Allorecognition System Research Articles

The greenbeard gene tgrB1 regulates altruism and cheating in Dictyostelium discoideum

Greenbeard genetic elements encode rare perceptible signals, signal recognition ability, and altruism towards others that display the same signal. Putative greenbeards have been described in various organisms but direct evidence for all the properties in one system is scarce. The tgrB1-tgrC1 allorecognition system of Dictyostelium discoideum encodes two polymorphic membrane proteins which protect cells from chimerism-associated perils. During development, TgrC1 functions as a ligand-signal and TgrB1 as its receptor, but evidence for altruism has been indirect. Here, we show that mixing wild-type and activated tgrB1 cells increases wild-type spore production and relegates the mutants to the altruistic stalk, whereas mixing wild-type and tgrB1-null cells increases mutant spore production and wild-type stalk production. The tgrB1-null cells cheat only on partners that carry the same tgrC1-allotype. Therefore, TgrB1 activation confers altruism whereas TgrB1 inactivation causes allotype-specific cheating, supporting the greenbeard concept and providing insight into the relationship between allorecognition, altruism, and exploitation.

The Bcvic1 and Bcvic2 vegetative incompatibility genes in Botrytis cinerea encode proteins with domain architectures involved in allorecognition in other filamentous fungi

Vegetative incompatibility is a fungal allorecognition system characterised by the inability of genetically distinct conspecific fungal strains to form a viable heterokaryon and is controlled by multiple polymorphic loci termed vic (vegetative incompatibility) or het (heterokaryon incompatibility). We have genetically identified and characterised the first vic locus in the economically important, plant-pathogenic, necrotrophic fungus Botrytis cinerea. A bulked segregant approach coupled with whole genome Illumina sequencing of near-isogenic lines of B. cinerea was used to map a vic locus to a 60-kb region of the genome. Within that locus, we identified two adjacent, highly polymorphic open reading frames, Bcvic1 and Bcvic2, which encode predicted proteins that contain domain architectures implicated in vegetative incompatibility in other filamentous fungi. Bcvic1 encodes a predicted protein containing a putative serine esterase domain, a NACHT family of NTPases domain, and several Ankyrin repeats. Bcvic2 encodes a putative syntaxin protein containing a SNARE domain; such proteins typically function in vesicular transport. Deletion of Bcvic1 and Bcvic2 individually had no effect on vegetative incompatibility. However, deletion of the region containing both Bcvic1 and Bcvic2 resulted in mutant lines that were severely restricted in growth and showed loss of vegetative incompatibility. Complementation of these mutants by ectopic expression restored the growth and vegetative incompatibility phenotype, indicating that Bcvic1 and Bcvic2 are controlling vegetative incompatibility at this vic locus.

The Hydractinia allorecognition system.

Hydractinia symbiolongicarpus is a colonial hydroid and a long-standing model system for the study of invertebrate allorecognition. The Hydractinia allorecognition system allows colonies to discriminate between their own tissues and those of unrelated conspecifics that co-occur with them on the same substrate. This recognition mediates spatial competition and mitigates the risk of stem cell parasitism. Here, I review how we have come to our current understanding of the molecular basis of allorecognition in Hydractinia. To date, two allodeterminants have been identified, called Allorecognition 1 (Alr1) and Allorecognition 2 (Alr2), which occupy a genomic region called the allorecognition complex (ARC). Both genes encode highly polymorphic cell surface proteins that are capable of homophilic binding, which is thought to be the mechanism of self/non-self discrimination. Here, I review how we have come to our current understanding of Alr1 and Alr2. Although both are members of the immunoglobulin superfamily, their evolutionary origins remain unknown. Moreover, existing data suggest that the ARC may be home to a family of Alr-like genes, and I speculate on their potential functions.

Cellular allorecognition and its roles in Dictyostelium development and social evolution.

