Evidence for selection at cytokine loci in a natural population of field voles (Microtus agrestis)

Individuals in natural populations are frequently exposed to a wide range of pathogens. Given the diverse profile of gene products involved in responses to different types of pathogen, this potentially results in complex pathogen-specific selection pressures acting on a broad spectrum of immune system genes in wild animals. Thus far, studies into the evolution of immune genes in natural populations have focused almost exclusively on the Major Histocompatibility Complex (MHC). However, the MHC represents only a fraction of the immune system and there is a need to broaden research in wild species to include other immune genes. Here, we examine the evidence for natural selection in a range of non-MHC genes in a natural population of field voles (Microtus agrestis). We concentrate primarily on genes encoding cytokines, signalling molecules critical in eliciting and mediating immune responses and identify signatures of natural selection acting on several of these genes. In particular, genetic diversity within Interleukin 1 beta and Interleukin 2 appears to have been maintained through balancing selection. Taken together with previous findings that polymorphism within these genes is associated with variation in resistance to multiple pathogens, this suggests that pathogen-mediated selection may be an important force driving genetic diversity at cytokine loci in voles and other natural populations. These results also suggest that, along with the MHC, preservation of genetic variation within cytokine genes should be a priority for the conservation genetics of threatened wildlife populations.     

The evolutionary landscape of cytosolic microbial sensors in humans

Host-pathogen interactions are generally initiated by host recognition of microbial components or danger signals triggered by microbial invasion. This recognition involves germline-encoded microbial sensors or pattern-recognition receptors (PRRs). By studying the way in which natural selection has driven the evolution of these microbial sensors in humans, we can identify genes playing an essential role and distinguish them from other, more redundant genes. We characterized the sequence diversity of the NOD-like receptor family, including the NALP and NOD/IPAF subfamilies, in various populations worldwide and compared this diversity with that of other PRR families, such as Toll-like receptors (TLRs) and RIG-I-like receptors (RLRs). We found that most NALPs had evolved under strong selective constraints, suggesting that their functions are essential and possibly much broader than previously thought. Conversely, most NOD/IPAF subfamily members were subject to more relaxed selective constraints, suggesting greater redundancy. Furthermore, some NALP genes, including NLRP1, NLRP14, and CIITA, were found to have evolved adaptively. We identified those variants conferring a selective advantage on some human populations as the most likely targets of positive selection. More generally, the strength of selection differed considerably between the major families of microbial sensors. Endosomal TLRs and most NALPs were found to evolve under stronger purifying selection than most NOD/IPAF subfamily members and cell-surface TLRs and RLRs, suggesting some degree of redundancy in the signaling pathways triggered by these molecules. This study provides novel perspectives and experimentally testable hypotheses concerning the relative biological relevance of the various families of microbial sensors in humans.     

Lack of genetic diversity across diverse immune genes in an endangered mammal,the Tasmanian devil (Sarcophilus harrisii)

The Tasmanian devil (Sarcophilus harrisii) is threatened with extinction due to the spread of devil facial tumour disease. Polymorphisms in immune genes can provide adaptive potential to resist diseases. Previous studies in diversity at immune loci in wild species have almost exclusively focused on genes of the major histocompatibility complex (MHC); however, these genes only account for a fraction of immune gene diversity. Devils lack diversity at functionally important immunity loci, including MHC and Toll‐like receptor genes. Whether there are polymorphisms at devil immune genes outside these two families is unknown. Here, we identify polymorphisms in a wide range of key immune genes, and develop assays to type single nucleotide polymorphisms (SNPs) within a subset of these genes. A total of 167 immune genes were examined, including cytokines, chemokines and natural killer cell receptors. Using genome‐level data from ten devils, SNPs within coding regions, introns and 10 kb flanking genes of interest were identified. We found low polymorphism across 167 immune genes examined bioinformatically using whole‐genome data. From this data, we developed long amplicon assays to target nine genes. These amplicons were sequenced in 29–220 devils and found to contain 78 SNPs, including eight SNPS within exons. Despite the extreme paucity of genetic diversity within these genes, signatures of balancing selection were exhibited by one chemokine gene, suggesting that remaining diversity may hold adaptive potential. The low functional diversity may leave devils highly vulnerable to infectious disease, and therefore, monitoring and preserving remaining diversity will be critical for the long‐term management of this species. Examining genetic variation in diverse immune genes should be a priority for threatened wildlife species. This study can act as a model for broad‐scale immunogenetic diversity analysis in threatened species.     

