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Frank SA. Immunology and Evolution of Infectious Disease. Princeton (NJ): Princeton University Press; 2002.

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Immunology and Evolution of Infectious Disease.

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Chapter 5Generative Mechanisms

In this chapter, I summarize the different ways in which parasites generate antigenic variants. The amount of new variation and the kinds of new variants influence antigenic polymorphism and the pace of evolutionary change (Moxon et al. 1994; Deitsch et al. 1997; Fussenegger 1997).

The first section describes baseline mutation rates and special hypermutation processes that raise rates above the baseline. Microbial mutation rates per nucleotide decline with increasing genome size, causing a nearly constant mutation rate per genome per generation of about 0.003. Genome-wide hypermutation can raise the mutation rate at all sites within the genome. Such mutator phenotypes probably have altered replication enzymes. Low frequencies of mutator phenotypes have been observed in stable populations of Escherichia coli, whereas fluctuating populations appear to maintain higher frequencies of mutators. In some cases, hypermutation may be targeted to certain genes by DNA repeats and other DNA sequence motifs that promote local replication errors.

The second section presents three common mechanisms that parasites use to change gene expression between antigenically variable copies of a gene, potentially allowing escape from immune recognition. Replication errors of short nucleotide repeats can alter regulatory sequences or disrupt translation of coding sequences. Gene conversion can copy variant genes from different genomic locations into a single expression site. Invertible pieces of DNA occasionally change the direction of nucleotide sequences, altering the expression of nearby genes. Different Plasmodium species have different families of antigenically variable surface molecules. The mechanisms by which Plasmodium species switch expression between antigenic variants are not fully understood.

The third section focuses on parasites that store antigenic variants within each genome. Some parasite genomes have dozens or hundreds of variants but express only one archival copy at a time. Intragenomic recombination between archived copies may create new variants. Studies of the spirochete Borrelia hermsii and the protozoan Trypanosoma brucei provide evidence of recombination between archival copies.

The fourth section lists different mechanisms that can mix genetic material between lineages of parasites to create new antigenic variants. Segregation brings together in one individual different chromosomes from distinct lineages. Intergenomic recombination mixes genetic sequences from different lineages. Horizontal transfer moves pieces of DNA from one individual into another by processes such as bacterial uptake of naked DNA from the environment.

The final section outlines promising topics of study for future research.

5.1. Mutation and Hypermutation

There are many different ways to measure mutation rates and many different processes that influence mutations (Drake et al. 1998). I focus in this section on errors in nucleotide replication that change the antigenic properties of the encoded molecule.

Baseline Mutation Rates

RNA virus populations typically have high frequencies of mutants and often evolve rapidly (Holland 1992; Knipe and Howley 2001). However, few studies have provided direct estimates of mutation rates. The limited data suggest relatively high mutation rates on the order of 10−4–10−5 per base per replication (Holland 1992; Coffin 1996; Preston and Dougherty 1996; Drake et al. 1998; Drake and Holland 1999).

Drake et al. (1998) summarized mutation rates for various microbes with DNA chromosomes (table 5.1). The table shows an amazingly consistent value of approximately 0.003 mutations per genome per generation. This value holds over genomes that vary in total size by four orders of magnitude; consequently the per base mutation rates also vary over four orders of magnitude.

Table 5.1. Mutation rates per replication in various microbes.

Table 5.1

Mutation rates per replication in various microbes.

Genome-wide Hypermutation

None of the microbes in table 5.1 face intense, constant selective pressure on antigens imposed by vertebrate immunity—it is unlikely that E. coli depends on antigenic variation to avoid clearance from its hosts. It would be interesting to know if pathogens under very intense selection by host immunity have higher baseline mutation rates than related microbes under less intense immune pressure.

