Neisserial surface variation: how and why? John Swanson, Robert J. Belland and Stuart A. Hill Rocky M o u n t a i n Laboratories, National Institutes of Health, Hamilton, Montana, USA Neisseria gonorrhoeae exhibits striking variablity in several of its surface components (pili, Opa proteins and lipooligosaccharide) in vivo and in vitro. Such flagrant variation of this mucosal pathogen's surface components contrasts sharply with changes in single surface components of blood-borne trypanosomes and borreliae. Despite these differences, similar molecular events are sometimes involved.

Current Opinion in Genetics and Development 1992, 2:805-811

Introduction Implicit in many studies of the genetic variation of pathogenic microbes is the assumption that the changes contribute to evasion or avoidance of the hosts immune system. The supposition is apt for trypanosomes and relapsing fever borreliae whose broad array of different surface coats interfere with the hosts ability to eradicate the parasitemic organism by means of immune mechanisms. Pathogens such as N. gonorrhoeae reside mainly at mucosal sites, are relatively less exposed to the hosts immune components, yet display impressive variability of their surface-exposed molecules. Although detectable as antigenic changes, they may relate more to altered function than to immune evasion or avoidance.

N. gonorrboeae exhibits striking variability of its pili, outer membrane Opa proteins, and lipooligosaccharide (LOS) both in vitro and in vivo. Here, our intent is to summarize recent, pertinent information on the genetic events underlying expression or structural changes of these surface components that have antigenic or functional significance. Briefly noted are selected, changeable surface components of borreliae, trypanosomes, mycoplasmas, and a few other pathogens for contrast and comparison.

Gonococcal pilus variation: gene conversion or transformation? How gonococci effect pilus phase (on/off) and antigenic variations is unsettled [1-4]. Early work pointed to classical RecA-dependent, non-reciprocal gene conversions, with unidirectional transfer of novel sequence segments from one of 20 + silent, partial pilin genes, pilS, into a single, pilin-expression locus, pile. The corresponding segment resident in pile was discarded during transfer but

the donor pilS was retained, unaltered [1 ]. Under special conditions, reciprocal exchange occurred between pile and the pi~S immediately downstream [5]. The continuous competence of gonococci coupled with their propensity to lyse and release DNA suggested that DNA transformation might contribute to pilin variation [6]. Data supporting this hypothesis includes: first, that pilE::CAT-operon fusions in transformation donorDNA integrate into the recipient pilE [7]; and second, that pilus phase-variation by gonococci is reduced by DNaseI treatment [5]. Accordingly, horizontal exchange of pil information and the consequent pilin changes are envisaged as resulting from DNA transformation [2,3]. Such transformation-mediated pilin variation would be relevant in vivo only in individuals infected simultaneously with two or more genetically distinct strains of gonococci [8.]. That transformation drives pilin variation is at odds with a number of observations: first, pilins vary gready in individuals infected with a single strain [9]; second, the relative instability at pile (changes in frequencies of approximately lxl03/ceU/generation) is not matched by any observable instability of an opa locus located 800bp downstream of pile [10"]; and third, transformation-defective gonococci exhibit frequencies and types of pile variation resembling those of transformation-competent organisms [11]. Transformation and pile phase-variation frequencies have recently been re-examined [12..]. Defined pile mutants (frameshift, nonsense and missence mutations) were assessed for reversion to pilus +, which heralded the correction of their respective lesions. There was no correlation between transformation (approximately 10-6/recipient) and reversion frequencies ( > 104/colonyforming unit/generation); reversion rates were also unchanged in the presence of DNaseI. Our recent results show that transformation with donor pilE::CAT fusions involves wholesale change of the recipient pile (i.e. pile exchange). This is distinct from transformation with

Abbreviations BC--basic copy; LOS~lipooligosaccharide; LPS---lipopolysaccharide; PMN~polymorphonudear leukocyte; SS~slipped-strand mispairing; URS--upstream repeat sequence; VMP--variable membrane protein; VSG--variable surface glycoprotein. (~) Current Biology Ltd ISSN 0959-437X