The social amoeba Dictyostelium discoideum is a tractable model organism to study cellular allorecognition, which is the ability of a cell to distinguish itself and its genetically similar relatives from more distantly related organisms. Cellular allorecognition is ubiquitous across the tree of life and affects many biological processes. Depending on the biological context, these versatile systems operate both within and between individual organisms, and both promote and constrain functional heterogeneity. Some of the most notable allorecognition systems mediate neural self-avoidance in flies and adaptive immunity in vertebrates. D. discoideum's allorecognition system shares several structures and functions with other allorecognition systems. Structurally, its key regulators reside at a single genomic locus that encodes two highly polymorphic proteins, a transmembrane ligand called TgrC1 and its receptor TgrB1. These proteins exhibit isoform-specific, heterophilic binding across cells. Functionally, this interaction determines the extent to which co-developing D. discoideum strains co-aggregate or segregate during the aggregation phase of multicellular development. The allorecognition system thus affects both development and social evolution, as available evidence suggests that the threat of developmental cheating represents a primary selective force acting on it. Other significant characteristics that may inform the study of allorecognition in general include that D. discoideum's allorecognition system is a continuous and inclusive trait, it is pleiotropic, and it is temporally regulated.

Enhanced hypovirus transmission by engineered super donor strains of the chestnut blight fungus, Cryphonectria parasitica, into a natural population of strains exhibiting diverse vegetative compatibility genotypes

Horizontal transmission of virulence attenuating hypoviruses of Cryphonectria parasitica is restricted by an allorecognition system termed vegetative incompatibility (vic). A super donor formulation of two engineered C. parasitica strains (SD328/SD82) with gene disruptions at four of six vic loci transmitted hypovirus to strains in the laboratory independent of vic genotype. We now report the transmission of hypovirus by the SD328/82 formulation to a diverse, natural C. parasitica population infecting American chestnut in a forest setting. Hypovirulent (HV) isolates were recovered from 94% of cankers treated with the hypovirus-infected SD328/82 formulation compared to 51% of cankers treated with a hypovirus-infected EU5/6 formulation (strains having the same vic genotypes as SD strains but lacking vic gene disruptions). Overall, the SD328/82 formulation transmitted hypovirus into more divergent vic genotypes compared to the EU5/6 formulation. These results demonstrate the SD328/82 formulation can serve as an enhanced hypovirus vector for highly divergent C. parasitica populations.

Species recognition in social amoebae

Aggregative multicellularity requires the ability of cells to recognise conspecifics. Social amoebae are among the best studied of such organisms, but the mechanism and evolutionary background of species recognition remained to be investigated. Here we show that heterologous expression of a single Dictyostelium purpureum gene is sufficient for D. discoideum cells to efficiently make chimaeric fruiting bodies with D. purpureum cells. This gene forms a bidirectional pair with another gene on the D. purpureum genome, and they are both highly polymorphic among independent wild isolates of the same mating group that do not form chimaeric fruiting bodies with each other. These paired genes are both structurally similar to D. discoideum tgrB1/C1 pair, which is responsible for clonal discrimination within that species, suggesting that these tgr genes constitute the species recognition system that has attained a level of precision capable of discriminating between clones within a species. Analysis of the available genome sequences of social amoebae revealed that such gene pairs exist only within the clade composed of species that produce precursors of sterile stalk cells (prestalk cells), suggesting concurrent evolution of a precise allorecognition system and a new 'worker' cell-type dedicated to transporting and supporting the reproductive cells.

Innate allorecognition by monocytic cells and its role in graft rejection.

Innate recognition of microbial products and danger molecules by monocytes and macrophages has been well established; this is mediated primarily by pattern-recognition receptors and is central to the activation of innate and adaptive immune cells required for productive immunity. Whether monocytes and macrophages are equipped with an allorecognition system that allows them to respond directly to allogeneic grafts is a topic of much debate. Recent studies provide compelling evidence that these cells can recognize allogeneic entities and that they mediate graft rejection via direct cytotoxicity and priming of alloreactive T cells. In addition, these studies have uncovered a mechanism of innate allorecognition based on detection of the polymorphic molecule signal regulatory protein α (SIRPα) on donor cells. Further understanding of innate allorecognition and its consequences would provide essential insight into allograft rejection and lead to better therapies for transplant patients.