Microevolutionary selection dynamics acting on immune genes of the green‐veined white butterfly,Pieris napi

Insects rely on their innate immune system to successfully mediate complex interactions with their microbiota, as well as the microbes present in the environment. Previous work has shown that components of the canonical immune gene repertoire evolve rapidly and have evolutionary characteristics originating from interactions with fast‐evolving microorganisms. Although these interactions are likely to vary among populations, there is a poor understanding of the microevolutionary dynamics of immune genes, especially in non‐Dipteran insects. Here, we use the full set of canonical insect immune genes to investigate microevolutionary dynamics acting on these genes between and among populations by comparing three allopatric populations of the green‐veined white butterfly, Pieris napi (Linné; Lepidoptera, Pieridae). Immune genes showed increased genetic diversity compared to genes from the rest of the genome and various functional categories exhibited different types of signatures of selection, at different evolutionary scales, presenting a complex pattern of selection dynamics. Signatures of balancing selection were identified in 10 genes, and 17 genes appear to be under positive selection. Genes involved with the cellular arm of the immune response as well as the Toll pathway appear to be enriched among our outlier loci, regardless of functional category. This suggests that the targets of selection might focus upon an entire pathway, rather than functional subsets across pathways. Our microevolutionary results are similar to previously observed macroevolutionary patterns from diverse taxa, suggesting that either the immune system is robust to dramatic differences in life history and microbial communities, or that diverse microbes exert similar selection pressures.     

Genome-wide polymorphisms show unexpected targets of natural selection

Natural selection can act on all the expressed genes of an individual, leaving signatures of genetic differentiation or diversity at many loci across the genome. New power to assay these genome-wide effects of selection comes from associating multi-locus patterns of polymorphism with gene expression and function. Here, we performed one of the first genome-wide surveys in a marine species, comparing purple sea urchins, Strongylocentrotus purpuratus, from two distant locations along the species' wide latitudinal range. We examined 9112 polymorphic loci from upstream non-coding and coding regions of genes for signatures of selection with respect to gene function and tissue- and ontogenetic gene expression. We found that genetic differentiation (F(ST)) varied significantly across functional gene classes. The strongest enrichment occurred in the upstream regions of E3 ligase genes, enzymes known to regulate protein abundance during development and environmental stress. We found enrichment for high heterozygosity in genes directly involved in immune response, particularly NALP genes, which mediate pro-inflammatory signals during bacterial infection. We also found higher heterozygosity in immune genes in the southern population, where disease incidence and pathogen diversity are greater. Similar to the major histocompatibility complex in mammals, balancing selection may enhance genetic diversity in the innate immune system genes of this invertebrate. Overall, our results show that how genome-wide polymorphism data coupled with growing databases on gene function and expression can combine to detect otherwise hidden signals of selection in natural populations.     

Differential evidence of natural selection on two leading sporozoite stage malaria vaccine candidate antigens

Experimental malaria vaccines based on two sporozoite stage candidate antigens of Plasmodium falciparum, the circumsporozoite protein (CSP) and thrombospondin-related adhesive protein (TRAP), have undergone clinical trials of efficacy. The relevance of naturally existing polymorphism in these molecules remains unknown. Sequence polymorphism in the genes encoding these antigens was studied in a Gambian population (sample of 48 trap and 44 csp gene sequences) to test for signatures of selection that would result from naturally acquired immunity. Allele frequency distributions were analyzed and compared with data from another population (in Thailand). Patterns of non-synonymous and synonymous polymorphism in P. falciparum and in Plasmodium vivax were compared with divergence from related species. Results indicate that polymorphism in TRAP is under strong selection for amino acid sequence diversity and that allele frequencies are under balancing selection within the Gambian P. falciparum population. There was no such evidence for CSP, calling into question the idea that most polymorphisms in this gene are under immune selection. There was a weak trend for regions known to encode T cell epitopes to have slightly higher indices suggesting balancing selection. Overall, the results predict more allele-specific immunity to TRAP than to CSP and should be considered in design and efficacy testing of vaccine candidates based on these antigens.     