High genome-wide mutation rates arise in bacteria by spontaneous mutator mutations, in which the mutator alleles raise the error rate during replication (Drake et al. 1998). The mutator alleles probably are various DNA replication and repair enzymes. Ten or more genes of E. coli can develop mutator mutations. Assuming that each gene has about 1,000 bases, then the overall mutation rate of mutator loci is 10 × 1,000 × 5 × 10−10 ≈ 10−6–10−5, based on the per base mutation rate in table 5.1. Some mutations will be nearly neutral; others will cause extremely high mutation rates and will never increase in frequency.

Typical E. coli cultures accumulate mutator mutants at a frequency of less than 10−5 (Mao et al. 1997), probably because most mutations are deleterious and therefore selection does not favor increased mutation rates. However, mutators can be strongly favored when the competitive conditions and the selective environment provide opportunities for the mutators to generate more beneficial mutations than the nonmutators (Chao and Cox 1983; Mao et al. 1997). In this case, mutators increase because they are linked with a higher frequency of beneficial mutations.

Although mutators are typically rare in freshly grown laboratory cultures, hospital isolates of E. coli and Salmonella enterica sometimes have mutator frequencies above 10−2 (Jyssum 1960; Gross and Siegel 1981; LeClerc et al. 1996). Extensive serial passage in the laboratory can also lead to high frequencies of mutators (Sniegowski et al. 1997). Thus, it appears that rapid change of hosts or culture conditions can increase the frequency of mutators 1,000-fold relative to stable environmental conditions. As Drake et al. (1998) point out, theory suggests that mutators can speed adaptation in asexual microbes (Leigh 1970; Moxon et al. 1994; Taddei et al. 1997). It would be interesting to compare naturally occurring frequencies of mutators in stable and rapidly changing selective environments.

DNA damage induces the SOS response of E. coli (Walker 1984). This response causes higher mutation rates even in the undamaged parts of the genome. Radman (1999) argues that this stress-induced mutagenesis is an adaptation to generate variability in the face of challenging environments. But it is not clear whether the special replication enzymes induced by SOS serve primarily to replicate DNA under difficult conditions, albeit with high mutation, or whether certain aspects of SOS are particularly designed to raise mutation above the minimum level that could be achieved efficiently during emergency. In any case, it is interesting to consider whether some microbes facultatively induce increased genome-wide mutation when challenged by host immunity.

Localized Hypermutation

Targeting mutations to key loci would be more efficient than raising the genome-wide mutation rate. Various mechanisms can increase the mutation rate over short runs of nucleotides (Fussenegger 1997; Ripley 1999). For example, Streptococcus pyogenes coats its surface with a variable M protein, of which eighty antigenically distinct variants are known (Lancefield 1962; Fischetti 1991). The amino acid sequence of the M6 serotype revealed repeats in three regions of the protein (Hollingshead et al. 1986, 1987). Region 1 has five repeats of 42 bp, each repeat containing two nearly identical 21 bp repeats; region 2 has five 75 bp repeats; and region 3 has two repeats of about 81 bp. In regions 1 and 2, the two outermost repeats vary slightly in sequence from the three identical repeats in the interior.

Sequence analysis of variant M proteins suggests that mutations occur by generating both gains and losses of the duplications. These mutations probably arise by intragenic recombination between the DNA repeats, but may be created by slippage during replication. Slippage mutations over repeated DNA lead to gain or loss in the number of repeats and occur at frequencies much higher than typical replication errors (Charlesworth et al. 1994). The repeats of the M protein are multiples of 3 bases; thus changes in repeat number do not cause frameshift mutations. Some of the repeats vary slightly in base composition, so recombinations can alter sequence composition as well as total length.

Fussenegger (1997) reviews several other cases of bacterial cell-wall proteins that have repeated sequences, most of which occur in multiples of 3 bp. Repeats are often associated with binding domains for other proteins or polysaccharides (Wren 1991), so perhaps the ability to generate variable-length domains provides an advantage in attachment to host tissues or in escape from host immunity.

Other mutational mechanisms besides repeats may increase local mutation rates (Ripley 1999). For example, when double-stranded DNA splits to be replicated, the complementary bases on a single strand may bind to each other to form "hairpin" structures. Such hairpins may increase errors in replication. Caporale (1999) suggests that certain genomic regions, such as antigenic sites, may have DNA base compositions that promote higher mutation rates.

