Pilin Antigenic Variation Occurs Independently of the RecBCD Pathway in Neisseria gonorrhoeae

Abstract

Type IV pilus expression has been strongly implicated in the virulence of Neisseria gonorrhoeae, the causative agent of gonorrhea. In Neisseria, these pili undergo frequent antigenic variation (Av), which is presumed to allow reinfection of high-risk groups. Pilin Av is the result of RecA-mediated recombination events between the gene encoding the major pilin subunit (pilE) and multiple silent pilin locus (pilS) copies, utilizing a RecF-like recombination pathway. The role of RecBCD in pilin Av has been controversial. Previous studies measuring pilin Av in recB and recD mutants in two independent strains of N. gonorrhoeae (MS11 and FA1090) by indirect methods yielded conflicting results. In addition, these two laboratory strains have been suggested to express very different DNA repair capabilities. We show that the FA1090 and MS11 parental strains have similar abilities to repair DNA damage via UV-induced DNA damage, nalidixic acid-induced double-strand breaks, and methyl methanesulfonate-induced alkylation and that RecB and RecD are involved in the repair of these lesions. To test the role of the RecBCD pathway in pilin Av, the rate and frequency of pilin Av were directly measured by sequencing the pilE locus in randomly selected piliated progeny of both MS11 and FA1090 in recB and recD mutants. Our results definitively show that recB and recD mutants undergo pilin Av at rates similar to those of the parents in both strain backgrounds, demonstrating that efficient pilin Av is neither enhanced nor inhibited by the RecBCD complex.Homologous recombination is a widely conserved process used by all organisms to repair DNA damage and to mediate genetic transfer. Usually RecA or a RecA homologue is required for homologous recombination, but the other processing enzymes used to prepare DNA for recombination differ between organisms. Generally, double-stranded DNA (dsDNA) breaks are repaired by the RecBCD complex in gram-negative organisms and the AddAB complex in gram-positive organisms, although exceptions exist in which AddAB has been found in some proteobacteria (1,4,29,33,56). Single-stranded DNA (ssDNA) gaps are typically repaired by the RecF pathway, which is ubiquitous among the Bacteria and is part of a universal step in recombinational repair (31). While the RecBCD and RecF pathways have been extensively studied in Escherichia coli, they are less well understood in the humanspecific pathogen Neisseria gonorrhoeae. N. gonorrhoeae is a gram-negative bacterium that has been isolated only from infected humans and is believed to exist naturally only within the human population. Like E. coli, N. gonorrhoeae has a RecA homologue and possesses most of the genes that define the RecBCD and RecF pathways of E. coli (27).In E. coli, the RecBCD exonuclease begins to degrade DNA at the site of the break, and upon reaching a particular sequence, a chi site, the RecD component is inactivated, leaving RecBC to act as a helicase, processively unwinding the DNA, yielding ssDNA, which is the substrate for RecA (reviewed in refere...

Frequency and Rate of Pilin Antigenic Variation of Neisseria meningitidis

“…N. meningitidis strain FAM18, which lacks a well-defined G-4 quartet and encodes class II pilin (6,18), undergoes pilin Av at a rate not detectable by the assay described here and is unlikely to undergo antigenic variation at all. These data clearly show that major differences in strain-to-strain pilin Av rates exist, an observation previously made for N. gonorrhoeae (10). The greatly reduced level of pilin Av in FAM18 suggests that this strain is not dependent upon pilin Av for its survival and spread from host to host and that other class II pilin gene-expressing strains (4) must have a relationship with the host immune system different from that of class I pilin-expressing strains.…”

“…The pilS copies share significant regions of homology with pilE yet lack a promoter or ribosome-binding site and the initial 5Ј coding sequence. Pilin Av relies on RecA and the RecF-like recombination pathway to catalyze gene conversion, resulting in an altered pilE sequence, carrying part of the pilS donor, and the original unaltered pilS sequence (8, 9).While the frequency of pilin Av has been measured in N. gonorrhoeae (5,10,12), this process has never been quantified in N. meningitidis. Two sequenced strains were picked to measure pilin Av: serogroup B strain MC58 (sequence type complex), isolated from an invasive infection (14), and serogroup C strain FAM18 (ST-11 complex), which was isolated from a patient with septicemia (1).…”

“…While the frequency of pilin Av has been measured in N. gonorrhoeae (5,10,12), this process has never been quantified in N. meningitidis. Two sequenced strains were picked to measure pilin Av: serogroup B strain MC58 (sequence type complex), isolated from an invasive infection (14), and serogroup C strain FAM18 (ST-11 complex), which was isolated from a patient with septicemia (1).…”

“…The pilin Av sequencing assay was performed as described previously (5,10,12) with slight modifications. Briefly, FAM18 and MC58 were grown on solid GCB with 1 mM IPTG, allowing for the expression of RecA, for 22 h and 12.5 h, respectively, which was estimated to produce 20 generations.…”

DNA Recombination Strategies During Antigenic Variation in the African Trypanosome

“…Indeed, N. gonorrhoeae antigenic variation appears to be a rather defined form of HR, because it only uses a sub-pathway of RecA-dependent HR initiated by a RecF-like machinery (134), and not RecBCD (135).…”