355 ISJ 13: 355-373, 2016 ISSN 1824-307X REVIEW Tuning the host-pathogen relationship through evolution with a special focus on the echinoid Sp185/333 system LC Smith1, MR Coscia2 1Department of Biological Sciences, George Washington University, Washington DC, USA 2Institute of Protein Biochemistry, National Research Council of Italy, Naples, Italy Accepted November 3, 2016 Abstract Diversification of immune genes in host organisms that are in deadly arms races with pathogens has resulted in a wide range of approaches by which the host survives. Well known examples of adaptive immunity in vertebrates include somatic recombination of the immunoglobulin gene family and assembly of the variable lymphocyte receptors. The CRISPR-Cas system in bacteria and archaea is also considered adaptive. For invertebrates that survive in the absence of adaptive immunity, innate immune diversity is accomplished based on functions of clusters of immune genes such as FREPs, VCBPs, C1qs, TLRs, and R genes. Single copy gene diversity can be accomplished through extensive alternative splicing or increases in alleles in populations. The Sp185/333 gene family in the purple sea urchin has multiple levels in which to generate immune diversity. These include clustered Sp185/333 genes, genomic instability in regions harboring the genes, predicted mRNA editing, expression of broad repertoires of Sp185/333 proteins, and post translational modifications. The Sp185/333 proteins have anti-pathogen activities and an individual recombinant protein can bind to multiple foreign cells and molecular patterns. The underlying characteristics of gene clusters, many with repeats, that are present in unstable genomic regions is common to a number of these examples, and is likely of central importance for organisms that survive solely on innate immunity. Key Words: Ig; TcR; VLR; DSCAM; FREPs; VCBP; TLR; Fu/HC; R genes; Sp185/333 Introduction When Alice went through the looking glass, she joined the Red Queen in the Queen’s race. During the race when Alice complained about running so hard for so long and getting nowhere, the Queen said to her, “it takes all the running you can do, to keep in the same place” (Carroll, 1871) (Fig. 1). Although this statement seems as crazy as both Wonderland and the Red Queen appeared to be, the idea has been employed as a hypothesis for evolution. The Red Queen’s race is analogous to the immunological arms race or coevolution between the diversity of an immune system and its effectiveness in protecting the long-lived host vs. the continuously changing virulence of pathogens that have (most often) much shorter life spans (Dawkins and Krebs, 1979). This host-pathogen arms race is driven by the need to adapt constantly to survive for ___________________________________________________________________________ Corresponding author: L. Courtney Smith Department of Biological Sciences Science and Engineering Hall Suite 6000, 800 22nd St NW W ashington DC 20052 E-mail: csmith@gwu.edu both the host and the pathogen, which in turn shapes the virulence and success of the pathogens as well as the detection and effector mechanisms of the host immune system (Haldane, 1949; Van Valen, 1973). The host-pathogen battle drives molecular evolution and affects all ranges of life including plants and animals and their pathogens, in addition to bacteria and their bacteriophages (Sironi et al., 2015). An example of this process in action is illustrated by an elegant study illustrating the experimental evolution of the bacterium Pseudomonas fluorescens SBW25 in the presence of its pathogen, bacteriophage Φ2 (Patterson et al., 2010). The outcome of this arms race demonstrates that evolutionary changes occur at higher rates when the host and pathogen coevolve together, and drives greater genotype diversification in both Pseudomonas and the phage compared to the bacteria alone or to the phage after the host cells have been killed by the phage. As expected, the bacteria show greater genetic diversity, and remarkably the phage genes that function in infectivity are also selected to change more rapidly. Because the number of genes that are involved both in pathogen virulence and host immunity is finite, 356 Fig. 1 Alice and the Red Queen in the Red Queen’s race. They both run as fast as they can but remain in the same place. Illustration by Sir John Tenniel from “Through the Looking-Glass” (Carroll, 1871). different approaches to diversification are incorporated into a few genes for increasing pathogen virulence or are incorporated into host immune genes to gain an advantage over the adversary in the arms race. Here, we review a range of examples of how a pathogen or a host changes or increases diversity of genes that function in the arms race. We end by focusing on the immune diversity in the Sp185/333 system of the purple sea urchin. Pathogen diversification for altered or increased virulence There is a wide range of how microbes generate genomic diversity to alter their phenotype to overcome a host immune system and/or to improve avoidance tactics to hide from host attack (Deitsch et al., 2009). Microbial pathogens adapt constantly to avoid mechanical clearance, circumvent barriers to invasion, avoid recognition and destruction by the host immune system, and/or alter or derail the host immune system with the aim of overwhelming the host response to result in a successful infection. The success in pathogen variation is measured by fitness or success in infection and proliferation leading to subsequent infection of other hosts. DNA recombination is employed by both bacterial and eukaryotic pathogens, and can move coding regions into an area of the genome that allows expression (Deitsch et al., 2009). This process can fuse intact genes or fragments of genes to generate chimeric sequences, or can move a promoter near a gene (or vice versa) that does not have a functional promoter, leading to altered expression and/or altered protein sequences. This can change not only whether a gene is expressed but can also change the level of expression. Antigenic variation or hypervariability, which includes phase variation, is the expression of a particular antigen or set of antigens sequentially over time that alters the pathogen surface phenotype and results in temporary avoidance of the host immune response until the next surface antigen is expressed (Fig. 2). Antigenic variation can be accomplished by changes in gene expression in single or multi-copy genes that are members of expanded families. Modifications to gene sequences within families can result from gene