Evolution of Immune Proteins in Insects

BackgroundGene duplication followed by functional divergence has long been hypothesized to be the main source of molecular novelty. Convincing examples of neofunctionalization, however, remain rare. Snake venom phospholipase A2 genes are members of large multigene families with many diverse functions, thus they are excellent models to study the emergence of novel functions after gene duplications.ResultsHere, I show that positive Darwinian selection and neofunctionalization is common in snake venom phospholipase A2 genes. The pattern of gene duplication and positive selection indicates that adaptive molecular evolution occurs immediately after duplication events as novel functions emerge and continues as gene families diversify and are refined. Surprisingly, adaptive evolution of group-I phospholipases in elapids is also associated with speciation events, suggesting adaptation of the phospholipase arsenal to novel prey species after niche shifts. Mapping the location of sites under positive selection onto the crystal structure of phospholipase A2 identified regions evolving under diversifying selection are located on the molecular surface and are likely protein-protein interactions sites essential for toxin functions.ConclusionThese data show that increases in genomic complexity (through gene duplications) can lead to phenotypic complexity (venom composition) and that positive Darwinian selection is a common evolutionary force in snake venoms. Finally, regions identified under selection on the surface of phospholipase A2 enzymes are potential candidate sites for structure based antivenin design.

Seminal fluid proteins show striking effects on reproduction, involving manipulation of female behavior and physiology, mechanisms of sperm competition, and pathogen defense. Strong adaptive pressures are expected for such manifestations of sexual selection and host defense, but the extent of positive selection in seminal fluid proteins from divergent taxa is unknown. We identified adaptive evolution in primate seminal proteins using genomic resources in a tissue-specific study. We found extensive signatures of positive selection when comparing 161 human seminal fluid proteins and 2,858 prostate-expressed genes to those in chimpanzee. Seven of eight outstanding genes yielded statistically significant evidence of positive selection when analyzed in divergent primates. Functional clues were gained through divergent analysis, including several cases of species-specific loss of function in copulatory plug genes, and statistically significant spatial clustering of positively selected sites near the active site of kallikrein 2. This study reveals previously unidentified positive selection in seven primate seminal proteins, and when considered with findings in Drosophila, indicates that extensive positive selection is found in seminal fluid across divergent taxonomic groups.

Much effort and interest have focused on assessing the importance of naturalselection, particularly positive natural selection, in shaping the human genome.Although scans for positive selection have identified candidate loci that may beassociated with positive selection in humans, such scans do not indicate whetheradaptation is frequent in general in humans. Studies based on the reasoning ofthe MacDonald–Kreitman test, which, in principle, can be used toevaluate the extent of positive selection, suggested that adaptation isdetectable in the human genome but that it is less common than in Drosophila orEscherichia coli. Both positive and purifying naturalselection at functional sites should affect levels and patterns of polymorphismat linked nonfunctional sites. Here, we search for these effects by analyzingpatterns of neutral polymorphism in humans in relation to the rates ofrecombination, functional density, and functional divergence with chimpanzees.We find that the levels of neutral polymorphism are lower in the regions oflower recombination and in the regions of higher functional density ordivergence. These correlations persist after controlling for the variation in GCcontent, density of simple repeats, selective constraint, mutation rate, anddepth of sequencing coverage. We argue that these results are most plausiblyexplained by the effects of natural selection at functionalsites—either recurrent selective sweeps or backgroundselection—on the levels of linked neutral polymorphism. Naturalselection at both coding and regulatory sites appears to affect linked neutralpolymorphism, reducing neutral polymorphism by 6% genome-wide and by11% in the gene-rich half of the human genome. These findings suggestthat the effects of natural selection at linked sites cannot be ignored in thestudy of neutral human polymorphism.

Breast milk is the preferred food for the newborns and considered as complete ‘edible immune system’. In addition, breast milk is a source of innate and adaptive proteins which not only protects the naïve infant gastrointestinal tract from enteropathogens but also aid in the appropriate initial colonization of gut microbiota. In this study, we analyzed antimicrobial immune proteins in the milk from C57BL/6 dams at several time points, and observed distinctive patterns in the immune protein levels throughout the lactation period. The presence of innate immune proteins serum amyloid A (SAA), CD14, and notably lipocalin-2 (Lcn2) were observed in high quantities with Lcn2 and SAA present at microgram levels suggesting a potential niche for these proteins during neonatal immune development. Moreover, low dose LPS administration to dams significantly increased some of these proteins in the milk. In addition, adaptive immune proteins immunoglobulins (Ig) A and G with IgG were present in milk at higher quantities compared to IgA at day 5 post-delivery. Interestingly, class-switching occurs by day 15 indicating a selective adaptive immune preference towards IgA, which could be due to gut colonization of newborns with more complex gut microbiota with aging and IgA being a major gut homeostatic factor. Additionally, milk from IgA-deficient dams allowed for robust proliferation of E. coli compared to wild-type milk further signifying the potential involvement of IgA during the early colonization of microbes in the neonatal gut. Collectively, our findings provide insight in the various immune proteins with antimicrobial activity may play a major role in the initial microbial colonization in the gut, which has long-lasting consequences on the host. Supported by NIH R01-CA219144, NCI Diversity Supplement (CA219144-05S1)

