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. Author manuscript; available in PMC: 2012 Sep 1.
Published in final edited form as: Mol Microbiol. 2011 Aug 4;81(5):1136–1143. doi: 10.1111/j.1365-2958.2011.07773.x

Focusing homologous recombination: pilin antigenic variation in the pathogenic Neisseria

Laty A Cahoon 1, H Steven Seifert 1,*
PMCID: PMC3181079  NIHMSID: NIHMS325060  PMID: 21812841

Summary

Some pathogenic microbes utilize homologous recombination to generate antigenic variability in targets of immune surveillance. These specialized systems rely on the cellular recombination machinery to catalyze dedicated, high-frequency reactions that provide extensive diversity in the genes encoding surface antigens. A description of the specific mechanisms that allow unusually high rates of recombination without deleterious effects on the genome in the well characterized pilin antigenic variation systems of Neisseria gonorrhoeae and Neisseria meningitidis is presented. We will also draw parallels to selected bacterial and eukaryotic antigenic variation systems, and suggest the most pressing unanswered questions related to understanding these important processes.

Keywords: Neisseria, pili, antigenic variation, recombination, gene conversion

Introduction

Antigenic variation is the process by which pathogenic organisms provide multiple versions of a surface antigen to evade host immune selection. It is often grouped with the related process of phase variation, which generates two phases of expression, either between two forms of an antigen or between a phase ON and phase OFF state. Site specific recombination between inverted recognition sites is the most common mechanism to switch between two forms of an antigen, while ON/OFF phase variation generally occurs by changes in repetitive DNA sequence repeat numbers in the promoter or within the phase variable gene itself (Bayliss, 2009). We will not be focusing on phase variation in this review.

The bacteria Neisseria gonorrhoeae and Neisseria meningitidis are strict human pathogens that colonize mucosal surfaces. N. gonorrhoeae generally colonizes the urogenital tract and is the causative agent of the sexually transmitted infection, gonorrhea which usually presents as urethritis in men and cervicitis in women. N. meningitidis usually colonizes the nasopharynx without incidence but can cause a serious septicemia and/or bacterial meningitis. N. gonorrhoeae and N. meningitidis express three major variable surface antigens that promote bacterial survival. These antigenic variation systems alter the lipooligosaccharide (LOS), the opacity (Opa) family of outer membrane proteins, and the pilus. LOS variants can selectively subvert host immune defense mechanisms and have been found to escape monoclonal antibody bactericidal killing (van Vliet et al., 2009, Bayliss et al., 2008). Opa outer membrane proteins mediate adherence to host cells and certain variants can promote cellular invasion additionally Opa protein expression can increase resistance to complement-mediated bacteriolysis (Makino et al., 1991, Bos et al., 1997). The LOS and Opa outer membrane protein each rely on ON/OFF phase variation of multiple genes to generate a more diverse repertoire of antigenic variants (Stern et al., 1986, Danaher et al., 1995, Jennings et al., 1995).

In contrast, pilin antigenic variation occurs as a result of non-reciprocal DNA recombination between one of multiple pilS silent storage loci and the pilin expression locus, pilE (Hagblom et al., 1985). Pilin is the major subunit of the Neisserial type IV pilus apparatus and pili are essential for establishing infection (Cohen & Cannon, 1999, Exley et al., 2009, Lauer et al., 1993, Robertson et al., 1977). The pilus assists in cellular adherence, bacterial aggregation and can mediate twitching motility and natural DNA transformation (Rudel et al., 1992, Sparling, 1966, Wolfgang et al., 1998, Park et al., 2001). A great deal of mechanistic insight into bacterial antigenic variation has been derived from study of the pathogenic Neisseria species (reviewed in Kline et al., 2003) and this review will highlight the unique molecular processes required for pilin antigenic variation in the pathogenic Neisseria, while drawing parallels with other pathogenesis-associated antigenic variation systems.

Recombination-based Antigenic Variation Systems

Recombination-associated antigenic variation systems have been identified in both prokaryotic and eukaryotic pathogens, and genomic sequencing suggests there may be many other organisms that encode antigenic variation systems (Table 1). Yet with the exception of Neisseria, Borrelia, and Trypanosoma, very little is known about the molecular factors and mechanisms required to generate antigenic variability by DNA recombination and less is known about these systems than has been determined in the pathogenic Neisseria. Members of the bacterial genus Borrelia are the causative agents of the multi-system disorder Lyme disease which is transmitted to humans through infected ticks during a blood meal. Members of the Trypanosoma genus are eukaryotic parasites transmitted through insect vectors that can cause fatal human diseases such as sleeping sickness and Chagas disease. The pathogenic Neisseria, Borrelia and Trypanosoma genus all evade the host immune response by high frequency antigenic variation of surface structures.