Mechanisms and applications of immune stimulatory CpG oligodeoxynucleotides

Immune stimulation has been widely recognized as an undesirable side effect of certain antisense oligodeoxynucleotides (ODN) which can interfere with their therapeutic application. It is now clear that these dose-dependent immune stimulatory effects primarily result from the presence of an unmethylated CpG dinucleotide in particular base contexts ('CpG motif). The sequence-specific immune activation is not just an experimental artifact, but is actually a highly evolved immune defense mechanism whose actual 'goal' is the detection of microbial nucleic acids. In contrast to vertebrate DNA, in which CpG dinucleotides are 'suppressed' and are highly methylated, microbial genomes do not generally feature CpG suppression or methylation [1]. Immune effector cells such as B cells, macrophages, dendritic cells, and natural killer cells appear to have evolved pattern recognition receptors (PRR) that by binding the microbe-restricted structure of CpG motifs, trigger protective immune responses. Although the specific immune activation appears to have a variety of potential therapeutic applications, it is generally undesirable in antisense ODN. Immune stimulation may be avoided in antisense oligos by the selection of CpG-free target sequences, by the use of ODN backbones that do not support immune stimulation, or by selective modifications of the cytosine in any CpG dinucleotides.     

Innate recognition of microbial-derived signals in immunity and inflammation

Microbes generate a vast array of different types of conserved structural components called pathogen-associated molecular patterns(PAMPs),which canbe recognized by cells of the innate immune system.This recognition of "nonself" signatures occurs through host pattern recognition receptors(PRRs),suggesting that microbial-derived signals are good targets for innate immunity to discriminate between self- and nonself.Such PAMP-PRR interactions trigger multiple but distinct downstream signaling cascades,subsequently leading to production of proinflammatory cytokines and interferons that tailor immune responses to particular microbes.Aberrant PRR signals have been associated with various inflammatory diseases and fine regulation of PRR signaling is essential for avoiding excessive inflammatory immune responses and maintaining immune homeostasis.In this review we summarize the ligands and signal transduction pathways of PRRs and highlight recent progress of the mechanisms involved in microbe-specific innate immune recognition during immune responses and inflammation,which may provide new targets for therapeutic intervention to the inflammatory disorders.     

鱼类模式识别受体的研究进展

天然免疫（innate immunity）是基于对病原微生物成分的非克隆性识别而启动的快速防御反应。天然免疫系统可通过胚系编码的模式识别受体（pattern-recognition receptors,PRR）识别恒定不变的病原基元,即病原相关分子模式（pathogen-associated molecular patterns,PAMPs）,启动信号级联转导,最终PRRs信号激活宿主免疫和前炎性基因的表达,引发针对所识别病原的免疫反应。目前PRRs主要分为5类,即C-型Lectins、Toll样受体（Toll-like receptors,TLRs）、视黄酸诱导基因I样受体（retinoic acid inducible gene I-like receptors,RLRs）、包含核苷酸结合区和亮氨酸富集区蛋白（the nucleotide-binding domain,leucine-rich repeatcontaining proteins,NLRs,也称NOD样受体）和最近发现的AIM样受体（absent in melanoma（AIM）-like receptors,ALRs）。近年来,随着5种鱼类基因组序列草图的完成,大量鱼类PRRs基因被发现,一些PRRs的配体特异性及其相关信号途径正在逐渐明晰。为此,将对鱼类Toll样受体（TLRs）、视黄酸诱导基因I样受体（RLRs）和NOD样受体（NLRs）的研究进展进行综述。     

Overlapping Patterns of Rapid Evolution in the Nucleic Acid Sensors cGAS and OAS1 Suggest a Common Mechanism of Pathogen Antagonism and Escape