Apart from the well-known case of repeats and replication slippage, no evidence at present associates antigenic sites with higher replication errors. But this would certainly be an interesting problem to study further. One could, for example, focus on associations between mutation rate and nucleotide sequence. Comparison would be particularly interesting between epitopes that evolve rapidly and conserved regions of antigenic molecules that evolve slowly. Such comparison may help to identify aspects of nucleotide composition that promote higher error rates in replication.

5.2. Stochastic Switching between Archival Copies

Many pathogens change critical surface molecules by switching expression between alternative genes. Three types of switch mechanisms commonly occur: replication errors that turn expression on or off, gene conversion into fixed expression sites, and invertible promoters that change the direction of transcription.

Regulatory Switches by Replication Errors of Short Repeats

Short, repeated nucleotide sequences often lead to high error rates during replication. Repeats have recurring units typically with 1–5 bases per unit. Short, repeated DNA sequences probably lead to replication errors by slipped-strand mispairing (Meyer 1987; Levinson and Gutman 1987; Charlesworth et al. 1994). Errors apparently arise when a DNA polymerase either skips forward a repeat unit, causing a deletion of one unit, or slips back one unit, producing a one-unit insertion.

Gene expression can be turned on or off by insertions or deletions. Inserted or deleted repeats within the coding sequence cause frameshift mutations that prevent translation and production of a full protein. For example, the eleven opacity genes of Neisseria meningitidis influence binding to host cells and tissue tropism. These genes each have between eight and twenty-eight CTCTT repeats, which can disrupt or restore the proper translational frame as the number of repeats changes (Stern et al. 1986; Stern and Meyer 1987). The limited repertoire of eleven genes and the crude on-off switching suggest that variable expression has more to do with altering cell tropism than with escape from host immunity (Fussenegger 1997).

On-off switches can also be created by short repeats in transcriptional control regions. Bordetella pertussis controls expression of two distinct fimbriae by transcriptional switching (Willems et al. 1990). Fimbriae are bacterial surface fibers that attach to host tissues. Particular cells produce both, only one, or neither of the fimbrial types. Sequences of about 15 C nucleotides in the transcriptional promoters of each of the two genes influence expression. The actual length of the poly-C sequence varies, probably by slipped-strand mispairing during replication. The length affects transcription of the attached gene. Thus, by the stochastic process of replication errors, the individual loci are turned on and off. Again, this sort of switching may have more to do with tissue tropism than with escape from immune recognition.

Gene Conversion

Some pathogens store many variant genes for a surface antigen, but express only one of the copies at any time. For example, there may be a single active expression site at which transcription occurs. Occasionally, one of the variant loci copies itself to the expression site by gene conversion—a type of intragenomic recombination that converts the target without altering the donor sequence. The genome preserves the archival library without change, but alters the expressed allele.

The spirochete Borrelia hermsii has approximately thirty alternative loci that encode an abundant surface lipoprotein (Barbour 1993). There is a single active expression site when the spirochete is in mammalian hosts (Barbour et al. 1991). The expression site is changed by gene conversion to one of the variant archival copies at a rate of about 10−4–10−3 per cell division (Stoenner et al. 1982; Barbour and Stoenner 1985). A small number of antigenic variants dominate the initial parasitemia of this blood-borne pathogen. The host then clears these initial variants with antibodies. Some of the bacteria from this first parasitemia will have changed antigenic type. Those switches provide new variants that cause a second parasitemia, which is eventually recognized by the host and cleared. The cycle repeats several times, causing relapsing fever.

The protozoan Trypanosoma brucei has hundreds of alternative loci that encode the dominant surface glycoprotein (Barry 1997; Pays and Nolan 1998). Typically, each cell expresses only one of the alternative loci. Switches in expression occur at a rate of up to 10−2 per cell division (Turner 1997). The switch mechanism is similar to that in Borrelia hermsii—gene conversion of archival copies into a transcriptionally active expression site. T. brucei has approximately twenty alternative transcription sites, of which only one is usually active. Thus, this parasite can also change expression by switching between transcription sites. It is not fully understood how different transcription sites are regulated.