Table 1.

Controlled DNA Recombination Associated Antigenic Variation Systems

Species Gene # of Expression Sites # of Storage Loci Reference
Prokaryotic Pathogens Neisseria gonorrhoeae pilE 1 19 (Hamrick et al., 2001)
Neisseria meningitidis pilE 1 8 (Tettelin et al., 2000)
Borrelia burgdorferi vlsE 1 15 (Zhang et al., 1997)
Borrelia hermsii vsp/vlp 1 ~59 (Dai et al., 2006)
Anaplasma marginale msp2/msp3 1 ~10 (Brayton et al., 2001)
Mycoplamsa genitalium mgpB/mgpC 1 9 (Iverson-Cabral et al., 2007)
Treponema pallidum tprK 1 47 (Centurion-Lara et al., 2004)
Eukaryotic Pathogens Trypanosoma brucei vsg 14–23 ~2000 (McCulloch & Horn, 2009)
Babesia bovis ves1 150 ~24 (Brayton et al., 2007)
Pneumocystis carinii msg 73 1 (Keely & Stringer, 2009, Edman et al., 1996)
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The pathogenic Neisseria, and Borrelia species each encode one expression locus, pilE and vlsE, respectively; whereas the eukaryotic Trypanosoma species encode 14–23 telomeric vsg expression sites (McCulloch & Horn, 2009). The expression loci share regions of conserved homology with the silent loci which are themselves flanked by regions of variability. In N. gonorrohoeae, an antigenic variation frequency of 0.13 recombination events per cell with a rate of 4×10−3 events per cell per generation has been reported, whereas N. meningitidis displays an antigenic variation frequency and rate of 0.03 events per cell and 1.6×10−3 events per cell per generation, respectively (Criss et al., 2005, Helm & Seifert, 2010). Similarly, Trypanosoma brucei shows a rate of 1×10−3 events per cell per generation (Turner & Barry, 1989). The mechanism by which the silent donor copy is chosen for recombination at the expression locus remains unknown. In N. gonorrohoeae there appears to be a preference for certain pilS donor copies more than others while in T. brucei there is a hierarchy of preference where telomeric vsg copies are activated first, followed by subtelomeric and lastly pseudogene vsg copies (Criss et al., 2005, Morrison et al., 2005).

Novel Aspects of Neisseria pilin antigenic variation

How the DNA recombination events leading to pilin antigenic variation are initiated in the pathogenic Neisseria was unknown until recently. A transposon-based genetic screen identified insertions in a region of DNA upstream of the expressed pilE locus that was required for pilin antigenic variation, but did not alter pilin expression (Sechman et al., 2005, Kline et al., 2007). A subsequent targeted genetic screen identified 11 GC base pairs in this pilE upstream region that when individually mutated absolutely inhibited pilin antigenic variation and a 12th GC base pair (designated G3) that when mutated retained a residual level of pilin antigenic variation (Figure 1A&C) (Cahoon & Seifert, 2009). Interestingly, a mutation of a neighboring GC base pair (designated G0) alone had no effect on pilin antigenic variation but mutating G0 in addition to G3 resulted in a complete loss-of-function, suggesting that G0 can partially substitute for G3 (Figure 1A&C) (Cahoon & Seifert, 2009). The organization of these 12 GC base pairs conforms to a guanine quartet or guanine quadruplex (G4) forming sequence (Figure 1A&B) (Cahoon & Seifert, 2009). Biophysical studies confirmed that the G-rich sequence forms a G4 structure in vitro and that point mutations that disrupt pilin antigenic variation, also disrupt the structure (Cahoon & Seifert, 2009). Growth of N. gonorrhoeae on N-methyl mesoporphyrin IX (NMM), which interacts specifically with G4 structures but not with double stranded or single stranded DNA (Ren & Chaires, 1999), inhibited the frequency of pilin antigenic variation by specifically depressing the recombination of some but not all pilS copies (Cahoon & Seifert, 2009). The molecular basis for this selective inhibition is not known. Additionally, point mutations that interfere with pilin antigenic variation and the G4 structure prevented single stranded nicks from being detected in the G4 forming sequence and the opposite C-rich strand, while treatment with NMM decreased the nicks detected specifically on the G-rich strand but not the C-rich strand (Cahoon & Seifert, 2009). We concluded from this study that for antigenic variation to occur this G4 structure must form and this was the first instance of a G4 structure being implicated in a prokaryotic biological process. We predict that other molecular processes of prokaryotes will also be influenced by G4 structures. It is yet to be determined when and how the pilE G4 structure forms, whether formation is regulated, and if proteins required for pilin antigenic variation bind and process the structure (Figure 2). Since the pilE G4 forming sequence is only localized upstream of the pilE but not near any pilS loci, and since the pilE G4 forming sequence is located a few hundred base pairs away from where recombination occurs, it is likely that there is considerable processing of DNA after the G4 structure forms to yield recombination at pilE.

