Recognition is also a necessary prerequisite for pathogen clearance via cellular immunity, and several gene families have been identified that encode membrane-bound phagocytic receptors. Phagocytosis is also promoted by 'tagging' of microbes with extracellularly secreted opsonins. Several genes encoding both phagocytic receptors and opsonins show evidence of adaptive amino acid evolution within the genus Drosophila (Sackton et al., 2007) and frequent genomic turnover within Drosophila and between Drosophila and other insects (Figure 13.2; Evans et al., 2006; Sackton et al., 2007; Waterhouse et al., 2007; Zou et al, 2007). In Drosophila, recognition genes are significantly more likely to show evidence of positive selection than genes with signalling or microbicidal functions (Sackton et al, 2007). This difference is largely driven by recognition genes that trigger the cellular response, with nine of 10 recognition genes that yield significant evidence of positive selection having been either experimentally confirmed to be involved in phagocytosis or homologous to known phagocytosis genes. Specifically, these are genes encoding thioester-containing proteins (TEPs) (Jiggins and Kim, 2006; Sackton et al., 2007), the Eater and Nimrod families (Sackton et al., 2007), the class C scavenger receptors (Lazzaro, 2005), and the CD36 homologue epithelial membrane protein (emp) (Sackton et al., 2007).
TEPs have been directly implicated as opso-nins mediating the cellular clearance of microbes including bacteria and malaria-causing Plasmodium in Drosophila and Anopheles (Levashina et al., 2001; Blandin et al, 2004; Stroschein-Stevenson et al, 2006). Proteolytic cleavage of a hypervariable spacer, or 'bait', domain exposes the thioester motif, which then covalently binds microbes and labels them for phagocytosis. TEPs appear to be hotspots of adaptation in several species. In D. melanogaster, there are six Tep genes, four of which have intact thioester domains and thus are likely to function as opsonizing agents (Blandin and Levashina, 2004). One of four of the intact Teps show evidence of adaptive divergence between D. melanogaster and D. simulans and three show evidence for directional selection in the melanogaster species group (Table 13.1; Jiggins and Kim, 2006; Sackton et al., 2007). Interestingly, one of the adaptively evolving Tep genes is constitutively expressed at higher levels in European than African populations of D. melanogaster, suggesting that expression of this Tep may be locally adapted (Hutter et al, 2008). Tep genes in mosquitoes and the more distantly related crustacean Daphnia also show evidence of adaptive amino acid evolution (Little et al., 2004; Little and Cobbe, 2005). In all cases, positively selected amino acid mutations are overrepresented in the bait domain that is cleaved to expose the thioester motif. It is unknown whether the proteases that cleave TEPs are produced by host or pathogen, so it is not yet possible to say whether adaptation in this domain is due to co-evolution with pathogen proteases or with pathogen molecules that interfere with host proteolysis.
Tep gene families are expanded in mosquitoes, with 13 Tep genes found in the Anopheles gambiae genome and eight in the Aedes aegypti genome (Christophides et al, 2002; Waterhouse et al, 2007). The expansions in size of the Tep gene family appear to have been independent in each of these two taxa and potentially reflect elevated pressure on cellular immunity. The A. gambiae Tepl gene is segregating for two sharply divergent alleles, one of which, when homozygous, confers absolute resistance to experimental infection with the rodent malaria Plasmodium berghei (Blandin et al, 2004; Baxter et al, 2007). Individuals homozygous for the susceptible allele sustain robust P. berghei infections. These two alleles differ by multiple amino acid substitutions, including several that are clustered around the thioester domain. It is currently unclear which substitutions cause the phenotypic differences in susceptibility, or whether it is an epistatic pheno-type involving substitution in multiple domains of the protein. Both alleles are found at high frequencies in natural populations (Obbard et al, 2008), suggesting selective forces maintain these two alleles in the wild. This system provides a tantalizing opportunity to understand the mechanisms
Table 13.1 Evolutionary genetics of the Tep gene family of phagocytic recognition molecules in Drosophila.