Invertible Sequences

E. coli stores two alternative fimbriae genes adjacent to each other on its chromosome (Abraham et al. 1985). A promoter region between the two genes controls transcription. The promoter triggers transcription in only one direction, thus expressing only one of the two variants. Occasionally, the promoter flips orientation, activating the alternative gene. The ends of the promoter have inverted repeats, which play a role in the recombination event that mediates the sequence inversion. Salmonella uses a similar mechanism to control flagellum expression (Silverman et al. 1979).

Moraxella species use a different method to vary pilin expression (Marrs et al. 1988; Rozsa and Marrs 1991). The variable part of the pilin gene has alternate cassettes stored in adjacent locations. Inverted repeats flank the pair of alternate cassettes, causing the whole complex occasionally to flip orientation. The gene starts with an initial constant region and continues into one of the cassettes within the invertible complex. When the complex flips, the alternate variable cassette completes the gene. Several bacteriophage use a similar inversion system to switch genes encoding their tail fibers, which determine host range (Kamp et al. 1978; Iida et al. 1982; van de Putte et al. 1984).

Fussenegger (1997) reviews other invertible-sequence mechanisms. These low-diversity switches provide only a limited advantage against immunity because, even if the switch rates were low, an infection would soon contain all variants at appreciable abundance. Thus, these switch mechanisms may serve mainly to generate alternative attachment variants.

Other Archival Switch Mechanisms

Plasmodium species are a diverse and polyphyletic group of protozoans that cause malarial symptoms in vertebrate hosts. Antigenic variation appears to be common and to be caused by diverse mechanisms. I briefly summarize three examples.

Infection and reproduction in host erythrocytes determine the buildup of parasite numbers within the host (Mims et al. 1998). P. falciparum expresses the var gene within erythrocytes. The gene product, PfEMP1, moves to the surface of the host cell, where it influences cellular adhesion and recognition by host immunity (Deitsch and Wellems 1996). The var genes are highly diverse antigenically (Su et al. 1995). Each parasite exports only one var type to the erythrocyte surface, but a clone of parasites switches between var types (Smith et al. 1995). Switching leads to a diverse population of PfEMP1 variants within a host and even wider diversity among hosts.

The mechanism of var switching is not known. It appears that many var loci are transcribed during the first few hours after erythrocyte infection, but only a single var gene is active when PfEMP1 is translated and moved to the erythrocyte surface (Q. Chen et al. 1998; Scherf et al. 1998). Switching between var loci does not depend on the mechanism of gene conversion found in Borrelia hermsii and Trypanosoma brucei. It may be that some mechanism shuts down expression of all but one locus without modifying the DNA sequence. Scherf et al. (1998) suggest that switches in gene expression do not depend on DNA sequence changes in promoter regions or changes in transcription factors. They argue that regional changes in chromatin structure may control variable expression.

Preiser et al. (1999) found antigenic variation at another stage in the Plasmodium life cycle (see also Barnwell 1999). The parasitic forms that invade erythrocytes are called merozoites. In a rodent malaria, P. yoelii, the merozoite surface molecule p235 plays a role in attachment or invasion of erythrocytes. There are at least eleven and perhaps as many as fifty discrete genes that encode variants of p235 (Borre et al. 1995). Within an erythrocyte, the parasite develops a multinucleate stage and then divides into new merozoites that burst the host cell. Preiser et al. (1999) found that each of the separate nuclei transcribe a different p235 gene. They suggest that upon division into separate merozoites, each merozoite presents a different p235 protein on its surface. Thus, each clone produces antigenically diverse progeny. The various p235 molecules may facilitate invasion of different classes of erythrocytes.

Other Plasmodium species express surface proteins that are distantly related to p235, but in those cases the surface molecules do not arise from an antigenically diverse, multicopy gene family (Barnwell 1999). Some of the Plasmodium species have diverged tens of millions of years ago, so it is not surprising that they have different strategies for attachment, immune evasion, and antigenic variation.