A diverse subset of pattern recognition receptors (PRRs) detects pathogen-associated nucleic acids to initiate crucial innate immune responses in host organisms. Reflecting their importance for host defense, pathogens encode various countermeasures to evade or inhibit these immune effectors. PRRs directly engaged by pathogen inhibitors often evolve under recurrent bouts of positive selection that have been described as molecular ‘arms races.’ Cyclic GMP-AMP synthase (cGAS) was recently identified as a key PRR. Upon binding cytoplasmic double-stranded DNA (dsDNA) from various viruses, cGAS generates the small nucleotide secondary messenger cGAMP to signal activation of innate defenses. Here we report an evolutionary history of cGAS with recurrent positive selection in the primate lineage. Recent studies indicate a high degree of structural similarity between cGAS and 2’-5’-oligoadenylate synthase 1 (OAS1), a PRR that detects double-stranded RNA (dsRNA), despite low sequence identity between the respective genes. We present comprehensive comparative evolutionary analysis of cGAS and OAS1 primate sequences and observe positive selection at nucleic acid binding interfaces and distributed throughout both genes. Our data revealed homologous regions with strong signatures of positive selection, suggesting common mechanisms employed by unknown pathogen encoded inhibitors and similar modes of evasion from antagonism. Our analysis of cGAS diversification also identified alternately spliced forms missing multiple sites under positive selection. Further analysis of selection on the OAS family in primates, which comprises OAS1, OAS2, OAS3 and OASL, suggests a hypothesis where gene duplications and domain fusion events result in paralogs that provide another means of escaping pathogen inhibitors. Together our comparative evolutionary analysis of cGAS and OAS provides new insights into distinct mechanisms by which key molecular sentinels of the innate immune system have adapted to circumvent viral-encoded inhibitors.     

Variable SNP density in aspartyl-protease genes of the malaria parasite Plasmodium falciparum

An analysis of the diversity of the aspartyl proteases of Plasmodium falciparum, known as plasmepsins (PMs), was completed in view of their possible role as drug targets. DNA sequence polymorphisms were identified in nine pm genes including their non-coding (introns and 5' flanking) sequences. All genes contained at least one single nucleotide polymorphism (SNP). Extensive microsatellite diversity was observed predominantly in non-coding sequences. All but one non-synonymous polymorphism (a conservative substitution) were mapped to the surface of the predicted protein, contradicting a possible role in enzymatic activity. The distribution of SNPs was found to be non-random among pm genes, with pm6 and pm10 having significantly higher SNP densities, suggesting they were under selection. For pm6 the majority of the SNPs were in introns and some of these may contribute to splice site variation. SNPs were found at a high density in both the coding and non-coding sequences of pm10. Recombination was important in generating additional diversity at this locus. Although direct selection for pm10 mutations could not be ruled out, the presence of balancing selection and a high density of SNPs in non-coding sequence led us to propose that another gene under selection may be influencing the diversity in the region. By sequencing short DNA tags in a 200 kb region flanking pm10 we show that a cluster of antigen genes, known to be under diversifying selection, may contribute to the observed diversity. We discuss the importance of diversity and local selection effects when choosing drug targets for intervention strategies.     

Toll样受体（TLR）介导的天然免疫间的相互调节

模式识别受体（PRR）在宿主细胞识别与抵御微生物病原体中起到了重要作用。Toll样受体（TLR）是研究比较清楚的一类PRR,可以识别多种病原体成份,启动天然免疫反应。此外,近来发现了几类其他模式识别受体,如C型凝集素受体（CLR）,核苷酸寡聚结合域（NOD）样受体（NLR）和视黄酸诱导基因I（RIG—I）样受体（RLR）,表明机体的天然免疫反应受到多种机制的精密调控。本文着重综述TLR与其他PRR在识别病原体和介导天然免疫信号通路间的相互关系。     

Arabidopsis thaliana Genes Encoding Defense Signaling and Recognition Proteins Exhibit Contrasting Evolutionary Dynamics

The interplay between pathogen effectors, their host targets, and cognate recognition proteins provides various opportunities for antagonistic cycles of selection acting on plant and pathogen to achieve or abrogate resistance, respectively. Selection has previously been shown to maintain diversity in plant proteins involved in pathogen recognition and some of their cognate pathogen effectors. We analyzed the signatures of selection on 10 Arabidopsis thaliana genes encoding defense signal transduction proteins in plants, which are potential targets of pathogen effectors. There was insufficient evidence to reject neutral evolution for 6 genes encoding signaling components consistent with these proteins not being targets of effectors and/or indicative of constraints on their ability to coevolve with pathogen effectors. Functional constraints on effector targets may have provided the driving selective force for the evolution of guard proteins. PBS1, a known target of an effector, showed little variation but is known to be monitored by a variable guard protein. Evidence of selection maintaining diversity was present at NPR1, PAD4, and EDS1. Differences in the signatures of selection observed may reflect the numbers of effectors that target a particular protein, the presence or absence of a cognate guard protein, as well as functional constraints imposed by biochemical activities or interactions with plant proteins.     