Current hypotheses of gene duplicate divergence propose that surviving members of a gene duplicate pair may evolve, under conditions of purifying or nearly neutral selection, in one of two ways: with new function arising in one duplicate while the other retains original function (neofunctionalization [NF]) or partitioning of the original function between the 2 paralogs (subfunctionalization [SF]). More recent studies propose that SF followed by NF (subneofunctionalization [SNF]) explains the divergence of many duplicate genes. In this analysis, we evaluate these hypotheses in the context of the large monosaccharide transporter (MST) gene families in Arabidopsis and rice. MSTs have an ancient origin, predating plants, and have evolved in the seed plant lineage to comprise 7 subfamilies. In Arabidopsis, 53 putative MST genes have been identified, with one subfamily greatly expanded by tandem gene duplications. We searched the rice genome for members of the MST gene family and compared them with the MST gene family in Arabidopsis to determine subfamily expansion patterns and estimate gene duplicate divergence times. We tested hypotheses of gene duplicate divergence in 24 paralog pairs by comparing protein sequence divergence rates, estimating positive selection on codon sites, and analyzing tissue expression patterns. Results reveal the MST gene family to be significantly larger (65) in rice with 2 subfamilies greatly expanded by tandem duplications. Gene duplicate divergence time estimates indicate that early diversification of most subfamilies occurred in the Proterozoic (2500-540 Myr) and that expansion of large subfamilies continued through the Cenozoic (65-0 Myr). Two-thirds of paralog pairs show statistically symmetric rates of sequence evolution, most consistent with the SF model, with half of those showing evidence for positive selection in one or both genes. Among 8 paralog pairs showing asymmetric divergence rates, most consistent with the NF model, nearly half show evidence of positive selection. Positive selection does not appear in any duplicate pairs younger than approximately 34 Myr. Our data suggest that the NF, SF, and SNF models describe different outcomes along a continuum of divergence resulting from initial conditions of relaxed constraint after duplication.

Plant pathogens utilize a portfolio of secreted effectors to successfully infect and manipulate their hosts. It is, however, still unclear whether changes in secretomes leading to host specialization involve mostly effector gene gains/losses or changes in their sequences. To test these hypotheses, we compared the secretomes of three host-specific castrating anther smut fungi (Microbotryum), two being sister species. To address within-species evolution, which might involve coevolution and local adaptation, we compared the secretomes of strains from differentiated populations. We experimentally validated a subset of signal peptides. Secretomes ranged from 321 to 445 predicted secreted proteins (SPs), including a few species-specific proteins (42 to 75), and limited copy number variation, i.e., little gene family expansion or reduction. Between 52% and 68% of the SPs did not match any Pfam domain, a percentage that reached 80% for the small secreted proteins, indicating rapid evolution. In comparison to background genes, we indeed found SPs to be more differentiated among species and strains, more often under positive selection, and highly expressed in planta; repeat-induced point mutations (RIPs) had no role in effector diversification, as SPs were not closer to transposable elements than background genes and were not more RIP affected. Our study thus identified both conserved core proteins, likely required for the pathogenic life cycle of all Microbotryum species, and proteins that were species specific or evolving under positive selection; these proteins may be involved in host specialization and/or coevolution. Most changes among closely related host-specific pathogens, however, involved rapid changes in sequences rather than gene gains/losses.IMPORTANCE Plant pathogens use molecular weapons to successfully infect their hosts, secreting a large portfolio of various proteins and enzymes. Different plant species are often parasitized by host-specific pathogens; however, it is still unclear whether the molecular basis of such host specialization involves species-specific weapons or different variants of the same weapons. We therefore compared the genes encoding secreted proteins in three plant-castrating pathogens parasitizing different host plants, producing their spores in plant anthers by replacing pollen. We validated our predictions for secretion signals for some genes and checked that our predicted secreted proteins were often highly expressed during plant infection. While we found few species-specific secreted proteins, numerous genes encoding secreted proteins showed signs of rapid evolution and of natural selection. Our study thus found that most changes among closely related host-specific pathogens involved rapid adaptive changes in shared molecular weapons rather than innovations for new weapons.