805

806 Prokaryotesand lower eukaryotes plasmid pilE::CAT fusions that lack 3' flanking-sequence homology to the resident pilE, which show segmental pilE replacements resembling spontaneous pilE changes (SA Hill et aL, unpublished data). Extensive flanking-sequence homologies clearly favor pilE exchange, and it is unclear whether pilS sequence transforms into pilE in a chromosomal context. Transformation-mediated and intracellular gene conversion events are quite distinguishable; the former replaces pilE in toto while the latter brings partial or complete pilS sequence into pilE. The limited diversity of pilE 'hypervariable' regions in unrelated gonococcal isolates suggests that pilus function constrains the extent of pilin variation and is consistent with the concept of horizontal transfer for p a information in vivo [8.]. A recent report [13"] suggests that functional selection drives pilin variation when pilC is switched-off, as described later.

The changing surfaces of borreliae and trypanosomes Changes in expression of variable membrane protein (VMP ) in Bon'elia hermsii and variable surface glycoprotein (VSG) in Trypanosoma brucei have several similarities to the intragenic conversions of neisserial pilin variation [14,15]. The VMP and VSG systems include a single telomeric expression locus and multiple silent intact gene copies. Silent VSG genes of trypanosomes (approximately 1000) reside in non-telomeric locations; only the silent basic copy (BC) is adjacent to an upstream repeat sequence (URS) [16.]. The BC contains more unique sequence than downstream silent genes, and sequence divergence in four silent VSG genes has been shown to be greater in their amino-terminal coding regions [17"]. Most silent VMP genes of B. hermsii are also intact, but partial silent copies are also present (T Kitten and A Barbour, personal communication). VMP silent genes reside on linear plasmids maintained in ratios of 1:1 (T Kitten and A Barbour, personal communication) and are activated through promoter addition [18]. Upstream and downstream repeat elements are thought to guide silent VMP copies into the expression site, analogous to the VSG gene system of trypanosomes. The repeats downstream of VMP genes resemble the "Sma/Cla repeat" (BI Restrepo et al., personal communication), which has been suggested to be important in neisserial pilin gene variation. Both t7. hermsii VMPs and trypanosomal VSGs are changed by gene conversions, with intact silent genes replacing the previously expressed gene. Partial gene replacements that create VMP chimaeras occur in 17. hermsii infecting immunodeficient mice (T Kitten and A Barbour, personal communication), suggesting that immune mechanisms may counterselect against such partial VMP gene replacements. Trypanosomes spontaneously switch VSG genes at a rate of 10.6- 107/celggeneration [14], while B. hermsii spawns serotypic variants at a rate of 10 .3- 10-4/cell/generation [19]. Programmed gene re-

arrangements are seen in both systems [20]; in T. brucei, the proximity of the BC to the URS dictates it preferred usage, with succeeding silent genes being expressed relative to sequence differences in their conserved upstream portions [16.,17.]. It has been suggested that the ability of old and new VSG molecules to form heterodimers that prevent the appearance of trypanosomes naked of VSG are also involved in determining the order of trypanosomal VSG gene expression [21,22]. The telomeric location common to VMP and VSG expression sites suggests that it confers special attributes: telomeres in yeast are sites for cellular nucleases [23"]. Recombination may initiate at telomeric expression as a result of the actions of site-specific DNases [24]. Antigenic variation in Borrelia burgdorferi differs from that in B. hermsii. The major outer surface proteins of B. burgdo~feri, OspA and OspB, are encoded by tandem ospA and ospB genes, which have considerable sequence identity [25]. Osp variants can arise through single base deletions that produce frameshift mutations and truncated Osp proteins, or through deletions involving homologous sequences common to ospA and ospB, resulting in the creation of chimeric ospA-ospB genes (Rosa et aL, personal communication). Such Osp variations have analogues in pile of gonococci with respect to the deletion of sequences between repeated regions [26,27]. Variation in a third B. her,nsii surface antigen, OspC, occurs by an unknown mechanism [28].