Candidate targets of balancing selection in the genome of Staphylococcus aureus

Signatures of balancing selection can highlight polymorphisms and functions that are important to the long-term fitness of a species. We performed a first genome-wide scan for balancing selection in a bacterial species, Staphylococcus aureus, which is a common cause of serious antimicrobial-resistant infections of humans. Using a sliding window approach, the genomes of 16 strains of S. aureus, including 5 new genome sequences presented here, and 1 outgroup strain of S. epidermidis were scanned for signatures of balancing selection. A total of 195 short windows were investigated based on their extreme values of both Tajima's D (>2.03) and π/K ratios (>0.12) relative to the rest of the genome. To test the unusualness of these windows, an Approximate Bayesian Computation framework was used to select a null demographic model that better accounted for the observed data than did the standard neutral model. A total of 186 windows were demonstrated to be unusual under the null model and, thus, represented candidate loci under balancing selection. These 186 candidate windows were located within 99 candidate genes that were spread across 62 different loci. Nearly all the signal (97.2%) was located within coding sequences; balancing selection on gene regulation apparently occurs through the targeting of global regulators such as agr and gra/aps. The agr locus had some of the strongest signatures of balancing selection, which provides new insight into the causes of diversity at this locus. The list of candidate genes included multiple virulence-associated genes and was significantly enriched for functions in amino acid and inorganic ion transport and metabolism and in defense mechanisms against innate immunity and antimicrobials, highlighting these particular functions as important to the fitness of this pathogen.     

Identifying footprints of directional and balancing selection in marine and freshwater three-spined stickleback (Gasterosteus aculeatus) populations

Natural selection is expected to leave an imprint on the neutral polymorphisms at the adjacent genomic regions of a selected gene. While directional selection tends to reduce within-population genetic diversity and increase among-population differentiation, the reverse is expected under balancing selection. To identify targets of natural selection in the three-spined stickleback ( Gasterosteus aculeatus ) genome, 103 microsatellite and two indel markers including expressed sequence tags (EST) and quantitative trait loci (QTL)-associated loci, were genotyped in four freshwater and three marine populations. The results indicated that a high proportion of loci (14.7%) might be affected by balancing selection and a lower proportion (2.8%) by directional selection. The strongest signatures of directional selection were detected in a microsatellite locus and two indel markers located in the intronic regions of the Eda-gene coding for the number of lateral plates. Yet, other microsatellite loci previously found to be informative in QTL-mapping studies revealed no signatures of selection. Two novel microsatellite loci ( Stn12 and Stn90 ) located in chromosomes I and VIII, respectively, showed signals of directional selection and might be linked to genomic regions containing gene(s) important for adaptive divergence. Although the coverage of the total genomic content was relatively low, the predominance of balancing selection signals is in agreement with the contention that balancing, rather than directional selection is the predominant mode of selection in the wild.     

Comparative genomics provides evidence for the 3-hydroxypropionate autotrophic pathway in filamentous anoxygenic phototrophic bacteria and in hot spring microbial mats

Stable carbon isotope signatures of diagnostic lipid biomarkers have suggested that Roseiflexus spp., the dominant filamentous anoxygenic phototrophic bacteria inhabiting microbial mats of alkaline siliceous hot springs, may be capable of fixing bicarbonate via the 3-hydroxypropionate pathway, which has been characterized in their distant relative, Chloroflexus aurantiacus. The genomes of three filamentous anoxygenic phototrophic Chloroflexi isolates (Roseiflexus sp. RS-1, Roseiflexus castenholzii and Chloroflexus aggregans), but not that of a non-photosynthetic Chloroflexi isolate (Herpetosiphon aurantiacus), were found to contain open reading frames that show a high degree of sequence similarity to genes encoding enzymes in the C. aurantiacus pathway. Metagenomic DNA sequences from the microbial mats of alkaline siliceous hot springs also contain homologues of these genes that are highly similar to genes in both Roseiflexus spp. and Chloroflexus spp. Thus, Roseiflexus spp. appear to have the genetic capacity for carbon dioxide reduction via the 3-hydroxypropionate pathway. This may contribute to heavier carbon isotopic signatures of the cell components of native Roseiflexus populations in mats compared with the signatures of cyanobacterial cell components, as a similar isotopic signature would be expected if Roseiflexus spp. were participating in photoheterotrophic uptake of cyanobacterial photosynthate produced by the reductive pentose phosphate cycle.     