Natural Killer (NK) Cell Receptors and their Role in Pregnancy and Abortion

Existing data suggest that decidual Natural Killer Cells (dNK CD3-CD56brightCD16dim/-) are important in early pregnancy (local response to pathogens, control of trophoblast invasion, uterine vascular remodeling), while in ongoing pregnancy they contribute to the acceptance of the embryo through various immunoregulatory mechanisms. In the so-called alloimmune abortions, CD3-CD56brightCD16dim/-NK cells are decreased in favor of CD3-CD56dimCD16bright NK cells, which are toxic for trophoblast. Most of the activating and inhibitory receptors regulating dNK function belong to the highly polymorphic KIR (Killer Immunoglobulin-like) family. KIRs have as ligands the only HLA molecules expressed on extravillous cytotrophoblast (HLA-C, -E, -G). The interactions of maternal KIR receptors with fetal HLA-C molecules provide an immunogenetic allorecognition system. If inhibitory KIRs recognize their specific ligands, they inhibit dNK activation for trophoblast damage. Otherwise, dNK are allowed to develop anti-trophoblast activity. Given the differences in both the KIR repertoire and the HLA-C allotypes among unrelated individuals, each pregnancy presents a different combination of maternal KIR receptors on dNK and self and non-self HLA-C allotypes on trophoblast. Current studies provide evidence for differences between the combinations arising in successful pregnancies to those found in cases of abortions.

Allorecognition, via TgrB1 and TgrC1, mediates the transition from unicellularity to multicellularity in the social amoeba Dictyostelium discoideum.

The social amoeba Dictyostelium discoideum integrates into a multicellular organism when individual starving cells aggregate and form a mound. The cells then integrate into defined tissues and develop into a fruiting body that consists of a stalk and spores. Aggregation is initially orchestrated by waves of extracellular cyclic adenosine monophosphate (cAMP), and previous theory suggested that cAMP and other field-wide diffusible signals mediate tissue integration and terminal differentiation as well. Cooperation between cells depends on an allorecognition system comprising the polymorphic adhesion proteins TgrB1 and TgrC1. Binding between compatible TgrB1 and TgrC1 variants ensures that non-matching cells segregate into distinct aggregates prior to terminal development. Here, we have embedded a small number of cells with incompatible allotypes within fields of developing cells with compatible allotypes. We found that compatibility of the allotype encoded by the tgrB1 and tgrC1 genes is required for tissue integration, as manifested in cell polarization, coordinated movement and differentiation into prestalk and prespore cells. Our results show that the molecules that mediate allorecognition in D. discoideum also control the integration of individual cells into a unified developing organism, and this acts as a gating step for multicellularity.

Friend or foe? How an invertebrate distinguishes self from non-self in a natural histocompatibility interaction. (TRAN3P.912)

Abstract The existence of sophisticated self/non-self systems is not limited to vertebrates. Several colonial marine invertebrates have evolved systems that distinguish between conspecifics via cell-cell contact. This phenomenon, known as allorecognition, occurs when colonies encounter each other as they grow across their substrate. Compatible colonies fuse or co-exist, while incompatible colonies reject and often aggressively compete for space. In all taxa studied to date, allorecognition phenotypes are determined by highly polymorphic loci, which ensure that colonies are only compatible with themselves or close kin. How allorecognition molecules achieve this specificity remains unknown. Allorecognition in Hydractinia is controlled by at least two genes, alr1 and alr2, which encode highly polymorphic transmembrane proteins similar to immunoglobulin superfamily molecules. Colonies with matching alleles at alr1 and alr2 fuse, while colonies with no matching alleles reject. Using in vitro assays with recombinant proteins, we demonstrate that alr1 alleles bind to themselves but not to other alr1 alleles. We suggest that, in vivo, compatibility between colonies is also determined by allele-specific homophilic binding of alr proteins. Since fusion is rare in nature and single populations contain hundreds of unique alr alleles, the Hydractinia allorecognition system appears to be based on a biophysical mechanism with unprecedented allelic diversity and specificity.