Cauxin is a novel urinary protein recently identified in the domestic cat that regulates the excretion of felinine, a pheromone precursor involved in sociochemical communication and territorial marking of domestic and wild felids. Understanding the evolutionary history of cauxin may therefore illuminate molecular adaptations involved in the evolution of pheromone-based communication, recognition, and mate selection in wild animals. We sequenced the gene encoding cauxin, CES7, in 22 species representing all major felid lineages, and multiple outgroups and showed that it has undergone rapid evolutionary change preceding and during the diversification of the cat family. A comparison between feline cauxin and orthologous carboxylesterases from other mammalian lineages revealed evidence of strong positive Darwinian selection within and between several cat lineages, enriched at functionally important sites of the protein. The higher rate of radical amino acid replacements in small felids, coupled with the lack of felinine and extremely low levels of cauxin in the urine of the great cats (Panthera), correlates with functional divergence of this gene in Panthera, and its putative loss in the snow leopard. Expression studies found evidence for several alternatively spliced transcripts in testis and brain, suggesting additional roles in male reproductive fitness and behavior. Our work presents the first report of strong positive natural selection acting on a major urinary protein of nonrodent mammals, providing evidence for parallel selection pressure on the regulation of pheromones in different mammalian lineages, despite the use of different metabolic pathways. Our results imply that natural selection may drive rapid changes in the regulation of pheromones in urine among the different cat species, which in turn may influence social behavior, such as territorial marking and conspecific recognition, therefore serving as an important mechanism for the radiation of this group of mammals.

The role of adaptation in the divergence of lineages has long been a central question in evolutionary biology, and as multilocus sequence data sets have become available for a wide range of taxa, empirical estimates of levels of adaptive molecular evolution are increasingly common. Estimates vary widely among taxa, with high levels of adaptive evolution in Drosophila, bacteria, and viruses but very little evidence of widespread adaptive evolution in hominids. Although estimates in plants are more limited, some recent work has suggested that rates of adaptive evolution in a range of plant taxa are surprisingly low and that there is little association between adaptive evolution and effective population size in contrast to patterns seen in other taxa. Here, we analyze data from 35 loci for six sunflower species that vary dramatically in effective population size. We find that rates of adaptive evolution are positively correlated with effective population size in these species, with a significant fraction of amino acid substitutions driven by positive selection in the species with the largest effective population sizes but little or no evidence of adaptive evolution in species with smaller effective population sizes. Although other factors likely contribute as well, in sunflowers effective population size appears to be an important determinant of rates of adaptive evolution.

Schizophrenia poses an evolutionary-genetic paradox as it exhibits strongly negative fitness effects (early mortality and decreased fecundity), yet it persists at a prevalence of approximately 1% worldwide. Evidence from several studies have suggested that schizophrenia is evolved and maintained in part as a maladaptive byproduct of recent positive selection and adaptive evolution in human beings. However, inconsistent results have been also proposed, challenging the recent positive selection theory to explain the high population frequency of schizophrenia-associated alleles. Here, we used public domain data to locate signatures of positive selection based on genetic diversity, derived allele frequency, differentiation between populations, and long haplotypes at schizophrenia-associated single nucleotide polymorphisms (SNPs) and randomly selected SNPs (as negative controls). We found evidence for positive selection at 10 out of the 105 schizophrenia-associated SNPs, while 5 of these SNPs involved positive selection for the protective allele. Taken together, the absence of widespread positive selection signals at the schizophrenia-associated SNPs, along with the fact that half of the positive selection favored the protective allele, provide little evidence supporting the positive selection theory in schizophrenia.

AIM2-like receptors (ALRs) are a family of nucleic acid sensors essential for innate immune responses against viruses and bacteria. We performed an evolutionary analysis of ALR genes (MNDA, PYHIN1, IFI16, and AIM2) by analyzing inter- and intraspecies diversity. Maximum-likelihood analyses indicated that IFI16 and AIM2 evolved adaptively in primates, with branch-specific selection at the catarrhini lineage for IFI16. Application of a population genetics–phylogenetics approach also allowed identification of positive selection events in the human lineage. Positive selection in primates targeted sites located at the DNA-binding interface in both IFI16 and AIM2. In IFI16, several sites positively selected in primates and in the human lineage were located in the PYD domain, which is involved in protein–protein interaction and is bound by a human cytomegalovirus immune evasion protein. Finally, positive selection was found to target nuclear localization signals in IFI16 and the spacer region separating the two HIN domains. Population genetic analysis in humans revealed that an IFI16 genic region has been a target of long-standing balancing selection, possibly acting on two nonsynonymous polymorphisms located in the spacer region. Data herein indicate that ALRs have been repeatedly targeted by natural selection. The balancing selection region in IFI16 carries a variant with opposite risk effect for distinct autoimmune diseases, suggesting antagonistic pleiotropy. We propose that the underlying scenario is the result of an ancestral and still ongoing host–pathogen arms race and that the maintenance of susceptibility alleles for autoimmune diseases at IFI16 represents an evolutionary trade-off.