Figure 1. The pilin expression locus guanine quartet (pilE G4).

Figure 1

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A. The pilE G4 motif and surrounding DNA sequence. Mutation of GC bps (boxed in black) completely blocks pilin antigenic variation while mutation of G3 (boxed in grey) has some residual activity. The DNA element underlined in black forms a guanine quartet (G4 or G-quadruplex) motif. When G3 is mutated a second G4 motif can form (underlined in grey) using the G0 base instead (See Figure 1C)). B. Cartoon of a guanine quartet structure. An all parallel G4 structure composed of the pilE G4 sequence is shown where guanines are numbered as indicated in Figure 1A. C. Kinetic pilus-dependent colony phase variation assay: This assay measures the average number of visible pilus-dependent colony morphology changes occurring over time and is a surrogate measure of antigenic variation. Mutation of G0 has no effect on phase variation while mutation of G3 shows some residual activity. Mutation of both G0 and G3 shows a loss of phase variation comparable to the G4 mutant strain (G4mt, G12→A). Error bars represent the standard error of the mean of 10 colonies. Asterisk indicates P<0.05 as measured by two-tailed Student’s T test.

Figure 2. A working model for N. gonorrhoeae pilin antigenic variation.

Figure 2

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Shown is the pilin expression locus (pilE) and upstream region. We propose that initiation of pilin antigenic variation begins with the formation of the pilE G4 structure which may be mediated by structure-specific binding proteins. Once a single stranded nick occurs either by a G4 specific nuclease or by a replication stall, (the latter is shown), RecQ and/or Rep unwind the pilE G4 structure and RecJ degrades the single stranded DNA substrate. Then homologous pairing occurs between pilE and a pilS donor. Next, RecOR assists RecA-mediated strand exchange that is modulated by RecX and/or RdgC. Then the Holliday junction DNA recombination intermediate is processed by RecG and RuvAB which is then resolved by RuvC.

For a G4 structure to form duplex DNA must first be melted, and therefore it is possible that the pilE G4 structure forms either during transcription and/or replication. Moreover, since the activation energy of G4 formation provides a barrier to formation it is also likely that proteins aid in the formation of the structure. Interestingly, G4 structure formation has been suggested to be a prerequisite to recombination in immunoglobin class-switching another high frequency genetic diversification system (Dempsey et al., 1999). In contrast, T. brucei vsg antigenic variation appears to be initiated by a double strand break adjacent to the 70 base pair repeats in the active expression site (Boothroyd et al., 2009). However, this may not be the only mechanism of initiation in T. brucei since mutation of Mre11, an enzyme involved in double strand break repair, impairs homologous recombination but not vsg switching (Tan et al., 2002, Robinson et al., 2002).

In the pathogenic Neisseria, the homologous recombination events leading to pilin antigenic variation are non-reciprocal. The pilS donor copy is never lost while the pilE locus undergoes gene conversion. Models which account for both gene conversion at the pilE locus and genomic conservation at the pilS loci include the unequal crossing over model, the hybrid intermediate model, and the half cross over model (reviewed in Kline et al., 2003). The ability to perform high frequency gene conversion in the context of a bacterial chromosome and to conserve the pilS sequences is proposed to be facilitated by the polyploid nature the bacteriam, which suggests that the pathogenic Neisseria may have evolved polypoidy to accomplish non-reciprocal high frequency DNA recombination at pilE without genetic loss of the pilS donor sequences (Tobiason & Seifert, 2006, Tobiason & Seifert, 2010). N. gonorrhoeae has a single copy of pilE and 19 unique pilS cassettes located in six discrete chromosomal loci while N. meningitidis possess 8 pilS cassettes in a single locus upstream of pilE (Tettelin et al., 2000, Hamrick et al., 2001). The divergence in genomic organization and increased number of pilS cassettes in N. gonorrhoeae compared to N. meningitidis may account for the higher frequency and rate of pilin antigenic variation observed in N. gonorrhoeae or may be due to a need for more antigenic diversity within a sexually transmitted infection.