conversion or recombination among similar genes, and slipped strand mispairing followed by excision and repair (Deitsch et al., 2009; Oren et al., 2016). Variation in expression among genes that are members of an expanded family that have variable sequences is a very effective means for avoiding or “out running” immune recognition by the host. Furthermore, changes in gene expression within a gene family may be related to variations in the environment, and success in infecting a host that may be based on which genes within the family are expressed and under what circumstances. An example of this type of immune avoidance is the variable surface glycoprotein (VSG) that changes expression over time on the surface of trypanosome parasites, which are the pathogens that cause African sleeping sickness (Horn, 2014) (Fig. 2). Using a large pool of mosaic VSG genes, the parasite is able to escape host antibody recognition and attack by continuosly changing epitopes on its protective extracellular coat. Over time, expression switches to an antigenically different VSG by silencing the initially expressed gene and activating the transcription of a new VSG, or by recombining a VSG from another region in the genome into the active transcription site. 357 Fig. 2 VSG gene expression in Trypanosome parasites switches over time. This simplified scheme of antigenic phase variation over time in trypanosome infection illustrates changes in the version of VSG proteins (red, green, blue) on the parasite surface that enables it to avoid the immune response temporarily. Once the host immune response is directed to the expressed VSG (dotted lines), parasites with a variant VSG expression survive, take over the population, and are the source of the relapsing parastaemia. This figure is modified from (Horn, 2014). Antigenic variation also occurs in bacteria and is characterized by multiple copies of genes that are slightly different, such as when variation is limited to a specific region of the gene sequence. Changes in antigens can be controlled by 1) switch mechanisms that regulate independent expression, 2) the expression of a single gene while other members of the family are silenced, and 3) continuosly changing which gene copy is expressed (Finlay and McFadden, 2006). Antigenic variation is also employed by viruses. The accumulation of mutations within viral genes that encode epitopes that are bound by antibodies is a process of antigenic drift followed by antibody-based selection, which gives rise to increased success in viral infectivity and is directly related to antigenic changes to the viruses. This is particularly frequent in the case of RNA viruses in which antigenic variation depends mostly on the higher mutational frequency of RNA replicases (Lauring et al., 2013). The strategies of the human immunodeficiency virus (HIV) to escape the host immune system is a combination of a high mutation rate (3x10-5 nucleotides per viral replication cycle) plus retroviral recombination to alter viral recognition by host cytotoxic T lymphocytes and binding by antibodies (Hamelaar, 2012). Furthermore, conformational masking and glycan shielding of the viral envelope protein and its unfolding only upon entry into a cell serve to neutralize the host antibody response (Kwon et al., 2015). Antigenic variations are a good means by which to avoid, at least temporarily, the host immune response and are employed by a wide range of pathogens and parasites. A common feature of genes that function in immune avoidance is the expansion of genes in pathogens that encode effector proteins that disrupt the host immune response. This has been documented repeatedly for bacterial pathogens of higher plants (Macho and Zipfel, 2015) and is also the means by which Shigella infects humans (Ashida et al., 2015) and Bacillus thuringiensis infects Caenorhabditis elegans (Schulte et al., 2013). For some plant pathogens and for Shigella, microbes inject effector peptides into the host cell cytoplasm that disrupt or derail the innate immune response of the host. Similarly, for both Bacillus and Shigella, the effector peptides block pathogen specific responses in nematodes and humans, respectively, and allow the pathogen to proliferate successfully. In addition to changes in genes encoded in the genome, prokaryotes also carry virulence genes on plasmids. A Bacillus pathogen of C. elegans has a plasmid with variable numbers of crystal toxin genes with altered sequence diversity (Schulte et al., 2010) in addition to increased gene diversity in the genome (Schulte et al., 2013) in response to interactions with the host. Genes encoding the effectors and other virulence genes are greatly expanded in the genomes of these pathogens when they are directly involved in the arms race and adapt to the co-adaptations of the host immune system. There is a wide range of means by which horizontal transfer can be used by microbes to change their genotypes and phenotypes that can be beneficial for increased virulence capabilities and proliferation success. Mating, or bacterial conjugation, is a key mechanism for the horizontal transfer of DNA, including virulence genes, among microbes (de la Casa-Esperon, 2012; Cabazon et al., 2015). Transfer is mediated by the formation of the Type IV secretion system by Gram negative bacteria and mating pair formation results in membrane fusion and the transfer of DNA from the donor to recipient cell. This complex is employed in “double duty” activity because it is also used to inject virulence factors into eukaryotic host cells to increase bacterial survival and proliferation. Non- mating horizontal or lateral DNA transfer is another 358 means for altering the genome of a pathogen (de la Casa-Esperon, 2012). Microbes can acquire DNA either from other cells of the same species, or from cells of different species that can modify the means by which the altered microbe survives in the environment, in a host, or both. The outcome of DNA acquisition can increase the pathogen diversity and alter the interactions between host and pathogen. Natural competence to take up DNA from the environment rather than directly from other cells can also transform the capabilities of microbes. Horizontal DNA transfer is frequent in marine Vibrios not only among the cells but also based on the uptake of environmental DNA (Sun et al., 2013). Acquired DNA that includes genes encoding proteins with functions that improve fitness to survive a host immune response are likely to be selected for, maintained, and passed on to daughter cells. Bacteriophage infection can also result in