Allorecognition, Germline Chimerism, and Stem Cell Parasitism in the Colonial Ascidian, Botryllus schlosseri

Many marine invertebrates have the ability to combine tissues with conspecifics and form chimeras. This ability is usually accompanied by the presence of a polymorphic self/non-self recognition system (allorecognition) that allows integration of closely related individuals, but blocks interactions between those more distantly related. The presence of a discriminatory allorecognition system suggests that there are costs and benefits to chimerism that are correlated to relatedness, but the nature of these costs and benefits is still poorly understood. Interestingly, allorecognition is found throughout the animal kingdom, with examples in nearly all the multicellular phyla; however, the role of allorecognition in these different species is not always clear. In addition, the molecular mechanisms underlying allorecognition in different species are not related, suggesting a unique origin for each species. This brings up the question as to whether there is a universal need for self/non-self recognition processes among the metazoa, or if these systems evolve in response to different selective pressures. We are studying allorecognition and chimerism in the colonial ascidian, Botryllus schlosseri. Allorecognition takes place at the tips of the extracorporeal vasculature, and can result in a parabiosis (fusion), or a rejection reaction that blocks vascular fusion. Fusion has both hypothesized benefits as well as well-documented costs. This article will focus on characterizing the costs and benefits of fusion and chimerism in Botryllus, and put them into the broader context of the role of allorecognition in the metazoa.

Sperm proteases and extracellular ubiquitin-proteasome system involved in fertilization of ascidians and sea urchins.

Ascidians (primitive chordates) are hermaphroditic animals that release spermatozoa and eggs almost simultaneously, but some species, including Halocynthia roretzi, show strict self-sterility. In H. roretzi, a 70-kDa vitelline coat (VC) protein consisting of 12 EGF-like repeats, named HrVC70, appears to be a promising candidate for the self/nonself-recognition (or allorecognition) system during gamete interaction. After spermatozoon recognizes the VC as nonself, sperm 700-kDa extracellular ubiquitin-conjugating enzyme complex appears to ubiquitinate Lys234 of HrVC70, and the ubiquitinated HrVC70 is degraded by the sperm 26S proteasome that is located on the sperm head surface. This novel ubiquitin-proteasome system enables spermatozoa to penetrate through the VC. Sperm trypsin-like proteases, acrosin and spermosin, also participate in fertilization, probably as sperm-side 'movable' binding proteins to the VC.

Molecular evolution of a polymorphic HSP40-like protein encoded in the histocompatibility locus of an invertebrate chordate

Allorecognition, the ability to distinguish self from non-self, occurs in most organisms. Despite the ubiquity of the allorecognition process, the genetic basis for allorecognition remains unexplored in most taxa outside vertebrates and flowering plants. The allorecognition system in the colonial ascidian Botryllus schlosseri is a notable exception. We have recently identified a polymorphic gene within the fuhc locus that may play a role in allorecognition. The encoded protein, called Hsp40-L, is a Type II member of the J-protein family which usually functions as a co-chaperone with Hsp70. While many of the residues that interact with Hsp70 are conserved in Hsp40-L, it may not be a housekeeping protein because it is surprisingly polymorphic and expressed in the ampullae, the site of allorecognition. While the majority of the Hsp40-L protein appears to evolve under purifying selection, a section of the C-terminal region likely experiences balancing/directional selection, characteristic of other allorecognition proteins.

Allorecognition triggers autophagy and subsequent necrosis in the cnidarian Hydractinia symbiolongicarpus.

Transitory fusion is an allorecognition phenotype displayed by the colonial hydroid Hydractinia symbiolongicarpus when interacting colonies share some, but not all, loci within the allorecognition gene complex (ARC). The phenotype is characterized by an initial fusion followed by subsequent cell death resulting in separation of the two incompatible colonies. We here characterize this cell death process using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and continuous in vivo digital microscopy. These techniques reveal widespread autophagy and subsequent necrosis in both colony and grafted polyp assays. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays and ultrastructural observations revealed no evidence of apoptosis. Pharmacological inhibition of autophagy using 3-methyladenine (3-MA) completely suppressed transitory fusion in vivo in colony assays. Rapamycin did not have a significant effect in the same assays. These results establish the hydroid allorecognition system as a novel model for the study of cell death.

Expression of putative immune response genes during early ontogeny in the coral Acropora millepora.

