H. Steven Seifert

An Alternative DNA Structure is Necessary for Pilin Antigenic Variation in Neisseria gonorrhoeae

Taking Shape DNA recombination mechanisms enable certain pathogens to modify the proteins on their outer surfaces by rearranging their genes and so avoid repeated detection by the immune system. Cahoon and Seifert (p. 764) have found that antigenic variation of a single genetic locus in the human pathogen Neisseria gonorrhoeae is triggered by a specific cis-acting DNA element. This 16–base pair DNA sequence formed an unusual DNA structure in vitro; a guanine quartet (G4), which has been implicated in only a few other biological processes. The G4 forming sequence is required for processing the gene conversion reaction leading to antigenic variation. These findings have implications both for understanding mechanisms of DNA recombination and its role in microbial pathogenesis. A guanine quartet DNA structure regulates antigenic variation in Neisseria gonorrheae. Pathogens can use DNA recombination to promote antigenic variation (Av) of surface structures to avoid immune detection. We identified a cis-acting DNA sequence near the antigenically variable pilin locus of the human pathogen, Neisseria gonorrhoeae. This 16–base pair guanine (G)–rich sequence was required for pilin Av and formed a guanine quartet (G4) structure in vitro. Individual mutations that disrupted the structure also blocked pilin Av and prevented nicks required for recombination from occurring within the G4 region. A compound that binds and stabilizes G4 structures also inhibited pilin Av and prevented nicks from occurring on the G-rich strand. This site constitutes a recombination initiation sequence/structure that directs gene conversion to a specific chromosomal locus.

Differential roles of homologous recombination pathways in Neisseria gonorrhoeae pilin antigenic variation, DNA transformation and DNA repair.

Neisseria gonorrhoeae (Gc) pili undergo antigenic variation when the amino acid sequence of the pilin protein is changed, aiding in immune avoidance and altering pilus expression. Pilin antigenic variation occurs by RecA‐dependent unidirectional transfer of DNA sequences from a silent pilin locus to the expressed pilin gene through high‐frequency recombination events that occur at limited regions of homology. We show that the Gc recQ and recO genes are essential for pilin antigenic and phase variation and DNA repair but are not involved in natural DNA transformation. This suggests that a RecF‐like pathway of recombination exists in Gc. In addition, mutations in the Gc recB, recC or recD genes revealed that a Gc RecBCD pathway also exists and is involved in DNA transformation and DNA repair but not in pilin antigenic variation.

A variable genetic island specific for Neisseria gonorrhoeae is involved in providing DNA for natural transformation and is found more often in disseminated infection isolates

Neisseria gonorrhoeae (the gonococcus) is the causative agent of the sexually transmitted disease gonorrhoea. Most gonococcal infections remain localized to the genital tract but, in a small proportion of untreated cases, the bacterium becomes systemic to produce the serious complication of disseminated gonococcal infection (DGI). We have identified a large region of chromosomal DNA in N. gonorrhoeae that is not found in a subset of gonococcal isolates (a genetic island), in the closely related pathogen, Neisseria meningitidis or in commensal Neisseria that do not usually cause disease. Certain versions of the island carry a serum resistance locus and a gene for the production of a cytotoxin; these versions of the island are found preferentially in DGI isolates. All versions of the genetic island encode homologues of F factor conjugation proteins, suggesting that, like some other pathogenicity islands, this region encodes a conjugation‐like secretion system. Consistent with this hypothesis, a wild‐type strain released large amounts of DNA into the medium during exponential growth without cell lysis, whereas an isogenic strain mutated in a peptidoglycan hydrolase gene (atlA) was drastically reduced in its ability to donate DNA for transformation during growth. This genetic island constitutes the first major discriminating factor between the gonococcus and the other Neisseria and carries genes for providing DNA for genetic transformation.

Characterization of the pilF—pilD pilus‐assembly locus of Neisseria gonorrhoeae

Expression of Type IV pili by the bacterial pathogen Neisseria gonorrhoeae appears to be essential for colonization of the human host. Several N. gonorrhoeae gene products have been recently identified which bear homology to proteins involved in pilus assembly and protein export in other bacterial systems. We report here the isolation and characterization of transposon insertion mutants in N. gonorrhoeae whose phenotypes indicate that the N. gonorrhoeae pilF and pilD gene products are required for gonoccocal pilus biogenesis. Mutants lacking the pilD gene product, a pre‐pilin peptidase, were unable to process the pre‐pilin subunit into pilin and thus were non‐piliated. pilF mutants processed pilin but did not assemble the mature subunit. Both classes of mutants released S‐pilin, a soluble, truncated form of the pilin subunit previously correlated with defects in pilus assembly. In addition, mutants containing transposon insertions in pilD or in a downstream gene, orfX, exhibited a severely restricted growth phenotype. Deletion analysis of pilD indicated that the poor growth phenotype observed for the pilD transposon mutants was a result of polar effects of the insertions on orfX expression. orfX encodes a predicted polypeptide of 23 kDa which contains a consensus nucleotide‐binding domain and has apparent homologues in Pseudomonas aeruginosa, Pseudomonas putida, Thermus thermophilus, and the eukaryote Caenorhabditis elegans. Although expression of orfX and pilD appears to be transcriptionally coupled, mutants containing transposon insertions in orfX expressed pili. Unlike either pilF or pilD mutants, orfX mutants were also competent for DNA transformation.