Iron Availability and Pilin Antigenic Variation

The pathogenic Neisseria undergo pilin antigenic variation under both laboratory conditions (Gould et al., 1944, Scherp & Fitting, 1949) and within the human host but whether differing environmental factors that are encountered during infection might influence the frequency of pilin antigenic variation has not been explored (Seifert et al., 1994, Rytkonen et al., 2004). In contrast, B. burgdorferi vlsE antigenic variation only occurs during mammalian infections which suggest that host factors and/or environmental cues are required for the process (Ohnishi et al., 2003). In N. gonorrhoeae, the frequency of pilin antigenic variation has been measured under several environmental conditions that are predicted to fluctuate during the course of infection or have been shown to influence N. gonorrhoeae cellular processes (Serkin & Seifert, 2000). Of the conditions tested (carbon source, temperature, aromatic amino acid availability, oxygen availability and iron availability), only iron limitation was found to significantly alter the frequency of pilin antigenic variation suggesting that iron is limited in some locations in the host (Serkin & Seifert, 2000). Under iron limiting conditions, a 5–9 fold increase in pilin antigenic variation was observed suggesting low iron availability in the host environment signals when an increase in pilin antigenic variation might be advantageous (Serkin & Seifert, 2000). We speculate that a stimulation of the frequency of pilin antigenic variation would most likely occur when N. gonorrhoeae are about to transit to a new host because a new repertoire of antigenic variants would not be recognized by a previously elicited host response. It is also possible that pilin antigenic variation is stimulated to provide pilus phase variants for functional purposes in the host.

Protein Factors Required for Pilin Antigenic Variation

N. gonorrhoeae pilin antigenic variation is a specialized recombination system that occurs via a RecF-like pathway of homologous recombination, which utilizes enzymes that participate in general recombination and repair pathways and enzymes that are non-pathway specific (Skaar et al., 2002, Kline & Seifert, 2005b, Mehr et al., 2000, Mehr & Seifert, 1998, Mehr & Seifert, 1997, Sechman et al., 2005, Stohl & Seifert, 2001, Koomey et al., 1987) (Table 2). In N. gonorrhoeae, pilin antigenic variation is totally dependent on RecA (Koomey et al., 1987), whereas B. burgdorferi vslE antigenic variation is RecA independent (Liveris et al., 2008) and T. brucei encodes five RecA orthologues (Rad51 and four Rad51 paralogues) but only some Rad51 paralogues participate in vsg antigenic variation (Conway et al., 2002, Robinson et al., 2002, McCulloch & Barry, 1999, Proudfoot & McCulloch, 2005, Proudfoot & McCulloch, 2006). In N. gonorrhoeae, RecA is tempered by the RecA modulator RecX which enhances pilin antigenic variation, and T. brucei Rad51 is mediated by BRCA2 during vsg antigenic variation (Stohl & Seifert, 2001, Gruenig et al., 2010, Hartley & McCulloch, 2008). Interestingly, the Neisseria RecX protein is a potent inhibitor of the RecA/DNA nucleoprotein filment, even though recX mutants show decreased frequencies of pilin antigenic variation (Gruenig et al., 2010, Stohl et al., 2003). These findings suggest that for RecA to be effective in promoting pilin antigenic variation it must limit its ability to promote heteroduplex formation. The reason why there is a constraint on RecA filament length is unknown.

Table 2.

Protein Factors Required For Pilin Antigenic Variation

Protein Associated Function Extent of pilin antigenic variation deficiency Reference
RecA recombinase complete loss (Koomey et al., 1987)
RecX RecA modulator some residual activity (Stohl & Seifert, 2001)
RdgC RecA modulator some residual activity (Mehr et al., 2000)
RecOR recombinase, assists in RecA- mediated strand exchange complete loss (Mehr & Seifert, 1998, Sechman et al., 2005)
RecJ 5′→3′ single strand exonuclease partial loss (Hill & Grant, 2002, Skaar et al., 2002)
RecQ 3′→5′ helicase partial loss (Mehr & Seifert, 1998)
Rep 3′→5′ helicase, replication restart partial loss (Kline & Seifert, 2005b)
RecG 3′→5′ helicase, branch migration of Holliday junctions some residual activity (Sechman et al., 2006, Sechman et al., 2005)
RuvAB 5′→3′ helicase, branch migration of Holliday junctions some residual activity (Sechman et al., 2006)
RuvC resolvase, resolution of Holliday junctions some residual activity (Sechman et al., 2006)
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Although unlike N. gonorrhoeae, B. burgdorferi does not require RecA for vslE antigenic variation, both organisms require the Holliday junction helicase RuvAB to accomplish antigenic variation (Dresser et al., 2009, Lin et al., 2009, Sechman et al., 2006). There may be other functional similarities between N. gonorrhoeae, B. burgdorferi, and T. brucei antigenic variation systems but further investigation is needed to define additional protein factors involved in the process in the latter two species.