BackgroundCorals, like many other marine invertebrates, lack a mature allorecognition system in early life history stages. Indeed, in early ontogeny, when corals acquire and establish associations with various surface microbiota and dinoflagellate endosymbionts, they do not efficiently distinguish between closely and distantly related individuals from the same population. However, very little is known about the molecular components that underpin allorecognition and immunity responses or how they change through early ontogeny in corals.Methodology/Principal FindingsPatterns in the expression of four putative immune response genes (apextrin, complement C3, and two CELIII type lectin genes) were examined in juvenile colonies of Acropora millepora throughout a six-month post-settlement period using quantitative real-time PCR (qPCR). Expression of a CELIII type lectin gene peaked in the fourth month for most of the coral juveniles sampled and was significantly higher at this time than at any other sampling time during the six months following settlement. The timing of this increase in expression levels of putative immune response genes may be linked to allorecognition maturation which occurs around this time in A.millepora. Alternatively, the increase may represent a response to immune challenges, such as would be involved in the recognition of symbionts (such as Symbiodinium spp. or bacteria) during winnowing processes as symbioses are fine-tuned.Conclusions/SignificanceOur data, although preliminary, are consistent with the hypothesis that lectins may play an important role in the maturation of allorecognition responses in corals. The co-expression of lectins with apextrin during development of coral juveniles also raises the possibility that these proteins, which are components of innate immunity in other invertebrates, may influence the innate immune systems of corals through a common pathway or system. However, further studies investigating the expression of these genes in alloimmune-challenged corals are needed to further clarify emerging evidence of a complex innate immunity system in corals.

High natural prevalence of a fungal prion

Prions are infectious proteins that cause fatal diseases in mammals. Prions have also been found in fungi, but studies on their role in nature are scarce. The proposed biological function of fungal prions is debated and varies from detrimental to benign or even beneficial. [Het-s] is a prion of the fungus Podospora anserina. The het-s locus exists as two antagonistic alleles that constitute an allorecognition system: the het-s allele encoding the protein variant capable of prion formation and the het-S allele encoding a protein variant that cannot form a prion. We document here that het-s alleles, capable of prion formation, are nearly twice as frequent as het-S alleles in a natural population of 112 individuals. Then, we report a 92% prevalence of [Het-s] prion infection among the het-s isolates and find evidence of the role of the [Het-s]/het-S allorecognition system on the incidence of infection by a deleterious senescence plasmid. We explain the het-s/het-S allele ratios by the existence of two selective forces operating at different levels. We propose that during the somatic stage, the role of [Het-s]/HET-S in allorecognition leads to frequency-dependent selection for which an equilibrated frequency would be optimal. However, in the sexual cycle, the [Het-s] prion causes meiotic drive favoring the het-s allele. Our findings indicate that [Het-s] is a selected and, therefore, widespread prion whose activity as selfish genetic element is counteracted by balancing selection for allorecognition polymorphism.

Allorecognition maturation in the broadcast-spawning coral Acropora millepora

Many sessile marine invertebrates discriminate self from non-self with great precision, but maturation of allorecognition generally takes months to develop in juveniles. Here, we compare the development of allorecognition in full-sibling, half-sibling and non-sibling contact reactions between newly settled juveniles of the broadcast-spawning coral Acropora millepora on the Great Barrier Reef (Australia). Absence of a rejection response showed that A. millepora lacks a mature allorecognition system in the first 2 months post-settlement. From thereon, incompatibilities were observed between juveniles, their level of relatedness (i.e. full-, half- and non-sibling status) governing the rate of allorecognition maturation. All contact reactions between non-siblings resulted in rejections by 3 months post-settlement, whereas the expression of allorecognition took at least 5 months between half-siblings and longer than 13 months for some full-siblings. Approximately 74 % of fused full-siblings (n = 19) persisted as chimeras at 11 months, thus maturation of allorecognition in this spawning coral appeared to be slower (>13 months) than in brooding corals (~4 months). We hypothesize that late maturation of allorecognition may contribute to flexibility in Symbiodinium uptake in corals with horizontal transmission, and could allow fusions and chimera formation in early ontogeny, which potentially enable rapid size increase through fusion.