In another example, P. vivax has an extensive family of variant vir genes, estimated to be present at 600–1,000 copies per haploid genome (del Portillo et al. 2001). The parasite expresses only a small subset of these genes in an infected erythrocyte. Del Portillo et al. (2001) expressed the vir gene product from two variant loci and tested the proteins for immunogenicity. Sera from twenty-five previously infected hosts provided a panel of antibodies to test for prior exposure to the vir gene products. One of the expressed proteins reacted with the serum from only one host, the other protein reacted with sera from two hosts. Thus, vir gene products are immunogenic, but each variant appears to be expressed rarely—the hallmarks of antigenic variation from a large archival library.

Sequences related to the vir family do not occur in P. falciparum or P. knowlesi, suggesting that these different lineages have evolved different families of variable antigens. The diversity of gene families in Plasmodium that play a role in antigenic variation provides an excellent opportunity for comparative, evolutionary studies.

5.3. New Variants by Intragenomic Recombination

Some parasites store many genetic variants for a particular surface molecule. Usually, each parasite expresses only one archival variant.

New variants of archival copies may be created by recombination. For example, Rich et al. (2001) found evidence for recombination between the archived loci of the variable short protein (Vsp) of Borrelia hermsii. They studied the DNA sequences of 11 vsp loci within a single clone. These vsp loci are silent, archival copies that can, by gene conversion, be copied into the single expression site. The genes differ by 30–40% in amino acid sequence, providing sufficient diversity to reduce or eliminate antigenic cross-reactivity within the host.

Rich et al. (2001) used statistical analyses of vsp sequences to infer that past recombination events have occurred between archival loci. Those analyses focus on attributes such as runs of similar nucleotides between loci that occur more often than would be likely if alleles diverged only by accumulating mutations within each locus. Shared runs can be introduced into diverged loci by recombination.

The archival antigenic repertoire of Trypanosoma brucei evolves rapidly (Pays and Nolan 1998). This species has a large archival library and multiple expression sites, but only one expression site is active at any time. New genes can be created within an active expression site when several donor sequences convert the site in a mosaic pattern (Pays 1989; Barbet and Kamper 1993). When an active expression site becomes inactivated, the gene within that site probably becomes protected from further gene conversion events (Pays et al. 1981; Pays 1985). Thus, newly created genes by mosaic conversion become stored in the repertoire. Perhaps new genes in silent expression sites can move into more permanent archival locations by recombination, but this has not yet been observed. Recombination between silent, archived copies may also occur, which, although each event may be relatively rare, could strongly affect the evolutionary rate of the archived repertoire.

These examples illustrate the scattered reports of recombination and the evolution of archived repertoires. These preliminary studies show the promise for understanding the interaction between mechanisms that create diversity and the strong forces of natural selection imposed by immune recognition. The combination of generative mechanisms and selection shapes the archival antigenic repertoire.

5.4. Mixing between Genomes

New antigenic variants can be produced by mixing genes between distinct lineages. This happens in three ways.

Segregation brings together chromosomes from different lineages. Reassortment of influenza A's neuraminidase and hemagglutinin surface antigens provides the most famous example (Lamb and Krug 2001). The genes for these antigens occur on two separate RNA segments of the genome—the genome has a total of eight segments. When two or more viruses infect a single cell, the parental segments all replicate separately and then are packaged together into new viral particles. This process can package the segments from different parents into a new virus.

New neuraminidase-hemagglutinin combinations present novel antigenic properties to the host. Rare segregation events have introduced hemagglutinin from bird influenza into the genome of human influenza (Webster et al. 1997). The novel hemagglutinins cross-reacted very little with those circulating in humans, allowing the new combinations to sweep through human populations and cause pandemics.

