Chapter 23 : Role of Phase and Antigenic Variation in Neisseria gonorrhoeae Colonization

Mechanisms of pilin antigenic variation and variation of pilus glycosylation. (A) Diagram of the pilin monomer, showing complete conservation at the N terminus and variability in the central and the C-terminal regions. Hypervariable (h-v) and semivariable (s-v) regions as defined by Hagblom et al. ( 94 ) are shaded; conserved regions are represented in white. The presence of a hypervariable region flanked by two cystines, which form a disulfide bond, was predicted from both the primary amino acid ( 216 ) and nucleotide ( 161 ) sequences. This hypervariable loop is the immunodominant region of the pilin ( 216 ), which suggests that immune pressure may have selected for the high degree of variation in this region. Posttranslational cleavage at amino acid 39 from the N terminus results in secreted (soluble) pilin (S pilin). (B) Schematic of the pilE gene and a pilS locus in N. gonorrhoeae and generation of a new pilE gene via nonreciprocal recombination. The number of copies per silent locus varies, with six copies being present in pilS1 of strain MS11 ( 93 ). Each copy contains six variable regions (minicassettes), which, when translated, correspond to the semivariable and hypervariable regions in the pilin protein. The pilE gene may receive a complete or partial pilS copy (as shown here). Therefore, over time, repeated recombination events into the pilE gene create a chimeric pilE gene composed of sequences from multiple pilS loci. The minicassettes are depicted here by shading, except for the ones within the pilE gene and the silent copy shown to be undergoing recombination, which are patterned. (C) Phase variation of pilus glycosylation in N. gonorrhoeae. Phase-variable expression of pilus glycosyltransferase (pgtA) can result in the presence or absence of galactose bound to an O-linked galactose N-acetylglucosamine molecule linked to a surface-exposed serine at position 63. The glycosylation state is also dependent on the presence of a serine residue at this position and therefore is also controlled by pilin antigenic variation. Only 11 of 17 pilin copies in the pilS loci of strain FA1019 encode a serine residue at this position ( 97 ). This finding suggests that antigenic variation may be a significant source of changes in pilus glycosylation.

Opa protein structure and function, and selection or induction of Opa protein expression during experimental murine genital tract infection. (A) Diagram of an opa gene showing the three regions of variability (SV, semivariable; HV1, and HV2, hypervariable regions 1 and 2) that define different opa alleles. The pentameric repeat responsible for phase variation is in the signal sequence-encoding region, with 7 to 28 copies present in different opa genes ( 54 , 238 ). (B) Cartoon depicting Opa-mediated adherence to and invasion of epithelial cells and nonopsonic uptake by phagocytes. (C) Opa phenotypes of vaginal isolates from three mice that demonstrated an increased percentage of Opa-positive variants among vaginal isolates following inoculation with a predominantly Opa-negative population of strain FA1090. More than 50% of the vaginal isolates expressed at least one Opa protein within 24 h following inoculation into the lower genital tract. Different Opa proteins predominated in different mice, with OpaB being most highly represented in mouse 3 and OpaI being mostly highly represented in mice 1 and 2. OpaI of this strain is known to bind HSPG, which is likely to be present in mice. OpaB does not bind HSPG but does bind to human CEACAM receptors (Guyer et al., Abstr. 101st Gen. Meet. Am. Soc. Microbiol. 2001). Gonococci that expressed more than one Opa protein simultaneously are represented by stripes.

Prototype N. gonorrhoeae LOS molecule, showing points of structural variation that lead to diversity in function. The basic structure and corresponding glycosyltransferase genes are based on references 15 and 83. Phase variation of LOS structure occurs due to frameshifts in a poly(G) region within lgtA (also called lsi2 [ 43 , 58 ]), lgtC, or lgtD ( 83 , 286 ) or a poly(C) region within lgtG ( 15 ). The synthesis of an alternative α chain, in which lgtC participates, occurs in only a minor population of gonococcal strains. Slippage of lgtA to an “off” position causes the production of a short-chain LOS species that confers high levels of serum resistance independent of growth in CMP-NANA ( 83 , 227 ). Expression of lgtA or lgtC increases serum sensitivity ( 227 ). In contrast, slippage of lgtG to an “on” position results in the formation of a highly bactericidal epitope on the β-chain ( 15 ). The addition of CMP-NANA via the action of sialyltransferase to the terminal galactose residue of the lacto-N-tetraose moiety is shown, a modification that results in several adaptive advantages, as described in the text. Phase variation of lgtA or lgtD can influence LOS sialylation by controlling the presence of the target species or by blocking the target residue, respectively ( 83 , 262 ). Sialylation blocks epithelial cell invasion mediated by interactions between the lacto-N-neotetraose moiety and the ASGP-R ( 99 , 262 ). Lectin-like interactions with gonococcal Opa proteins also rely on this tetrasaccharide species and are blocked by sialic acid ( 25 ).

Genetic control of the gonococcal Hg receptor and selection of “on” phase variants under certain circumstances in vivo. (A) Diagram of the Hg receptor (HpuAB) operon as described by Chen et al. ( 46 ), showing the poly(G) region in hpuA that is responsible for strand slippage during replication. The upper transcript is in frame; the lower transcript has lost a guanidine in the poly(G) tract and is out of frame. A putative Fur box is present upstream of the start site, which is likely to play a role in iron repression of transcription. (B) Representative graphs showing total recovery of gonococci versus recovery of Hg+ variants over time in a mouse with no influx of vaginal PMNs (top) versus a mouse that developed a PMN response during infection (bottom). The shaded area in the bottom graph corresponds to the period during which the frequency of Hg+ variants among vaginal isolates was significantly elevated over that of the inoculum. Numbers of vaginal PMNs were elevated during this period. Hg was detected on day 5 in vaginal washes from the mouse with inflammation; none was detected at any time point in mice without inflammation. (C) Cartoon depicting the circumstances during which Hg+ receptor expression may be advantageous. Selection for Hg+ variants by menstrual blood is supported by an analysis of endocervical isolates from women ( 4 ); whether the presence of Hg as an additional iron source in the female genital tract leads to increased virulence is not known. Selection for Hg+ variants on the development of a PMN influx in the lower genital tract of mice suggests that gonococci may capitalize on the introduction of Hg, which exudes into the lumen with other serum components, during inflammation ( 115 ). It is possible that Hg+ variants are also selected for during the bloodstream stage of disseminated infection; however, no evidence for this hypothesis has been reported.