the acquisition of bacterial DNA fragments that have been captured within the phage genome and can be transferred to other bacteria upon subsequent infections. “Infections of infections” also occur in the form of mobile genetic elements that use phages for transfer from one bacterial cell to another and can influence the pathogenicity and evolution of microbes (Penades et al., 2015). The wide variation and efficiency of microbial diversification in the arms race with their hosts, particularly given their short generation times, have driven the diversity mechanisms in the long-lived multicellular hosts. Host immune diversification Adaptive immune systems in vertebrates; V(D)J recombination The long-lived (relative to microbial pathogens) host response to constant genomic diversification and virulence changes in pathogens is to diversify the sequences of single copy genes in individual immune cells and/or to expand the copies of a gene followed by diversification of the sequences in the resulting gene family. Both approaches have been well documented in animals and the second approach has been particularly well employed in higher plants. Gnathosomes diversify their immunoglobulin (Ig) family genes, which function in both pathogen detection and response, by somatic recombination of variable, diversity and joining (V(D)J) segments to generate antigen binding sites in the variable exon of the rearranged single copy Ig and T cell Receptor (TcR) genes (Tonegawa, 1983; Davis et al., 1984). The enzymes of central importance in this process are the recombinase activating genes (RAGs) that function in “cutting and pasting” segments of the Ig gene family into a functional exon (Teng and Schatz, 2015) and the activation induced deaminase (AID) gene that is involved in class switching and affinity maturation of Ig genes (Muramatsu et al., 2000). However, other variations in Ig family proteins are known that are not involved in V(D)J recombination and show sequence diversity in the constant region. For example, the hinge region in IgM from an Antarctic fish (Trematomus bernacchii) is unique relative to the hinge regions of all other vertebrate Ig isotypes because it is much longer, is encoded by a 3′ extension of the Cµ2 exon, and is highly polymorphic Fig. 3 The origins of the adaptive immune system in vertebrates inferred from the TM regions of immune receptors. Preliminary evidence of a common origin is derived from the phylogenetic relationships among Agnathan VLRs, Gnathostome Igμ, TcR β and δ chains based on the consensus of their transmembrane region nucleotide sequences. Sequences were aligned by ClustalX (ver. 1.8) and the phylogenetic tree was constructed by the Neighbor-joining method with 100 bootstrap iterations. Positions with gaps were excluded from the alignment used to generate the tree. among individuals in the population (Coscia et al., 2012). Because a longer hinge region contributes to greater flexibility of the antibody binding sites, this may improve antigen binding capacity and effectiveness for IgM in this fish. Changes in the size of the hinge region may result from variations in the splice sites that alter the mRNA, or may be the result of non-synonymous substitutions at several specific sites that are under purifying selection compared to most other sites. Consequently, because the polymorphism mostly affects the hinge, which is long enough to be solvent exposed, it may protect this region from cleavage by pathogen proteases, such as those of the nematode parasites that cause a high incidence of infections in Antarctic fish (Coscia and Oreste, 1998; Coscia et al., 2000). In addition to differences among antibodies outside of the variable regions, there are also conserved features in antibodies that are present across a wide range of vertebrates. One of these features is the motif FXXXFXXS/TXXXS (X; any amino acid) in the Ig transmembrane (TM) region that can be considered a universal attribute of Ig molecules, and likely play a significant role in their function (Varriale et al., 2010). Interestingly, a 72 nucleotide sequence encoding the TM region includes 13 amino acids under purifying selection and show some identity with the TM regions from Agnathan variable lymphocyte receptor (VLR)A, VLRB, VLRC (see below) (Pancer et al., 2005), and murine TcR β and δ chains (Coscia and Oreste, unpublished) (Fig. 3). Nearly half of the positions in the VLR TM region are conserved including the 72 nucleotides. Although VLRs are phylogenetically unrelated to Igs, both classical antibodies from the Gnathostomes and VLRs from Agnathans show regions of diversity within the assembled or copied antigen binding regions, in addition to regions outside of the antigen binding sites. Furthermore, certain regions are conserved likely because of essential functions perhaps including interactions with TM regions of other proteins on the lymphocyte surface. Thus, the presence of a shared sequence 359 supports the notion that a common ancestor of the jawed and jawless vertebrates likely had both humoral and cellular based adaptive immunity. The apparent immunological “Big Bang” of adaptive immunity (Bernstein et al., 1996) and the sudden appearance of somatic recombination of the Ig gene family in higher vertebrates was thought to have arisen by a horizontal transfer of a transposon encoding recombinase, which was inserted into an ancestral Ig superfamily gene during the early evolution of jawed vertebrates (Bernstein et al., 1996; Agrawal et al., 1998; Hiom et al., 1998; Schluter et al., 1999; Flajnik, 2004). However, ProtoRAG genes with similarities to vertebrate RAGs and Transib transposases with putative “cut and paste” activity have been found in a range of invertebrates including the purple sea urchin (Strongylocentrotus purpuratus), hydra (Hydra magnipapillata), a sea anemone (Nematostella vectensis), and amphioxus (Branchiostoma floridae) in addition to related Transib transposons identified in the fruit fly (Drosophila melanogaster) and the mosquito (Anopheles gambiae) (reviewed in (Fugmann, 2010). Although these invertebrate gene sequences show similarities to ProtoRAG-like sequences, most are not affiliated with terminal inverted repeats, which are typical of transposases and are associated with binding and cutting activity. However, functional analysis of a Transib transposase from the corn earworm (Helicoverpa zea) demonstrated DNA cleavage and ligation that is similar to vertebrate RAG1 (Hencken et al., 2012). In the purple sea urchin (S. purpuratus), the RAG1 protein homologue, SpRag1-like, associates with RAG2 