Microbial antigenic variation mediated by homologous DNA recombination

Abstract

Pathogenic microorganisms employ numerous molecular strategies in order to delay or circumvent recognition by the immune system of their host. One of the most widely used strategies of immune evasion is antigenic variation, in which immunogenic molecules expressed on the surface of a microorganism are continuously modified. As a consequence, the host is forced to constantly adapt its humoral immune response against this pathogen. An antigenic change thus provides the microorganism with an opportunity to persist and/or replicate within the host (population) for an extended period of time or to effectively infect a previously infected host. In most cases, antigenic variation is caused by genetic processes that lead to the modification of the amino acid sequence of a particular antigen or to alterations in the expression of biosynthesis genes that induce changes in the expression of a variant antigen. Here, we will review antigenic variation systems that rely on homologous DNA recombination and that are found in a wide range of cellular, human pathogens, including bacteria (such as Neisseria spp., Borrelia spp., Treponema pallidum, and Mycoplasma spp.), fungi (such as Pneumocystis carinii) and parasites (such as the African trypanosome Trypanosoma brucei). Specifically, the various DNA recombination-based antigenic variation systems will be discussed with a focus on the employed mechanisms of recombination, the DNA substrates, and the enzymatic machinery involved.

Keywords: Mycoplasma; Neisseria; Pneumocystis; Treponema; Trypanosoma; gene conversion.

Fig. 1

Results of pilin antigenic variation. A starting piliated variant can produce piliated (P⁺) antigenic variants, under-piliated (P^+/−) antigenic colony morphology variants, or non-piliated (P⁻) colony morphology variants. The colony morphologies are shown in the light micrographs from (Swanson, 1978). P⁺↔P⁺ and P⁺↔P^+/− variants occur by gene conversion reactions between pilS copies and the pilE locus. P⁺↔P⁻ variation can occur by three distinct mechanisms: (a) gene conversion, (b) PilC variation, or (c) pilE deletion. Only (a) and (b) are reversible. Pili are shown as straight lines were a color change indicates an antigenic variation and or phase variation event. S-pilin is shown as circles with the same color as the pilin.

Fig. 2

Cartoon of the pilin loci of strain FA1090. Depicted in light blue are the five silent loci and the silent copies encoded in each. The pilE locus in shown in black (with the variable sequences highlighted in blue) and includes the single silent copy (the upstream silent locus) that is associated with the expressed gene. The conserved, non-coding Sma/Cla repeat is present at the 3′ end of each locus (black oval). In the middle is a detailed cartoon of the pilE gene with the constant region (C), semi-variable region (SV), cysteine region 1 (cys1), hypervariable loop (HV_L), cysteine region 2 (cys2), and the hypervariable tail (HV_T). Conserved DNA segments are shown in black and variable ones are shown in blue.

Fig. 3

Cartoon depicting the gene conversion reactions resulting in pilin phase and antigenic variation. A donor pilS copy and the recipient pilE gene are shown. Below, three out of the many potential recombinants are shown, each having a segment of microhomology at the ends of the new segment of DNA. In each case, the original pilS copy is retained. Conserved DNA/amino acid segments are shown in black and variable ones are shown either in red or yellow. The Sma/Cla repeat shown as a black oval is only present downstream of some silent copies (see Fig. 2).

Fig. 4

The guanine quadraplex (G4)-forming sequence is located upstream of the pilE gene. (a) The G4-forming sequence is located on the bottom strand of the DNA about 180 bp upstream of the pilE -10 sequence of the promoter (P). The different segments of the pilE are indicated and the Sma/Cla repeat is shown as a black oval. (b) Sequence of the G4-forming region. Shown in blue is the G₃ residue that, when mutated, produces an AVI phenotype that is lost when the G₀ residue is also mutated; a G₀ mutation has no phenotype by itself. The loop bases are shown in red and can be changed without altering pilin antigenic variation. (c) The parallel G4 structure as determined by NMR analysis (Kuryavyi V.V. et al., paper under review).

Fig. 5

Antigenic variation in Borrelia species. (a) In B. Burgdorferi, (parts of) variant, silent vls cassettes (vls2-vls16, in light blue), can be transferred to the vlsE ORF within the vls expression site by virtue of gene conversion. This process is dependent on the activity of RuvA and RuvB(Dresser et al., 2009; Lin et al., 2009}. The silent vls segments (only vls2 to vls6 are shown) are flanked by 17-bp direct repeats(in red). The promoter within the expression site, located upstream of the vlsE ORF, is indicated by a black triangle. (b) In B. hermsii, silent vsp and vlp genes can be transferred in their entirety to the expression site, thereby replacing the gene that was originally present. The gene replacements occur by gene conversion that involve sequences upstream (the upstream homologous sequence, UHS; in red) and downstream (the downstream homologous sequence, DHS; in blue) of the vsp/vlp ORFs.