Gonococcal Opa variation: illegitimate recombination A number of prokaryotic pathogens employ illegitimate modes of recombination [29-31] to change expression states of important structural genes at high frequencies. The genetic events utilizing these pathways are characteristically independent of the homologous recombination functions such as recA [29], but do not utilize site-specific recombinases. A unifying feature of the sequences involved is their highly repetitive nature. This characteristic is essential to regions undergoing high-frequency DNA replication errors such as slipped- strand mispairing (SSM) [32]. Neisserial opa genes switch their expression states by changing the number of direct pentameric repeat elements (CTI'CT) encoding tile signal peptide of the Opa protein [33-35]. Varying the number of repeat elements serves to either align or misalign the N-formyl methionine initiation codon with the codon for the amino-terminus of the exported protein, resulting in a change in the expression state of the gene [33]. Neisseria meningitidis has three or four opa loci while N. gonorrhoeae strains have as many as 11 [10",33,36]. Members of the opa repertoire in N. gonorrhoeae strain MS11 contain large regions of sequence homology surrounding three hypervariable segments that are predicted to be exposed on the surface [10"]. Genetic reassortment

Neisserial surface variation: how and why? Swanson, Belland, Hill 807 among opa genes is negligible when compared to that seen for neisserial pilin sequences. Phase-variation frequencies of opa genes, expected to be involved a priori, vary substantially in vitro and in viva Particular Opa. members clearly predominate among Opa + derivatives from particular Opa stocks [37,38]. Frequencies for the expression of particular Opa proteins relate to the number of repeat elements in their respective genes, with intermediate numbers (approximately 8-18) favoring higher switch-on frequencies (J Swanson and R Belland, unpublished data). Another important feature is promoter structure, with three different promoter motifs found among the 11 opa loci of MS11. Two have no consensus -35 region [10"], suggesting that additional regulatory factors are involved in opa transcription. Studies of the opa-switching mechanism in Escherichia coli using opa::phoA fusions have highlighted three points: first, phase-change frequencies increase with increased transcription [39]; second, predictions based on the SSM model do not hold for the observed change frequencies (R Belland and J Swanson, unpublished data); and third, the repeat region in each opa has the potential to form unusual non-B-DNA structures (H-DNA [39] and triplex molecules [R Belland, unpublished data] ) because of its purine/pyrimidine strand bias and mirror palindromic sequence. PilC expression has recently been described as influencing pilus phase variation by the induction of, or selection for, particular pilE sequence changes [40]. Outer-membrane PilC protein is involved in pilus assembly, and its production switches on/off in response to the number of residues in a stretch of G residues that occur in the signal peptide encoding portion of pilC. PilC is involved in pilin transport, and its absence is envisaged as a selective pressure that results in the generation of pilE sequences that encode soluble, secreted pilin molecules and thus relieve the intoxication of cells containing accumulated, unprocessed pilin molecules [13"]. Both opa and pibC variations in gonococci are independent of RecA [13",34]. Expression of opa and pilC elements is either on or off, unlike the translational switching of the licA gene in Haemophilus influenzae [41]. The number of direct tetrameric repeat elements (CAAT) in the 5' end of the licA open reading frame determines whether the LicA protein is produced at either low or high levels, or not at all [41]. The licA gene per se does not encode a structural surface protein, but rather determines the biosynthetic pathway utilized for the production of LOS [41,42]. Sequence changes outside the coding regions can also alter the expression state. Bordetella pertussiafim genes depend on the bvg locus for coordinated expression but still show phase variation as a result of insertions and deletions in a stretch of C residues upstream of the tim -10 region, within the promoter element [43]. Presumably the distance between the bvg responsive element, the AB-box, and the -10 region is critical for initiating transcription [43]. Because variation in the number of

C residues is the only identifiable difference between high- and low-level expression of tim, it seems probable that some form of illegitimate recombination is responsible, but whether these changes are dependent on recA is unclear. The size-variable surface lipoproteins of Mycoplasma hyorhinis show a similar variation in expression as a result of changes in the number of A residues upstream of the -10 regions of the v/pA, -B and -C genes [44.-]. Uniquely, the Vlp proteins also show considerable size variation as the result of changes in the numbers of repeated amino-acid segments in the surface-exposed carboxy-terminal sections of the proteins [45,46].