Until recently, whether the RecBCD pathway is involved in pilin antigenic variation was controversial. Initially recB, recC, and recD mutants were shown to have no difference in pilin antigenic variation when compared to the parental strain by a colony based PCR assay and by colony phase variation assay (Mehr & Seifert, 1998). Then two reports measuring pilin antigenic variation in recB and recD mutants yielded conflicting results showing either an inhibition or reliance on the RecBCD pathway in two different N. gonorrhoeae strains. However, the conclusions of these studies were based on assays that may have been influenced by the drastically reduced growth rate of the recB and recD mutants (Chaussee et al., 1999, Hill et al., 2007). Therefore, to definitively determine whether the RecBCD pathway plays a role in pilin antigenic variation, the rate and frequency of pilin antigenic variation was directly measured in the same two strains by sequencing the pilE locus in randomly selected piliated progeny of recB and recD mutants (Helm & Seifert, 2009). The recB and recD mutants showed similar rates and frequencies of pilin antigenic variation as the parental strains, conclusively demonstrating that N. gonorrhoeae pilin antigenic variation is absolutely independent of the RecBCD pathway of recombination (Helm & Seifert, 2009).

A Working Model

A working model for N. gonorrhoeae pilin antigenic variation can be proposed based on the knowledge we have gained about the processes and molecules required for this genetic diversification system (Figure 2). We propose that pilin antigenic variation begins with the formation of the G4 structure after the DNA duplex is melted, and that this formation is mediated by one or more unknown structure specific binding proteins. Since N. gonorrhoeae is polyploid, recombination may be initiated at pilE only on the chromosome carrying a formed pilE G4 structure, but this hypothesis remains to be directly tested. Once formed, the G4 structure could then be nicked by an endonuclease. Alternatively, a nick could be produced by a stalled replication fork on the leading strand since the pilE G4 structure is located on the lagging strand during DNA replication, inversion of the pilE G4 sequence orientation caused a complete loss of pilin antigenic variation, and DNA polymerases cannot replicate through a formed pilE G4 structure (Cahoon & Seifert, 2009). However, since the replication restart helicase PriA has no role in pilin antigenic variation (Kline & Seifert, 2005a), if the replication fork collapses, it is not restored through PriA. This nicked substrate could then be processed by the RecJ exonuclease and RecQ and/or Rep helicases (Cahoon & Seifert, 2009, Kline & Seifert, 2005b, Mehr & Seifert, 1998, Sechman et al., 2005, Skaar et al., 2002). Since Escherichia coli RecQ helicase has been shown to unwind G4 structures in vitro (Wu & Maizels, 2001), it is likely that the gonococcal RecQ helicase resolves the pilE G4 structure during or after antigenic variation. Then homologous pairing occurs between the pilS donor sequence and pilE, and RecOR assists RecA-mediated strand exchange which is modulated by RecX (Koomey et al., 1987, Mehr & Seifert, 1998, Sechman et al., 2005, Stohl & Seifert, 2001). RdgC, a DNA binding protein which is associated with recombination and replication fork repair may also temper RecA (Briggs et al., 2007, Mehr et al., 2000). The resultant DNA recombination intermediate is likely processed by the RecG and RuvAB helicases and then RuvC Holliday junction can resolve the structure (Sechman et al., 2006, Sechman et al., 2005). While these steps are all reasonable based on what we presently know about pilin antigenic variation, the actual detailed steps are likely to be more complex, and await further characterization for both the pathogenic Neisseria pilin antigenic variation system and other types of recombination-associated antigenic variation systems.

Acknowledgments

We would like to thank Adrienne Chen for careful reading of the manuscript, Vitaly Kuryavyi for modeling of the pilE G4 nucleotide orientation, and Ryan Smith for assistance with Adobe Photoshop. Our work is funded by NIH Grants RO1 AI044239 and R37 AI033493.

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