Intergenomic recombination occurs when chromosomes from different lineages exchange pieces of their nucleotide sequence. In protozoan parasites such as Plasmodium and Trypanosoma, recombination happens as part of a typical Mendelian cycle of outcrossing sex (Jenni et al. 1986; Conway et al. 1999). Recombination can occur in viruses when two or more particles infect a single cell. DNA viruses may recombine relatively frequently because they can use the host's recombination enzymes (Strauss et al. 1996). RNA viruses may recombine less often because the host lacks specific enzymes to mediate reciprocal exchange of RNA segments. However, many descriptions of RNA virus recombination have been reported (Robertson et al. 1995; Laukkanen et al. 2000; and see chapter 10). In all cases, even rare recombination can provide an important source for new antigenic variants.

Horizontal transfer of DNA between bacteria introduces new nucleotide sequences into a lineage (Ochman et al. 2000). Transformation occurs when a cell takes up naked DNA from the environment. Some species transform at a particularly high rate, suggesting that they have specific adaptations for uptake and incorporation of foreign DNA (Fussenegger et al. 1997). For example, Neisseria species transform frequently enough to have many apparently mosaic genes from interspecies transfers (Spratt et al. 1992; Zhou and Spratt 1992; Fussenegger et al. 1997), and N. gonorrhoeae has low linkage disequilibrium across its genome (Maynard Smith et al. 1993). Horizontal transfer also occurs when bacteriophage viruses carry DNA from one host cell to another or when two cells conjugate to transfer DNA from a donor to a recipient (Ochman et al. 2000).

5.5. Problems for Future Research

1. Selection of mutation rates

Intense immune pressure favors the generation of antigenic variants. However, variants, like any mutations, may have associated costs. To what extent have molecular attributes of antigenic genes been shaped by the costs and benefits of generating variants? Do microbes under intense immune pressure have higher genome-wide mutation rates compared with similar organisms that do not face immune attack? To what extent have nucleotide sequences of antigens been shaped by the tendency of particular motifs to generate replication errors—a form of local hypermutation?

2. Selection of mechanisms that control switching between archival variants

I described various mechanisms by which gene expression shifts between archived variants. The rate at which switches occur probably affects the parasite's ability to extend infection. If switches happen too quickly, then novel variants will not be expressed after the immune response develops against the many variants expressed early in infection. If switches happen too slowly, then the parasite may be cleared before the variants are expressed. Thus, natural selection can strongly influence the molecular details of the switch process in order to adjust the rate of change between variants. This can be tested by selecting in vitro for faster or slower switch rates. Does an evolutionary response occur in the switch rates? If so, how is the response accomplished at the molecular level?

One could also test the evolution of the switch rate in vivo, comparing situations that imposed different immune pressures on rates of change and on particular orders in which variants are expressed. Such studies allow one to relate the molecular mechanisms of switching to the adaptive significance of switching. Two general questions arise. To what extent do switch rates evolve to enhance parasite fitness? To what extent do mechanistic properties of switching constrain rates of change between variants?

3. Rates of diversification between archival copies

Some parasites have large families of variants archived within the genome. I described studies of Borrelia hermsii and Trypanosoma brucei in which intragenomic recombination between archival copies generated new variants. This calls attention to the rate at which new variants can be created and the rate of diversification between members of archival gene families.

Rich et al. (2000) argue that all of the antigenic variants of Plasmodium falciparum have arisen from a recent common ancestor. A more detailed study by Volkman et al. (2001) estimates that the most recent common ancestor lived less than ten thousand years ago. If this estimate is correct, then the diverse var family of antigenic variants must have evolved very rapidly. However, this conclusion remains contentious—Hughes and Verra (2001) argue that the P. falciparum lineage is much older.

It would be interesting to compare rates of diversification in these families of variants between the different Plasmodium species, Trypanosoma brucei, Borrelia hermsii, and other microbes with similar families of variants. It would also be interesting to compare P. falciparum with another malarial parasite of humans, P. vivax. As noted in the text, P. vivax has a family of antigenic variants that does not occur in P. falciparum. How does the history of variation compare in these two species?

Copyright © 2002, Steven A Frank.
Bookshelf ID: NBK2384
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