Molecular Evolution of Daphnia Immunity Genes: Polymorphism in a Gram-Negative Binding Protein Gene and an α-2-Macroglobulin Gene

Studies of DNA polymorphism have shown that some immune system genes of mammals and plants are exceptionally diverse, indicating that coevolution between these taxa and their parasites mediates positive selective sweeps and/or balancing selection. The genes of the arthropod immune system remain comparatively unstudied. We isolated two putative immune system genes from the cladoceran crustacean Daphnia and examined DNA sequence diversity. For one gene, encoding a putative gram-negative binding protein, we found evidence of only purifying selection, indicating that this gene is under strong functional constraint and that selection acts to eliminate amino acid variation. For another gene, encoding a putative -2-macroglobulin, we found evidence of positive selection, indicating the possible involvement of this gene in a host–parasite arms race. We discuss the assumed function of these genes and offer speculation regarding which components of the arthropod immune system might experience diversifying adaptive evolution.[Reviewing Editor: Dr. Martin Kreitman]     

Innate sensing of self and non-self RNAs by Toll-like receptors

Toll-like receptors (TLRs) have an important role in innate immunity in mammals by recognizing conserved microbial components that are known as pathogen-associated molecular patterns (PAMPs). Although the majority of these receptors sense pathogen components on the cell surface, a subset of them (TLR3, TLR7, TLR8 and TLR9) senses viral and bacterial nucleic acids in endosomal compartments. Of considerable interest is the recent finding that TLR7 and TLR8 can also recognize small interfering RNA (siRNA), which is the main effector in RNA interference. This immune activation by siRNAs can be abrogated by the 2'-ribose modification of uridines. Here, we discuss the recent developments that have expanded the understanding of self-non-self discrimination of RNAs by the innate immune system, and consider future directions for therapeutic applications of these findings.     

Conditional neutrality at two adjacent NBS-LRR disease resistance loci in natural populations of Arabidopsis lyrata

We examined patterns of nucleotide diversity at a genomic region containing two linked candidate disease resistance (NBS-LRR) genes in seven populations of the outcrossing plant Arabidopsis lyrata. In comparison with two adjacent control genes and neutral reference genes across the genome, the NBS-LRR genes exhibited elevated nonsynonymous variation and a large number of major-effect polymorphisms causing early stop codons and/or frameshift mutations. In contrast, analysis of synonymous diversity provided no evidence that the region was subject to long-term balancing selection or recent selective sweeps in any of the seven populations surveyed. Also in contrast with earlier surveys of one of these R genes, there was no evidence that the resistance genes or the major-effect mutations were subject to elevated differentiation between populations. We suggest that conditional neutrality in the absence of the corresponding pathogen, rather than long-term balancing selection or local adaptation, may in some circumstances be a significant cause of elevated functional polymorphism at R genes. In contrast with the R genes, analysis of diversity and differentiation at the flanking FERONIA locus showed high population divergence, suggesting local adaptation on this locus controlling male-female signalling during fertilization.     

Regulation of dendritic cell function through toll-like receptors

Higher animals establish host defense by orchestrating innate and adaptive immunity. This is mediated by professional antigen presenting cells, i.e. dendritic cells (DCs). DCs can incorporate pathogens, produce a variety of cytokines, maturate, and present pathogen-derived peptides to T cells, thereby inducing T cell activation and differentiation. These responses are triggered by microbial recognition through type I transmembrane proteins, Toll-like receptors (TLRs) on DCs. TLRs consist of ten members and each TLR is involved in recognizing a variety of microorganism-derived molecular structures. TLR ligands include cell wall components, proteins, nucleic acids, and synthetic chemical compounds, all of which can activate DCs as immune adjuvants.