The Type VI secretion system (T6SS) delivers a wide range of antibacterial effectors, including phospholipases of the Tle family. Here, we characterize Tle1 from Pseudomonas aeruginosa and demonstrate that it functions as a bactericidal toxin with moderate activity when associated to the membranes in the periplasm. Bacterial two-hybrid assays revealed specific protein-protein interactions within the tle1 locus, involving the immunity protein Tli1a, the chaperone/adaptor Tla1, and the spike protein VgrG4a. These interactions were independently validated by co-purification assays. Structural modeling with AlphaFold 3 produced a high-confidence ternary complex in which a VgrG4a trimer accommodates one Tle1 monomer and one Tla1 monomer. The three predicted interfaces (Tle1-Tla1, Tla1-VgrG4a, and Tle1-VgrG4a) were confirmed experimentally in vivo and important charged residues mediating these interfaces were identified. Furthermore, modeling of the Tle1-Tli1a complex suggests an inhibition mechanism that does not occlude the catalytic pocket. Consistently, Tli1a was localized to the outer membrane of P. aeruginosa , supporting in silico predictions of an outer membrane lipoprotein and positioning it ideally to neutralize periplasmic Tle1 activity. We did not observe the second candidate immunity protein, Tli1b, either in P. aeruginosa or in E. coli . Together, these findings elucidate the molecular interactions underlying Tle1 delivery and inhibition and highlight the role of Tli1a as a dedicated immunity protein that protects P. aeruginosa from self-intoxication.

This chapter introduces the major theories of molecular evolution, such as the neutral theory, the nearly neutral theory, and the selectionist theory. It discusses the major neutrality tests designed to test the neutral theory using population data. The bulk of the chapter discusses various Markov chain models of codon substitution and their use in detecting Darwinian selection driving the fast evolution of protein-coding genes, as indicated by accelerated nonsynonymous substitution rates. Branch models, site models, and branch-site models are all covered in detail. The chapter then discusses empirical findings in genome scans for positive selection: genes involved in host–pathogen antagonism or in male and female reproduction, as well as duplicated genes that have acquired new functions are most often found to be under Darwinian selection.

BackgroundDuring the modern human expansion, new environmental pressures may have driven adaptation, especially in genes related to the perception of ingested substances and their detoxification. Consequently, positive (adaptive) selection may have occurred in genes related to taste, and in those related to the CYP450 system due to its role in biotransformation of potentially toxic compounds. A total of 91 genes (taste receptors and CYP450 superfamily) have been studied using Hierarchical Boosting, a powerful combination of different selection tests, to detect signatures of recent positive selection in three continental human populations: Northern Europeans (CEU), East Asians (CHB) and Africans (YRI). Analyses have been refined with selection analyses of the 26 populations of 1000 Genomes Project Phase 3.ResultsGenes related to taste perception have not been positively selected in the three continental human populations. This finding suggests that, contrary to results of previous studies, different allele frequencies among populations in genes such as TAS2R38 and TAS2R16 are not due to positive selection but to genetic drift. CYP1 and CYP2 genes, also previously considered to be under positive selection, did not show signatures of selective sweeps. However, three genes belonging to the CYP450 system have been identified by the Hierarchical Boosting as positively selected: CYP3A4 and CYP3A43 in CEU, and CYP27A1 in CHB.ConclusionsNo main adaptive differences are found in known taste receptor genes among the three continental human populations studied. However, there are important genetic adaptations in the cytochrome P450 system related to the Out of Africa expansion of modern humans. We confirmed that CYP3A4 and CYP3A43 are under selection in CEU, and we report for the first time CYP27A1 to be under positive selection in CHB.

Weighing the evidence for adaptation at the molecular level

The ubiquitous and conserved cytosolic heat-shock proteins 90 (HSP90A) perform essential functions in the cell. To understand the evolutionary origin of HSP90A functional diversification, we analyzed the distribution of HSP90A family from 54 species representing the main eukaryotic lineages. Three independent HSP90A duplications led to the paralog subfamilies HSP90AA (heat-stress inducible) and HSP90AB (constitutive) and trace back to key time points during vertebrate, seed plant, and yeast evolution. HSP90AA and HSP90AB present divergent selection pressures, positive selection (PS), and signatures of functional divergence (FD) after duplication. The differential evolutionary patterns support different mechanisms for HSP90A functional diversification in vertebrates and seed plants. Mapping of PS and FD residues onto the HSP90A structure suggests the acquisition of novel and/or specialized client protein and/or cochaperone binding functions. We propose these residues as targets for further experimental studies of HSP90A proteins, reported to be capacitors of rapid evolutionary change, and targets for anticancer therapeutics.