Molecular models accounting for the gene conversion reactions mediating gonococcal pilin antigenic variation

The pilus antigenic variation (Av) system of Neisseria gonorrhoeae is one of several high‐frequency variation systems that utilize gene conversion to switch between numerous forms of an antigen on the cell surface. We have tested three predictions of the first models that explain the movement of DNA during pilin Av: (i) Av requires two recombinations at short regions of identity, (ii) circular intermediates exist that carry pilE/pilS hybrid loci and (iii) these pilE/pilS hybrid loci target the pilS sequences to a recipient pilE gene. We confirm that normal pilin Av utilizes recombination at very short regions of DNA sequence identity and that these recombination events can occur independent of homologous recombination functions. We have isolated covalently closed circular DNA molecules carrying hybrid pilin loci, but propose that an alternative hybrid molecule is the intermediate of pilin Av. Our most striking finding is that transformation of isolated pilE/pilS hybrid loci targets the pilS sequences of the hybrid to a recipient pilE at frequencies much higher than normal recombination frequencies. These results show that the different steps of a model that explains pilin Av can be separately tested to support the validity of these novel models that account for the high‐frequency gene conversions that mediate pilin Av.

QUESTIONS ABOUT GONOCOCCAL PILUS PHASE- AND ANTIGENIC VARIATION

Pathogenic organisms inhabit one of several defined locations within a host where temperature, pH, and nutrients are relatively constant. While the microorganism must adapt to different environments within the host, the host immune system is the most formidable predator that can limit the growth of a pathogen. Neisseria gonorrhoeae (the gonococcus, Gc) is the causative agent of gonorrhoea, and has evolved several systems for varying the antigenicity of different surface antigens, presumably to help evade the effects of the human immune system. The On/Off/On phase variation of surface structure expression also alters the antigenic characteristics of the bacterial cell surface. Antigenic variation of the major subunit of the pilus, pilin, occurs by unidirectional, homologous recombination between a silent locus and the expression locus. The silent loci lie from 1 to 900 kb from the expression locus in the chromosome yet all can donate their sequences to the expression locus. The genetic composition of the pilin loci of two Gc strains has been elucidated, and the types of changes that lead to altered forms of the pilus have been extensively characterized. However, little is known about the precise molecular mechanisms used to allow high‐frequency, non‐reciprocal, chromosomal recombination between pilin loci or about what regulates the process of maintaining chromosome fidelity.

The frequency and rate of pilin antigenic variation in Neisseria gonorrhoeae

The pilin antigenic variation (Av) system of Neisseria gonorrhoeae (Gc) mediates unidirectional DNA recombination from silent gene copies into the pilin expression locus. A DNA sequencing assay was developed to accurately measure pilin Av in a population of Gc strain FA1090 arising from a defined pilin progenitor under non‐selective culture conditions. This assay employs a piliated parental Gc variant with a recA allele whose promoter is replaced by lac‐regulatory elements, allowing for controlled induction of pilin Av. From this assay, the frequency of pilin Av was measured as 0.13 recombination events per cell, with a corresponding rate of pilin Av of 4 × 10−3 events per cell per generation. Most pilin variants retained the parental piliation phenotype, providing the first comprehensive analysis of piliated variants arising from a piliated progenitor. Sequence analysis of pilin variants revealed that a subset of possible recombination events predominated, which differed between piliated and non‐piliated progeny. Pilin Av exhibits the highest reported frequency of any pathogenic gene conversion system and can account for the extensive pilin variation detected during human infection.

The recX gene potentiates homologous recombination in Neisseria gonorrhoeae.

In the pathogen Neisseria gonorrhoeae (Gc), the RecA protein is necessary for DNA repair, DNA transformation and pilus antigenic variation. Many bacteria contain a gene, recX, which has been suggested to downregulate recA through an unknown mechanism. To investigate the possible role of recX in Gc, we cloned and insertionally inactivated the recX gene. The recX loss‐of‐function mutant showed decreases in pilus phase variation, DNA transformation and DNA repair ability compared with wild type. We were able to complement all these deficiencies by supplying a functional copy of recX elsewhere in the chromosome. The recX mutant still showed increases in pilus phase variation under conditions of iron starvation, and the recX mutant showed levels of RecA protein equivalent to wild type. Although the precise role of recX in recombination remains unclear, RecX aids all RecA‐related processes in Gc, and this is the first demonstration of a role for recX in homologous recombination in any organism.

Recombination, repair and replication in the pathogenic Neisseriae: the 3 R′s of molecular genetics of two human‐specific bacterial pathogens

Most of the detailed mechanisms that have been established for the molecular biological processes that mediate recombination, repair and replication of DNA have come from studies of the Escherichia coli paradigm. The human specific pathogens, Neisseria gonorrhoeae and N. meningitidis, are Gram‐negative bacteria that have some molecular processes that are similar to E. coli and others that appear to be divergent. We propose that the pathogenic Neisseriae have evolved a specialized collection of molecular mechanisms to adapt to life limited to human hosts. In this MicroReview, we explore what is known about the basic processes of DNA repair, DNA recombination (genetic exchange and pilin variation) and DNA replication in these human specific pathogens.

A bacterial siren song: intimate interactions between Neisseria and neutrophils

Neisseria gonorrhoeae and Neisseria meningitidis are Gram-negative bacterial pathogens that are exquisitely adapted for growth at human mucosal surfaces and for efficient transmission between hosts. One factor that is essential to neisserial pathogenesis is the interaction between the bacteria and neutrophils, which are recruited in high numbers during infection. Although this vigorous host response could simply reflect effective immune recognition of the bacteria, there is mounting evidence that in fact these obligate human pathogens manipulate the innate immune response to promote infectious processes. This Review summarizes the mechanisms used by pathogenic neisseriae to resist and modulate the antimicrobial activities of neutrophils. It also details some of the major outstanding questions about the Neisseria–neutrophil relationship and proposes potential benefits of this relationship for the pathogen.