homologues including SpRag2-like from the sea urchin and RAG2 from the bull shark, and it also binds to the mouse one-turn recombination signal sequence (Fugmann et al., 2006). Furthermore, when associated with RAG2, SpRAG1-like supports low levels of V(D)J recombination in vitro (Carmona et al., 2016). The ProtoRAG genes in amphioxus, Branchiostoma belcheri, are linked and oriented “head to head” as in other animals, are associated with terminal inverted repeats (unlike the sea urchin SpRAGL genes), and support DNA cleavage, transposition, and the ligation of terminal inverted repeats, albeit at low effeciency, which is similar to signal joint ligation in vertebrates (Huang et al., 2016). The identification of RAG gene pairs in basal, extant deuterostomes in addition to other invertebrates with Transib transposase genes indicates that the origins of the RAG enzyme mediated adaptive immune system in higher vertebrates may have resulted from vertical inheritance of the ancestral ProtoRAG transposases in invertebrates rather than by lateral transfer from a microbe to a basal vertebrate (Fugmann, 2010). Assembly of variable lymphocyte receptors The jawless vertebrates, hagfish and lampreys, have an alternative adaptive immune system that uses a completely different mechanism to diversify the VLR genes, which function as antibodies and TcRs in these fish (Pancer et al., 2005; Boehm et al., 2012). There are three VLR genes that encode VLRA, VLRB and VLRC that have functions similar to αβTcR, Ig, and γδTcR, respectively (reviewed in (Flajnik, 2014) (Table 1). The VLR genes are assembled in a “copy choice” or gene conversion- like mechanism that randomly selects leucine rich repeats (LRR) from flanking cassettes of LRR segments and copies them into and replaces the non-coding region of the single copy, incomplete, germline VLR gene (Nagawa et al., 2007; Rogozin et al., 2007; Boehm et al., 2012). The assembly employs short stretches of nucleotides that match between different LRRs, which can be located at any place within an individual LRR. The assembly activity takes place in the thymoid regions at the tips of the gill filaments for VLRA and VLRC (Bajoghli et al., 2011) and in the hematopoietic regions of the typhlosole and kidney for VLRB genes (reviewed in Boehm et al., 2012; Kasahara, 2015). Unlike the Ig system in higher vertebrates, VLR gene assembly does not use RAG encoded recombinases, but is mediated by two cytidine deaminases (CDAs) in the lamprey (Guo et al., 2009). Expression of the CDA1 gene is restricted to the tips of the gill filaments in association with VLRA and VLRC expression, whereas expression of CDA2 is associated with VLRB expression in the typhlosole (Bajoghli et al., 2011). Although the assembly results in a combinatorial set of full length LRRs in VLR genes that appears to maintain the reading frame (Nagawa et al., 2007; Rogozin et al., 2007), apoptotic lymphocytes are detected and selection of VLRA and VLRC is suggested based on non-random numbers of LRRs (Sutoh and Kasahara, 2014). The astounding parallels between the Ig/TcR system and the VLR system suggest the possibility of common origins of antigen diversification and mechanisms of lymphocyte functions in the jawed and jawless vertebrates that predates RAG activities (Table 1). Table 1 Parallels in the antigen receptors and types of lymphocytes in jawless and jawed vertebrates1 Jawless Vertebrates Jawed Vertebrates Antigen Receptor Cell Type Antigen Receptor Cell Type VLRA on the surface of T-like lymphocytes αβTcR on the surface of αβ T lymphocytes VLRC on the surface of T-like lymphocytes γδTcR on the surface of γδ T lymphocytes VLRB on the surface of and secreted from B- like lymphocytes BCR on the surface or and secreted from B lymphocytes 1modified from Kasahara and Sutoh (2014). 360 Fig. 4 CRISPR-Cas, the adaptive immune system in prokaryotes. The CRISPR locus is composed of CRISPR- associated (Cas) genes followed by a start transcription site (bent arrow), a leader (L) and a series of repeats (black triangles) separated by spacers (rectangles of various colors). Upon infection with a bacteriophage (shown at the top), a section of the phage DNA (green) is incorporated as a new spacer at the beginning of the CRISPR array. Upon subsequent infection by a phage with DNA sequence that is the same as the spacer, the expression of the CRISPR (cr)RNA that includes the “green” spacer results in a sequence match to the DNA of the second phage infection and results in cleavage and destruction of the invading DNA. This figure is from (Barrangou and Marraffini, 2014) and reprinted from Molecular Cell with permission from Elsevier. CRISPR-Cas adaptive immunity in prokaryotes A third version of adaptive immunity mediated by clustered regularly interspersed repeats (CRISPR) has been identified in about 50% of bacteria and most of the archaea (Sternberg et al., 2016). The CRISPR system functions to protect bacteria from bacteriophage infection and lysis using activities with similarities to RNA interference in higher animals and plants. The CRISPR region of the bacterial genome acquires and incorporates short stretches of bacteriophage DNA called spacers, which act as a mechanism to recognize bacteriophage DNA in subsequent infections (Fig. 4). This sequence similarity is employed to cleave the foreign DNA and RNA using the CRISPR associated (Cas) proteins (Jiang and Doudna, 2015; Marraffini, 2015). Although there are a range of variations in CRISPR systems in different bacterial and archaeal species, in general it is the prokaryote adaptive immune system that is effective in protection against bacteriophages and other foreign DNA such as mobile genetic elements (Barrangou and Marraffini, 2014). Because the spacers are incorporated into the genome, immunity to specific phages is heritable and adaptive, although it is Lamarckian rather than Darwinian and is not anticipatory. Although it was long believed that higher vertebrates were the only group to function with adaptive immunity, the VLR and CRISPR/Cas examples demonstrate that there are multiple mechanisms for adaptive self protection against the diversity of pathogens. Diversity of innate immunity in invertebrates Drosophila melanogaster Most animals and all plants survive solely on their innate immune functions. Drosophila melanogaster was one of the first model organisms that lent itself to analysis because of its well understood genetics, short life cycle, and easily modified genes for functional analysis to understand how the immune system responds to pathogenic threats and injury. There