Lipooligosaccharide variation The quantitatively dominant glycolipid in the gonococcal outer membrane is termed lipooligosaccharide to emphasize the truncation of its saccharide side chains as compared with the lipopolysaccharide (LPS) derived from many other Gram-negative bacteria. The composition and linkages of these oligosaccharide side chains vary. Any given gonococcal strain may synthesize several ( > 3-6) LOS molecules with differing side chains [47-49,50"]. Some LOS variants contain terminal GaI4GIcNAc that mimics the glycosphingolipids (gangliosides) on human erthrocytes [51]. Identical oligosaccharides occur in LPS/LOS of several other bacteria that infect mucosal surfaces (N. meningitidis, H. influenzae and I-L. ducreyii) [52"]. Little is known about the genetics of gonococcal LOS variation, but it is known to be RecAindependent and reversible (T Chen, B Belland and J Swanson, unpublished data). One lipooligosaccharideinvolved locus, /si-1, has been cloned, can transform gonococci to express the specified LOS molecules, and is thought to act as a transcriptional attenuator. No gross chromosomal rearrangement in the Zsi-1locus is apparent among LOS variants [53]. Another locus, /si-3, is thought to be necessary for the addition of "O-side chains" to the LOS core [54].

Clinical implications Gonococcal variation in vivo generates an impressive array of pilus, Opa and LOS phenotypes shed by individual males challenged intra-aurethrally with pilus + Opa cells [9,37,50"] (see Fig. 1). The pathogenetic implications of these variations are not completely clear, partly because of the lack of relevant laboratory infection models. Pflus + N.. gonorrhoeae and N. meningitidis attach more avidly to tissue-culture cells than pilus- organisms, and it is this function that probably accounts for the clear relationship between piliation and the virulence of gonococci in human male volunteers [9]. The expression of different pilins by pilus + neisseriae modifies their abilities to adhere to various tissue-culture cells in vitro [55",56].

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Fig. 1. Opa protein, pilin, and lipooligosaccharide (LOS) variations of organisms reisolated from a single male infected with strain MS11 gonococci (Gc}. Input gonococci were instilled into the subjects urethra and gonococci were then reisolated from the urine samples, and from one semen specimen, collected at intervals (in hours) thereafter. The Opa, pilin and LOS phenotypes of representative reisolates (selected from hundreds that were characterized) are shown by immunoblotting with monoclonal antibodies following electrophoresis of whole cell lysates. The anti-Opa and anti-pilin monoclonals react with all Opa and pilin molecules in this strain, while the anti-LOS monoclonal was specific for the particular LOS elaborated by input organisms. Organisms representing the input organisms and 2 h reisolates are identical, but after 22 h post-instillation, most reisolates express one, another, or several Opa proteins and many produce pilin that differ in apparent size from input cells. By 46 h some reisolates express a changed LOS that does not react with the monoclonal. The variation depicted here represents a fraction of that found among gonococcal reisolates from this and other experimentally infected males [9,371.

How Opa influences gonococcal pathogenicity is unclear. Pilus- gonococci that express certain Opa proteins show enhanced attachment to tissue-culture cells in vitro [57%58",59"] while others influence bacterial adherence and ingestion by neutrophils [60o,61]. Opa expression also affects clumping of gonococci [37], and this may contribute to their dissemination from an infected focus via ciliary, fluid-flow, peristaltic, and other forces operative on mucosal surfaces. Except for Opa influences on interactions between gonococci and neutrophfls, pilus and Opa variations have no clear relevance to immune avoidance or evasion; rather they appear to contribute to the gonococcus ability to colonize particular anatomical niches and to adhere to and b e c o m e internalized by epithelial cells. When males are experimentally infected with gonococci whose LOSa cannot be sialylated, variants producing a different LOSb (larger apparent size and capable of being sialylated) arise in some but not all subjects [62]; in some such males, variants arise concurrent with the onset of the symptoms (leukorrhea, dysuria) typical of gonorrheal disease [50"]. LOS variation may impact on both