Overview Upregulated in response to infection
McDonald-Kreitman test (Box 13.1)
Exceptionally elevated dN/dS between D. melanogaster and D. simulans clustered around the bait domain; elevated dN/dS in the melanogaster species subgroup Elevated dN/dS in the melanogastergroup with trend towards an excess of positively selected sites at the bait domain Elevated amino acid replacements across entire gene in D. melanogaster
Upregulated in response to infection
Required for efficient phagocytosis of the bacterium Escherichia coli
Elevated dN/dS in the melanogaster species subgroup
Elevated dN/dS across the entire gene in the melanogaster species group
Expression levels significantly higher in European than in African D. melanogaster populations
Required for efficient phagocytosis of the bacterium Staphylococcus aureus
Upregulated in response to infection
Not expressed; likely to be a pseudogene
Lacks a thioester domain
Required for efficient phagocytosis of the fungus Candida albicans
Elevated dN/dS in the melanogaster species group with an excess of positively selected sites at the bait domain Not significant
Reviewed in Blandin and Levashina (2004) Stroschein-Stevenson et al. (2006)
Jiggins and Kim (2006)
Sackton et al. (2007)
Jiggins and Kim (2006)
Hutter et al. (2008)
a Not significant indicates genes that were included in the referenced studies but not found to depart from the null expectation. Empty cells indicate that no information has been obtained.
that lead to the maintenance of immune response polymorphisms in a natural context.
Whole-genome comparisons within the genus Drosophila indicate that, in striking contrast to recognition molecules that trigger the humoral response, recognition molecules that initiate the cellular response show abundant evidence of adaptive evolution. Deeper investigation of the Tep gene family reveals that adaptive evolution extends beyond Drosophila to include mosquitoes and Daphnia, and demonstrates extant functional variation in a mosquito Tep gene. The signals of adaptive evolution suggest that these recognition molecules interact with evolutionarily labile pathogen motifs or that, like signalling molecules in humoral defence, they are potentially subject to interference by pathogen-produced proteins.
The diverse evolutionary trajectories of various genes in the insect immune response (Figure 13.2) can be interpreted in light of their molecular functions and interactions with pathogens. Pathogen-recognition molecules that stimulate the humoral response interact with highly conserved microbial cell-wall components. Although obligate pathogens are sometimes able to reduce their cell walls to escape detection, most microbes are evolution-arily constrained because they must also be able to persist in non-infectious environments. Similarly, there may be few ways in which microbes can evolve resistance to AMPs, especially when host insects simultaneously employ multiple peptides with distinct activities. If there is little adaptation in pathogens to escape host humoral recognition and antibiotic killing, then it may be expected that there would be little indication of adaptive amino acid evolution in the host genes over short evolutionary time. Both humoral recognition factors and AMPs exhibit rapid rates of genomic duplication and deletion, and in some taxa duplication is coupled with a burst of amino acid diversification that presumably increases breadth of function.
In contrast, signal transduction proteins in the humoral immune response are largely maintained in strict orthology across insect species, but frequently show indications of adaptive amino acid evolution within species. A hypothesized explanation is that the strong maintenance of orthology in these pathways makes them attractive targets for immune suppression by generalist pathogens. This may be a particularly successful strategy for microbes that are unable to evade or resist the recognition and microbicidal stages of humoral immunity. Gene duplication and diversification are not commonly observed here, perhaps because this is not a successful strategy for escaping pathogen interference. Genomic retention of a duplicated gene that can be manipulated by pathogens would be detrimental because host signalling function would be impaired. Instead, rapid fixation of amino acid 'escape' variants in signalling genes seems to be the most effective host strategy, and coordinate compensatory mutation in physically interacting proteins may amplify the signal of adaptive evolution in this functional category.
Recognition factors and opsonins in the cellular immune response evolve by adaptive amino acid evolution and frequent genomic turnover. In general, little is known about the specific activities and recognition profiles of these genes, making it difficult to interpret the evolutionary patterns in a functional context. The evolutionary genetics, however, do lead to functional predictions, including that the cellular recognition factors bind evolutionarily labile pathogen epitopes or are subject to pathogen interference, both of which could drive rapid amino acid evolution. At the moment, virtually nothing is known about the molecular evolution or population genetics of host genes that drive phagocytosis after pathogen recognition. Microbes are capable of manipulating host cells both to promote and inhibit phagocytic uptake (Schmid-Hempel, 2008), leading to the prediction that genes encoding the machinery of phagocytosis will, like genes in humoral signalling pathways, show abundant evidence of adaptive evolution.
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