are four major immune signaling pathways in Drosophila which are Toll, IMD, JAK/STAT and JNK (Stokes et al., 2015; Huang et al., 2016) that enable the fruit fly to translate the detection of pathogens to the production of anti-microbial peptides (AMPs) that control pathogens. The same signaling pathways are present in the common house fly, Musca domestica, with similar numbers of genes encoding proteins that function in the signaling pathways 361 (Armitage et al., 2015). However, compared to Drosophila the house fly genome shows a great expansion in the numbers of immune genes encoding recognition and effector proteins with increased sequence diversity. Clearly, the difference in life histories between the fruit fly and the house fly - living on and eating fruit vs. the well-known preferred habitats of the house fly on which it walks, eats, and lays eggs - is reflected in the diversity of the immune system in the house fly. For any given organism, the activities and diversity of immune function is the outcome of co-evolution with the types and diversity of the pathogens with which it shares the habitat, and its ability to keep pace with rapidly evolving microbes (reviewed in (Ghosh et al., 2011). DSCAM in arthropods One aspect of the Drosophila immune system, in addition to many other members of the arthropod phylum, is the gene encoding the Down Syndrome Cell Adhesion Molecule (DSCAM). DSCAM is present in more advanced arthropods as a single copy gene with duplications of specific exons (Fig. 5), or as a multicopy gene family in more basal arthropods that do not show exon expansions (Armitage et al., 2015; Brites and du Pasquier, 2015). DSCAM proteins in vertebrates and arthropods have 10 Ig domains and six fibronectin domains in the extracellular region, a transmembrane region, and a cytoplasmic tail that includes signaling motifs (Schmucker and Chen, 2009; Ng et al., 2014). The single copy DSCAM gene in insects generates significant sequence diversity in the encoded proteins (Armitage et al., 2015). Diversity is based on extensive expansion of exons that encode the 5′ portions of Ig domains 2 and 3 and all of Ig 7, which show mutually exclusive alternative splicing of the duplicated exons in the primary transcripts that encode each of these Ig domains (Fig. 5). Mutually exclusive alternative splicing of duplicated exons is based on insufficient distance between two duplicated exons, incompatible splicing signals located in regions between different sets of exons, secondary stem/loop structure of the DSCAM mRNA, and trans-acting factors that regulate splicing (Park et al., 2004; Kreahling and Graveley, 2005; Schmucker and Chen, 2009; Hemani and Soller, 2012; Spoel and Dong, 2012). The transcriptional outcome for this single copy gene is a theoretical 38,016 different proteins in Drosophila, about half of which are expressed in neuronal tissue and function in axon guidance, while the rest are expressed in hemocytes (Schmucker et al., 2000; Watson et al., 2005; Schmucker and Chen, 2009). The resulting sequence diversity in the three Ig domains defines the shape of the extracellular region that is based on interactions among the Ig domains, and influences interactions with particular binding targets. The DSCAM response in mosquito and shrimp immunity in response to pathogen challenge is to mount the DSCAM proteins on the hemocyte surface and to secrete or release DSCAM proteins into the hemolymph (Deitsch et al., 2009; Dong and Dimopoulos, 2009; Ng et al., 2014). Remarkably, the versions of DSCAM that are present in the hemolymph and on hemocytes after challenge show elevated specificity towards the infecting pathogen and function as opsonins to induce phagocytosis, clearance, and host protection, all of which lead to survival (Dong et al., 2006; Hung et al., 2013). DSCAM appears to be a major means by which the arthropods generate immune sequence diversity in response to pathogens. Overall, there are several means for generating sequence diversity in essentially single copy or very small families of genes in animals. These include somatic recombination of duplicated gene segments for Ig and TcR genes, copy choice or gene conversion assembly of duplicated LRR cassettes in the VLR genes, and extensive alternative splicing of duplicated exons in DSCAM genes in arthropods. Fig. 5 The DSCAM gene structure and alternative splicing. The exons in the DSCAM gene in Drosophila that encode the N-terminal portions of Ig domains 2 (red) and 3 (blue), plus all of Ig domain 7 (green) are duplicated in tandem in the gene (top). In addition, the exon encoding the transmembrane region (yellow) is duplicated. The duplicated exons undergo alternative splicing in the mRNA (middle), to produce a highly diversified set of proteins (bottom) that are expressed in neurons and hemocytes. This figure is from (Schmucker et al., 2000) and reprinted from Cell with permission from Elsevier. 362 Fig. 6 Diversity in the FREP gene families and the encoded proteins. The FREP genes (on the left) have one to seven exons and encode proteins (on the right) with one or two Ig superfamily (IgSF) domains or proteins with only the fibrinogen (FBG) domain, although some include the interceding region (ICR). Protein families listed to the right and are underlined produce mRNAs that are alternatively spliced. Color coding in the genes and the proteins is defined in the legend below; epidermal growth factor domain, EGF. This figure is from (Hanington and Zhang, 2011) with minor modifications and is reprinted from the Journal of Innate Immunity with permission from Karger Publishers. FREPs in gastropods Another approach for generating sequence diversity in immune systems is through gene duplication that generates clustered families of similar genes followed by sequence variation among gene family members, which is driven by pathogen pressure. One example is the freshwater gastropod mollusc, Biomphalaria glabrata, that is an intermediate host for trematode parasites and serves as a vector for terminal infection in birds or mammals including humans (Gordy et al., 2015; Macho and Zipfel, 2015). Snails have 14 families of fibrinogen related protein (FREP) genes that are expressed in response to infection and result in wide arrays of FREPs in their hemolymph (Adema et al., 1997). FREPs are composed of a signal sequence, one or two Ig domains, an interceding region of variable length and sequence, and a conserved fibrinogen related domain (Gordy et al., 2015) (Fig. 6). The gene structure shows great diversity including variations in the Ig exons, changes to the