Fig. 6

Structure of the T. pallidum tprK gene and mechanism of antigenic variation of the variable (V) regions of TprK. The tprK gene (shown at the top) represents the single expression site for the antigenic TrpK protein. Within the tprK ORF (in dark grey), seven discrete V regions are located. These regions, which vary in length from 32 to 91 bp, are termed V1 to V7(in blue) (Stamm & Bergen, 2000; LaFond et al., 2003; Centurion-Lara et al., 2004). At the 5′ and 3′ side of another tpr gene, i.e. tprD (shown at the bottom in light grey), sequence cassettes are located that represent diverse V region donor sequences, which can be either complete or partial. These donor cassettes can be copied and inserted into the tprK locus, creating variant V region sequences (Centurion-Lara et al., 2004). The V regions and V donor sequences have flanking as well as internal four-bp repeats that may play a role in recombination. During recombination at the tprK V regions, the donor V sequences remain unaltered. It was therefore hypothesized that V region diversification occurs by means of a gene conversion mechanism(Centurion-Lara et al., 2004).The tprK promoter is indicated by a black triangle.

Fig. 7

Structure of the M. pneumoniae P1 operon and predicted mechanism of antigenic variation of the P1 protein. The P1 operon contains three ORFs, i.e. MPN140, MPN141 and MPN142, the latter two of which encode antigenic surface proteins (P1 and P40/P90). MPN141 contains two variable DNA elements, a RepMP4 element (RepMP4-c; in blue) and a RepMP2/3 element (RepMP2/3-d; in red), which are not unique to the P1 operon: in total, eight RepMP4 variants and 10 RepMP2/3 variants are found dispersed throughout the M. pneumoniae genome (Table 1). The downstream MPN142 gene contains a RepMP5 element (in yellow); this element has eight counterparts in the genome. The RepMP sequences can be transferred (in part) to their homologous sequences within the P1 operon or to other homologous variants (Spuesens et al., 2009, 2011) by means of segmental gene conversion. To illustrate these gene conversion processes, a subset of RepMP2/3 elements is shown in different colors; sequences from these elements can be transferred to other RepMP2/3 elements in the genome, including the element that is located within MPN141. The P1 promoter is indicated by a black triangle.

Fig. 8

Antigenic variation in Pneumocystis species. The msg gene in the expression site (at the top) can be replaced periodically by any of the ~80 silent, donor msg genes (at the bottom) by means of homologous DNA recombination; this leads to the expression of a novel variant of the Msg protein at the fungal surface. The expression site contains a unique sequence, i.e. the upstream conserved sequence (UCS; in blue), located at the 5′ side of the msg ORF, which is flanked by a 24-bp conserved sequence (the conserved recombination junction element [CRJE]; the small red box). This CRJE, which may have a function in the recombination of msg sequences, is also present immediately upstream of all silent msg ORFs. The latter ORFs were also found to recombine with each other.

Fig. 9

Genomic location of the Variant Surface Glycoprotein (VSG) gene repertoire in Trypanosoma brucei. (a) The vast majority of the VSG genes and pseudogenes (more than 1500 in T. brucei 927) are located in extensive subtelomeric tandem arrays. These haploid regions are attached to the diploid chromosomal cores. The vast majority of the VSGs (more than 90%) are pseudogenes (ψ) (indicated with grey filled boxes), with functional VSGs indicated with filled coloured boxes. (b) A subset of the VSGs (more than 200) are located adjacent to the telomere repeats of small chromosomes including an abundant class of minichromosomes (of which there are about 100 in the cell). (c) About 15 VSGs are located adjacent to the telomeres of the VSG expression site transcription units (promoters indicated with flags). Only one VSG expression is transcribed at a time (indicated with an arrow).

Fig. 10

Mechanisms of Variant Surface Glycoprotein (VSG) switching in T. brucei. The large open boxes indicate trypanosomes. The active VSG gene is transcribed from a single active telomeric VSG expression site (ES), with the ES promoter indicated with a flag, and transcription with an arrow. (a) VSG switching mediated by duplicative gene conversion involves a silent VSG being copied into the active ES, thereby replacing the old VSG. (b) VSG switching mediated by segmental gene conversion involves the recombination of segments of multiple VSG genes and pseudogenes, resulting in the generation of a new mosaic VSG. (c) VSG switching mediated by telomere exchange involves a DNA cross-over on two telomeres. This inserts a previously silent telomeric VSG into the active ES, and moves the previously active VSG to a silent telomere. (d) VSG switching can be mediated through transcriptional control whereby a previously silent ES is activated and the active ES is silenced.

Fig. 11

Duplicative gene conversion can result in a silent VSG being copied into the active VSG expression site transcription unit. Above are indicated tandem arrays of silent VSG genes (coloured boxes) and pseudogenes (ψ) (grey filled boxes). The VSG expression site transcription unit is shown below, with the promoter indicated with a flag, and transcription with an arrow. VSGs are flanked upstream by characteristic 70 bp repeats (indicated with vertically hatched box). VSGs also contain highly conserved sequences at the 3′ end (indicated with a black box). Gene conversion can occur using these two areas of homology to copy a duplicate copy of a silent VSG into the active VSG expression site (indicated below).

Fig. 12

Segmental gene conversion of multiple VSG pseudogenes can result in the creation of a new functional chimeric VSG. Three different VSG pseudogenes are indicated above, with disruptions of the open reading frame indicated with arrow heads and vertical lines. Multiple successive gene conversion reactions can take place, resulting in the creation of a new functional VSG which is a mosaic of segments of the different VSG pseudogenes.