immune avoidance and other pathogenetic behaviors of gonococci. Important in this context is the sialylation of those LOS molecules with Gal4GlcNAc that occurs in vivo [63,64] and renders gonococci resistant to killing by human serum [65,66]; analogous serum-resistance is found for/-L, influenzaewith sialylated LOS [52"]. Resistance to killing by complement appears to be the result of sialicacid residues binding plasma factor-H which then combines with cell surface-bound complement component, C3b, leading to its rapid inactivation [67-69]. Sialylation of LOS may also modify interactions of gonococci with neutrophils [70-] and reduce their ingestion by epithelial cells (J van Putten, personal communication).

Conclusion The somewhat ill-defined relationships between variations in gonococci and their interactions with the hosts immune system contrast with the generally accepted notions about the VSGs and VMPs of trypanosomes and

Neisserial surface variation: how and why? Swanson, Belland, Hill 809 relapsing fever borreliae, respectively. The virulence of the latter two organisms relies heavily on their having changeable surface coats for their protection from host antibodies. Several facets of trypanosomal VSG changes need clarification. Trypanosomes cultivated in vitro express no VSG, so studies on its variability are limited to organisms in a mammalian host. Relatively little is known about VSG changes that might occur but are not amplified or modified by host immunity. Borreliae that cause relapsing fever, and which infect immunocompromised mice, express VMP chimeras not seen in intact, immune-competent animals. Although VMP changes resemble gonococcal pitE changes, their relationship to the appearance of dominant VMP serotypes in vivo is unclear. In spite of the similarities for some switching events in these different organisms, the multi-component variation of the gonococcal surface contrasts sharply with the relatively monotonic changes in VMP of B. hermsii and VSG of T. brucei.

by a Family of 11 Complete Genes. Mol Microbiol 1991, 5:1889-1901. Details the cloning and sequence analysis of the entire opa repertoire in strain MSll along with estimates of opa sequence divergence and evolutionary relatedness.

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DONELSONJE: DNA Rearrangements and Antigenic Variation in Afifican Trypanosomes. In Mobile DNA Edited by Berg DE, Howe MM. Washington DC: American Society for Microbiology; 1989:763-781.

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MANDRELLRE, GRIFFIS JM, IVbXCHERBA: Lipooligosaccharides (LOS) of Neisseria gonorrhoeae and Neisseria meningitidis have Components that are lmmunochemically Similar to Precursors of Human Blood Group Antigens. J Exp Ned 1988, 168:107-126.

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MANDRELLRE, MCLAUGHUN R, KWAIK YA, LESSE A, YAMASAKI R, GIBSON B, SPINOLASM, APICELLAMA: Lipooligosaccharides (LOS) of Some Haemophilus Species Mimic Human Glycosphingolipids, and some LOS are Sialylated. Infect hnmun 1992, 60:1322-1328. Demonstration that H. influenzae LOS is sialylated, reinforcing the notion that this is a significant mechanism, in addition to capsulation, employed by pathogens for coping with host defenses. 53.

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Neisserial surface variation: how and why? Swanson, Belland, Hill 57. •o

MAKiNOS-I, VAN PUTTENJPM, MEYERTF: Phase Variation of the Opacity Outer Membrane Protein Controls Invasion of Neisseria gonorrhoeae into Human Epithelial Cells. EMBO J 1991, 10:1307-1315. This paper clearly demonstrates the invasive capability of N. gonorrhoeae expressing a specific Opa protein. Importantly, the report demonstrates that, despite the sequence similarities found within the opa-gene family, specific members of the protein family impart important biological attributes to the organism that could potentially play a role in its virulence. SIMOND, REST RE: Escherichia coli Expressing a Neisseria gonorrhoeae Opacity-Associated Outer Membrane Protein Invade Human Cervical and Endometrial Epithelial Cell Lines. Proc Natl Acad Sci USA 1992, 89:5512-5516. Demonstration that an Opa protein fusion expressed in E. coli leads to the specific attachment and uptake of the recombinant strain by ME-180 human endocervical epithelial cells.

an Immunelectron Microscopic Analysis. J Infect Dis 1990, 162:506-512. 64.