interceding domain exons, deletion of the fibrinogen domain exon, or the absence of all exons except for the fibrinogen domain exon (Fig. 6). In addition to variations in the structure of the genes, FREP2, 3, 4, and 12 gene families in the germ line DNA of B. glabrata appear to be expanded and diversified in hemocyte DNA (Gordy et al., 2015). For example, the estimate for the number of germ line FREP13 genes is about four, whereas the number of different sequences of the first Ig coding region in hemocyte DNA is increased by about 10 fold and may be expanded by somatic recombination (Loker et al., 2004; Zhang et al., 2004; Hanington et al., 2010). The FREP gene families are interesting examples of small gene families that are expanded and diversified in the snail hemocytes (Adema, 2015), however the mechanism by which this occurs is not known. The FREPs highlight the concept that somatic diversification of genes encoding innate immune molecules can occur in (at least some) invertebrates, and therefore is not exclusive to vertebrates. 363 Immune diversity from expanded gene families and/or from expanded alleles per locus Variable region-chitin binding proteins A family of innate immune receptors, the variable region-containing chitin-binding proteins (VCBPs), are present in the marine Protochordates, Branchiostoma floridae and Ciona intestinalis, and encode proteins composed of a leader, two tandem Ig V-type domains, and a chitin-binding domain (Cannon et al., 2002, 2004; Dishaw et al., 2010, 2011); reviewed in Liberti et al. (2015). There are five VCBP genes (A-D) in C. intestinalis, that have little sequence diversity, however, two of the five genes in B. floridae, BfVCBP2 and BfVCBP5, show significant sequence diversity relative to the other three B. floridae genes and all of the VCBP genes in C. intestinalis. The diversity for BfVCBP2 and BfVCBP5 is based on hundreds of alleles in the population in which most of the diversity is in the N- terminal Ig V-type domain. However, there are also indels in non-coding regions and inverted repeats that may influence genomic instability in the region of these loci (Dishaw et al., 2010). Instability may drive sequence diversity that result from gene duplications, gene conversion, recombination and unequal crossing-over that would lead to significant diversity in allelic sequences in B. floridae populations. The V-type domains are likely the site of bacterial binding because both a full-length VCBP protein and a recombinant protein missing the chitin binding domain show similar binding characteristics (Dishaw et al., 2011). None of the four VCBP genes in C. intestinalis nor three of the VCBP genes in B. floridae (BfVCBP1, 3, and 4) show sequence diversity, suggesting that each recognizes stable non-self molecules. VCPB proteins are variably expressed in association with the stomach and intestinal epithelium, are expressed by celomocytes, and appear to mediate immune activities (Dishaw et al., 2011; Liberti et al., 2014). Interactions may result in gastrointestinal homeostasis between host and commensal gut microbes in addition to protection from pathogenic invasion. The VCBP gene family in the Protochordates provides an example of immune detection genes that are both conserved without sequence diversity and others that show significant diversity. Expanded gene families in plants and animals In many cases, genes encoding immune detection and response proteins are simply expanded in the genome of the host organism. This has been evaluated extensively for the variety of disease resistance (R) genes in higher plants (Spoel and Dong, 2012), and may in part be an outcome of shared sequences among R genes that encode the LRR region of the proteins that may drive recombination and meiotic mispairing (McDowell and Simon, 2008; Joshi and Nayak, 2013; Oren et al., 2016) in addition to ectopic duplications and gene recombination (Meyers et al., 2003; Kuang et al., 2004; Smith and Hulbert, 2005). Diversification of R genes also appears to be induced by contact with pathogens that activates mechanisms for non- homologous recombination among the R genes to diversify the sequences with potential to create new recognition capabilities for R proteins in progeny of the infected plant (Durrant et al., 2007). In addition to plants, significantly expanded gene copy numbers have also been identified in the genome of the Pacific oyster, Crassostrea gigas, particularly for complement homologues encoding C1q, fibrinogen domain containing proteins, and C-type lectin domain-containing proteins (Zhang et al., 2015). Gene family expansions for the TLR genes have been documented in sea urchins, B. floridae, and the annelid, Capitella capitata (reviewed in (Buckley and Rast, 2012) (Davidson et al., 2008)) in addition to expansions in a variety of other genes encoding pattern recognition receptors in a wide range or organisms (reviewed in (Buckley and Rast, 2015). Sequence diversity of single copy genes In addition to expansions in the number of members of gene families, some immune genes in invertebrates are present in only a few copies or are single copy yet have significant sequence diversity, which is based on large numbers of alleles with slightly different sequences in the population. One example is the set of linked genes in the fusion/histocompatibility (Fu/HC) locus in the compound tunicate, Botryllus schlosseri. The Fu/HC locus is composed of single copy genes that function in allograft recognition and rejection or fusion among individuals, which is an important life history trait for B. schlosseri. Natural allograft reactions occur commonly when these sessile invertebrates grow into contact with one another resulting in either fusion of their vasculature or rejection and tissue separation (Oka and Watanabe, 1957; Scofield et al., 1982). The responses to non- self are controlled by 1) two, very tightly linked candidate fuhc (cfuhc) genes that encode secreted and transmembrane Fu/HC proteins, 2) fester and uncle fester that encode putative receptors in non- self detection, 3) hsp40-l that encodes a chaperone protein, and 4) the Botryllus histocompatibility factor (bhf) that shows lower allelic polymorphism, encodes a cytoplasmic protein that is partially disordered and is associated with the cytoplasmic face of the plasma membrane (Fig. 7) (Voskoboynic Fig. 7 The Fu/HC locus in Botryllus schlosseri. The Fu/HC locus spans about 305 kb and the orientations and distances between the genes are indicated including the tight linkage between cfuhcsec and cfuhctm. Additional genes are present in this region including nine genes that match to homologues in other organisms with known function and seven with unknown function. This figure is modified from (Taketa and De Tomaso, 2015). 