Invertebrate immune diversity

The arms race between hosts and pathogens (and other non-self) drives the molecular diversification of immune response genes in the host. Over long periods of evolutionary time, many different defense strategies have been employed by a wide variety of invertebrates. We review here penaeidins and crustins in crustaceans, the allorecognition system encoded by fuhc, fester and Uncle fester in a colonial tunicate, Dscam and PGRPs in arthropods, FREPs in snails, VCBPs in protochordates, and the Sp185/333 system in the purple sea urchin. Comparisons among immune systems, including those reviewed here have not identified an immune specific regulatory “genetic toolkit”, however, repeatedly identified sequences (or “building materials” on which the tools act) are present in a broad range of immune systems. These include a Toll/TLR system, a primitive complement system, an LPS binding protein, and a RAG core/Transib element. Repeatedly identified domains and motifs that function in immune proteins include NACHT, LRR, Ig, death, TIR, lectin domains, and a thioester motif. In addition, there are repeatedly identified mechanisms (or “construction methods”) that generate sequence diversity in genes with immune function. These include genomic instability, duplications and/or deletions of sequences and the generation of clusters of similar genes or exons that appear as families, gene recombination, gene conversion, retrotransposition, alternative splicing, multiple alleles for single copy genes, and RNA editing. These commonly employed “materials and methods” for building and maintaining an effective immune system that might have been part of that ancestral system appear now as a fragmented and likely incomplete set, likely due to the rapid evolutionary change (or loss) of host genes that are under pressure to keep pace with pathogen diversity.

Urochordate Histoincompatible Interactions Activate Vertebrate-Like Coagulation System Components

The colonial ascidian Botryllus schlosseri expresses a unique allorecognition system. When two histoincompatible Botryllus colonies come into direct contact, they develop an inflammatory-like rejection response. A surprising high number of vertebrates' coagulation genes and coagulation-related domains were disclosed in a cDNA library of differentially expressed sequence tags (ESTs), prepared for this allorejection process. Serine proteases, especially from the trypsin family, were highly represented among Botryllus library ortholgues and its “molecular function” gene ontology analysis. These, together with the built-up clot-like lesions in the interaction area, led us to further test whether a vertebrate-like clotting system participates in Botryllus innate immunity. Three morphologically distinct clot types (points of rejection; POR) were followed. We demonstrated the specific expression of nine coagulation orthologue transcripts in Botryllus rejection processes and effects of the anti-coagulant heparin on POR formation and heartbeats. In situ hybridization of fibrinogen and von Willebrand factor orthologues elucidated enhanced expression patterns specific to histoincompatible reactions as well as common expressions not augmented by innate immunity. Immunohistochemistry for fibrinogen revealed, in naïve and immune challenged colonies alike, specific antibody binding to a small population of Botryllus compartment cells. Altogether, molecular, physiological and morphological outcomes suggest the involvement of vertebrates-like coagulation elements in urochordate immunity, not assigned with vasculature injury.

The Fetal Allograft Revisited: Does the Study of an Ancient Invertebrate Species Shed Light on the Role of Natural Killer Cells at the Maternal-Fetal Interface?

Human pregnancy poses a fundamental immunological problem because the placenta and fetus are genetically different from the host mother. Classical transplantation theory has not provided a plausible solution to this problem. Study of naturally occurring allogeneic chimeras in the colonial marine invertebrate, Botryllus schlosseri, has yielded fresh insight into the primitive development of allorecognition, especially regarding the role of natural killer (NK) cells. Uterine NK cells have a unique phenotype that appears to parallel aspects of the NK-like cells in the allorecognition system of B. schlosseri. Most notably, both cell types recognize and reject “missing self” and both are involved in the generation of a common vascular system between two individuals. Chimeric combination in B. schlosseri results in vascular fusion between two individual colonies; uterine NK cells appear essential to the establishment of adequate maternal-fetal circulation. Since human uterine NK cells appear to de-emphasize primary immunological function, it is proposed that they may share the same evolutionary roots as the B. schlosseri allorecognition system rather than a primary origin in immunity.