MANDRELLRE, LESSEAJ, SUGAIJV, SHERO M, GRIFFISJM, COLE A, PARSONSNJ, SMITH H, MORSE SA, APICELLAMAc In Vitro and In Vivo Modification of Neisseria gonorrhoeae Lipooligosaccharide Epitope Structure by Sialylation. J Exp Med 1990, 171:1649-1664.

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PARSONSNJ, ANDRADEJR(], PAI'EL PV, COLE JA, SMITH H: Sialylation of Lipopolysaccharide and Loss of Absorption of Bactericidal Antibody During Conversion of Gonococci to Serum Resistance by Cytidine 5'-Monophospho-N-AcetylNeuraminic Acid. Microb Pathog 1989, 7:63-72.

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HORSTMANNRE): Target Recognition Failure by the Nonspecific Defense System: Surface Constituents of Pathogens Interfere with the Alternative Pathway of Complement Activation. Infect Immun 1992, 60:721-727.

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MERI S, PANGBURNMK: Discrimination Between Activators and Nonactivators of the Alternative Pathway of Complement: Regulation via a Sialic Acid/Polyanion Binding Site on Factor H. Proc Natl Acact Sci USA 1990, 87:3982-3986.

58. •

59. •

WEELJFL, HOPMANCTP, VAN PUTTENJPM: In Situ Expression and Localization of Neisseria gonorrhoeae Opacity Proteins in Infected Epithelial Cells: Apparent Role of Opa Proteins in Cellular Invasion. J Exp Med 1991, 173:1395-1405. An electron microscopic examination of the attachment and uptake of N. gonorrhoeae expressing a specific Opa protein by cultured Chang conjunctival cells. 60. •

BELLANDRJ, CHEN T, SWANSONJ, FISCHER SH: Human Neutrophil Response to Recombinant Neisserial Opa Proteins. Mol Microbiol 1992, 6:1729-1737. A demonstration that seven different Opa protein fusions expressed in E. coli exhibit the same behavior in the presence of human polymorphonuclear leukocytes (PMNs) as N. gono~'hoeae MS11 expressing the analogous proteins; that is, certain proteins elicit a strong non-opsonic stimulation of PMNs while one particular Opa protein is non-reactive. 61.

FISCHERSH, REST RF: Gonococci Possessing Only Certain P.II Outer Membrane Proteins Interact with Human Neutrophils. Infect l m m u n 1988, 56:1574-1579.

62.

SWANSONJ: Some Affects of LOS and Opa on Surface Properties of Gonococci. In Proceedings of Hoe Seventh International Pathogenic Neisseria Conference. Berlin: Walter de Gmyter & Co.; 1991:391-396.

63.

APICELLAMA, MANDRELLRE, SHERO H, WILSON ME, GRIFFISS JM, BROOKS GF, LAMMELC, BREEN JF, RICE PA: Modification by Sialic Acid of Neisseria gonorrhoeae IApoollgosaccharide Epitope Expression in Human Urethral Exudates:

70. •

REST RE, FRANGIPANE JV: Growth of Neisseria gonorrhoeae in CMP-N-Acetylneuraminic Acid Inhibits Nonopsonic (Opacity-Associated Outer Membrane Protein-Mediated) Interactions with Human Neutrophils. Infect Immun 1992, 60:989-997. This, together with [67--69], is another interesting example of the affects of LOS sialylation on gonococcal behavior.

J swanson, RJ Belland, SA Hill, National Institutes of Health, National Institute of Allergy and Infectious Disease, Rocky Mountain laboratories, Laboratory of Microbial Structure and Function, Hamilton, Montana 59840, USA.

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