364 et al., 2013, Taketa et al., 2015; reviewed by (Taketa and De Tomaso, 2015)). Diversity of the polymorphic genes in the Fu/HC locus is based on up to hundreds of alleles in the population, of which the most polymorphic are the two cfuhc genes and fester, plus the hsp40-l region that encodes the C- terminus of the protein. Diversity for fester and uncle fester proteins can also be increased through alternative splicing. The examples of immune genes described above for both vertebrates and invertebrates illustrate multiple ways in which to achieve sequence diversity at immune loci, either through the assembly or copying of gene segments into a non-functional single copy gene rendering it functional, through the expansion of genes into clustered families of similar genes, through the presence of many alleles for single copy genes in a population, and/or through extensive alternative splicing. The Sp185/333 system in echinoids Sp185/333 mRNAs The variety of highly variable gene families has shown that different groups of organisms often use different genes with different diversification mechanisms to attain the same biological outcome of host protection from pathogens (Raftos and Raison, 2008; Ghosh et al., 2011). In the purple sea urchin, Strongylocentrotus purpuratus, for which the genome sequence is available (Sodergren et al., 2006), the numbers of immune genes and immune gene families with expanded members illustrate a surprising level of complexity for immune defense in this invertebrate (Hibino et al., 2006; Rast et al., 2006). Significant expansions are present in the TLR family (Buckley and Rast, 2012), the NOD and NALP families (Hibino et al., 2006; Rast et al., 2006; Buckley and Rast, 2015), the genes encoding scavenger receptors with cysteine rich domain (Pancer, 2000; Pancer, 2001), and small C-type lectin genes (Sodergren et al., 2006). Overall, the set of immune genes identified as homologues of those known from vertebrates indicates that the echinoid immune system is quite sophisticated (Hibino et al., 2006; Rast et al., 2006). Another immune response gene family are the Sp185/333 genes that are strongly upregulated in adult coelomocytes and larval blastocelar cells in response to challenge with marine bacteria and lipopolysaccharide (LPS) (Rast et al., 2000; Nair et al., 2005; Ho et al., 2016). The Sp185/333 sequences identified from a cDNA library screen of immune activated celomocytes using a subtracted probe (Nair et al., 2005) matched to only two sequences in GenBank; EST333 [(accession number; R62081 (Smith et al., 1996)] and DD185 [accession number; AF228877 (Rast et al., 2000)]. These two matches are the basis of a combined name for the gene family, Sp185/333, because the sequence provides no prediction for putative function. The paradox of the result is that many of the partial cDNA sequences do not match to each other, however, once alignments were undertaken by hand, patterns of recognizable short regions of sequence emerged called elements (Fig. 8A) (Terwilliger et al., 2006, 2007). Although this appears reminiscent of alternative splicing as in DSCAM from arthropods from sets of duplicated exons (Ng et al., 2014; Brites and du Pasquier, 2015), the Sp185/333 genes are small with only two exons, which rules out the possibility of alternative splicing as a mechanism for sequence diversity (Terwilliger et al., 2006). Careful alignments of genes amplified from three sea urchins show that the element structure of the cDNAs is entirely encoded by the second exon (Fig. 8A) (Buckley and Smith, 2007). Element patterns result from the mosaic of elements that are variably present and absent in the second exon, which impart significant sequence diversity to the mRNAs and deduced proteins. Although, individual elements are recognizable based on length and sequence, the same element from different genes may have slightly different sequences that adds to the diversity of genes and mRNAs with the same element pattern. Analysis of 121 unique Sp185/333 genes from three sea urchins shows that none share sequence among animals, although sequences of some element are shared among genes. In addition to elements, the second exon has six types of repeats that are present as two to four imperfect tandem repeats in the 5′ end of the second exon plus interspersed repeats at the 3′ end (Fig. 8A) (Buckley et al., 2008a). Computational predictions for the origins of the tandem repeats suggest three ancestral versions that underwent duplications, recombinations and deletions to generate the current structure of the 5′ end of the second exon. These characteristics that impart significant sequence diversity within the gene family plus increased expression in response to immunological challenge strongly suggest that these genes function in the immune response of the purple sea urchin (Smith, 2012). It is generally assumed that transcription is a high fidelity process producing mRNAs that reflect exactly the sequence of the coding regions in the genes that has likely been optimized by selection. Consequently, the identification of early stop codons and missense sequences in the Sp185/333 cDNAs were assumed to be the result of pseudogene expression (Terwilliger et al., 2007). However, comparisons among the mRNAs and genes from each of three sea urchins show, very few identical matches (Buckley et al., 2008b). The gene sequences show that pseudogenes (based on coding regions) are not present in the genome (Buckley and Smith, 2007) and that the early stop codons and frame shifts are only observed in the mRNAs. Alignments of genes and mRNAs with the same element pattern from the same sea urchin show that most of the differences are in positions of cytidine in the genes that corresponded to uracil in the mRNAs (Buckley et al., 2008b). Speculations on the basis for these changes include mRNA editing by a cytidine deaminase for the C to U changes, and perhaps polymerase (pol)µ for other nucleotide changes. Both cytidine deaminases and polµ genes are present in the sea urchin genome. It is noteworthy that the most common cDNA sequence element pattern is E2, which is edited at the same nucleotide in many cDNA sequences to generate a truncated protein (Fig. 8A) (Buckley et al., 2008b). 