fitness.Infections caused by Neisseria meningitidis (the meningococcus; MC) and Neisseria gonorrhoeae (the gonococcus; GC), pathogenic members of the genus Neisseria, are associated with significant morbidity and mortality in their exclusive human host. MC and GC residing on mucosal surfaces are exposed to DNA-damaging agents from a potent immune system and also suffer genotoxic stress from endogenous sources, predisposing factors for mutations (29,36). Mutator strains exhibit an increased spontaneous mutation rate compared to those commonly found in the corresponding wild-type species (17). Such a phenotype is often caused by heritable changes in components of the methyl-directed mismatch repair (MMR) pathway engaged in postreplication repair. However, Richardson et al. (41) demonstrated that only 39% of MC strains exhibiting elevated spontaneous mutation rates could be fully or partially complemented with wild-type mutS or mutL alleles and thus directly linked to defects in the MMR system. Conflicting evidence exists on the association of Dam methylase variants causing hypermutable neisserial strains with enhanced phasevariable capsule switching (4, 21, 40). Clearly, mechanisms other than MMR are implicated in MC mutator phenotypes. Associations between hypermutation and defects in MMR have not yet been reported in the close relative GC.One of the most frequent forms of oxidative DNA damage is the oxidation product of guanine, 7,8-dihydro-8-oxo-2Ј-deoxyguanosine (8oxoG) (8). The base excision repair (BER) pathway is probably the cell's major line of defense against the deleterious effects of such DNA damage (45). BER involves the release of modified base residues from DNA by DNA glycosylases that leave abasic (AP) sites in the DNA. The AP site may be further cleaved by an AP-lyase activity inherent ...

Mismatch Correction Modulates Mutation Frequency and Pilus Phase and Antigenic Variation in Neisseria gonorrhoeae

“…This is especially true for the pathogenic Neisseria strains, where mutators are frequently isolated from epidemic-associated, invasive strains of N. meningitidis compared to related strains that colonize the nasopharynx and rarely cause invasive disease (38). Disrupted genes in N. meningitidis that produce a mutator phenotype include mutS and mutL as well as the fpg and mutY glycosylases of base excision repair and 8-oxo-guanine repair pathways (6,37,38,53). The role of mutators in the natural history of Gc is less well understood, but we presume that mutator clinical isolates of Gc do exist and will be found to lack active MutS or MutL.…”

Genetic Interactions of DNA Repair Pathways in the PathogenNeisseria meningitidis

“…The DNA fragments were cloned into pBluescript SKϩ (pBSKϩ) (Stratagene), creating plasmids pH 44/76fpg, pM1080mutS, pM1080uvrA, and pMC58dinB. The plasmids pM1080mutY and pM1080mutY-kan r have been described previously (10 (48) into the respective genes in pBSKϩ (Fig. 1).…”

“…All plasmids were propagated in E. coli ER2566 (New England Biolabs). The DNA repair genes containing antibiotic resistance cassettes were transformed into the MC strain H44/76 as previously described (10). Since dinB does not contain the DNA uptake sequence required for MC transformation, a primer containing the DNA uptake sequence (TD113, 5Ј-GC GCCGTCTGAAGAATTCGCCCGATGCCGGCAG-3Ј) was employed to generate the pMC58dinB-Erm r template that was transformed into H44/76.…”

“…The MC H44/76 wild type and DNA repair mutants (Table 1) were propagated and assessed for spontaneous mutation frequencies by rifampin resistance selection as described previously (10). Rifampin inhibits RNA synthesis by binding specifically to the RNA polymerase ␤-subunit and thus prevents transcriptional initiation.…”

“…The only fully characterized MC DNA repair component to date is the BER DNA glycosylase MutY (10). MC homologues to other components of the BER pathway have been identified; however, the genes encoding DNA glycosylase Nei and endonuclease Nfo are missing (29).…”