365 Fig. 8 Diversity of the Sp185/333 system in sea urchins. A) The Sp85/333 mRNAs are encoded by the two exons in the genes that code for the leader (L) and the mature protein. The mRNAs are composed of a mosaic of blocks of sequence called elements that are shown as colored rectangles in this cartoon alignment. The combination of elements defines the element pattern that is named according to the distinctive and highly diverse sequence of element 15 (Terwilliger et al., 2006). Element 25 has three versions that are defined by the position of the stop codon. A very common edit site for mRNAs is in element 12 of the E2 element pattern (indicated) and changes a codon to a stop resulting in truncated proteins that are missing the his-rich region. Repeats are shown at the bottom and correspond to the locations of imperfect tandem repeats towards the 5ʹ end of the second exon and the imperfect interspersed repeats in the 3ʹ half of the second exon. This figure is modified from (Buckley and Smith, 2007). B) The gene family locus structure has tightly clustered genes within 34 kb that is positioned 6.1 kb from the end of the bacterial artificial chromosome (BAC) clone insert. Distances between the genes are indicated below. Each gene is surrounded by GA short tandem repeats (STRs) and segmental duplications that include three D1 genes (yellow, green, blue) of nearly identical sequence are surrounded by GAT STRs. The size of the segmental duplications are indicated by brackets above. This figure is modified from (Miller et al., 2010). C) The standard Sp185/333 protein structure has an N-terminal leader (red), a gly-rich region (orange), a his-rich region (blue) and a C-terminal region (gray). The recombinant fragments employed for binding analyses are indicated below. This figure is modified from (Smith and Lun, 2016). 366 Not only does this appear to be non-random, but edited E2 messages encoding truncated proteins are more commonly present prior to immune challenge, whereas after challenge, the preponderance of mRNAs encoding full-length proteins increases (Terwilliger et al., 2007; Sherman et al., 2015). Given the possibility of mRNA editing, the set of mRNAs expressed from a single gene may encode a set of similar but non-identical Sp185/333 proteins. In general, the diversity of the Sp185/333 gene family generates diverse mRNAs that increase diversity by mRNA editing to produce a broad array of Sp185/333 proteins. The Sp185/333 gene family The Sp185/333 gene family structure shows tightly clustered genes that are spaced apart by 3 to 12 kb (Miller et al., 2010). One cluster of six genes is present within 34 kb, with the peripheral genes oriented opposite relative to the internal genes (Fig. 8B). All genes are surrounded by GA short tandem repeats (STRs), or microsatellites, and the regions between the STRs, which includes the genes and flanking regions of the genes, show increased sequence conservation compared to regions that are distal to the GA STRs (Miller et al., 2010). This has been interpreted as a result of gene duplication that is mediated by the GA STRs. Sequence conservation among regions bounded by the GA STRs is also consistent with gene conversion in which sequence exchange is initiated by short regions of shared sequences within the coding regions of the genes and is terminated at the GA STRs. This type of sequence exchange may be similar to observations reported in yeast (Gendrel et al., 2000). The beneficial outcome of limited gene conversion would be sequence diversification of the genes while blocking sequence homogenization of entire gene clusters (Miller et al., 2010). In addition to GA STRs surrounding the genes, GAT STRs are positioned at the edges of three segmental duplications that are located in tandem, are of identical size, nearly identical sequence, and include duplicated genes of the D1 element pattern with almost identical sequence (Fig. 8B). The level of sequence identity suggests very recent GAT STR-mediated duplications within the Sp185/333 gene cluster, which correlates with D1 genes being the most common type based on random gene sequencing from three sea urchins (Buckley and Smith, 2007). Overall, the basis for Sp185/333 gene sequence diversity is likely based on 1) shared blocks of sequences among genes, 2) the presence of STRs within the Sp185/333 gene cluster, and 3) tight clustering of the genes, all of which may drive gene conversion, duplication, deletion and recombination (Miller et al., 2010; Oren et al., 2016). This complex family structure with a variety of repeats is very likely the basis for the poor assembly of the Sp185/333 gene family in the sea urchin genome sequence in which only six genes are present in a single cluster when 50 ± 10 genes per genome have been estimated (Smith, 2012). The Sp185/333 proteins Sequence diversity is a characteristic of the Sp185/333 gene family, which appears to be increased by mRNA editing, and infers great Sp185/333 protein diversity. The edited mRNAs encoding missense sequence has been suggested as another means for increasing protein diversity (Smith, 2012) as missense amino acids in Sp185/333 proteins are present in the sea urchin coelomic fluid (CF) as confirmed by mass spectrometry (Dheilly et al., 2009). The Sp185/333 proteins from the CF appear much larger than expected relative to sizes predicted from the cDNA sequences suggesting the possibility of multimerization (Brockton et al., 2008). This outcome is also observed for a recombinant Sp185/333 protein that appears as monomers, dimers and multimers. Sp185/333 proteins from all sea urchins show multimers, and the repertoires among individual sea urchins are quite different (Brockton et al., 2008; Dheilly et al., 2009; Sherman et al., 2015). When Sp185/333 proteins are evaluated by two dimensional (2D) Western blots, significant diversity is noted, and most of the proteins migrate to the acidic region during isoelectric focusing (Dheilly et al., 2009) (Fig. 9A). However, the presence of many histidines in the C- terminal region of the deduced protein sequences (see Fig. 8C) (Terwilliger et al., 2006) allows isolation of full-length Sp185/333 proteins from the CF of sea urchins by nickel affinity (Sherman et al., 2015; Lun et al., 2016). Consistent with the presence of multiple histidines, most of these nickel- isolated proteins are present in the basic region of a 2D Western blot (Sherman et al., 2015) (Fig. 9B). When arrays of Sp185/333 proteins are compared among sea urchins both before and after challenges with a variety of microbes, there are no similarities among the arrays either among animals in response to the same microbe or in an individual animal responding to the same microbe over multiple challenges (Sherman et al., 2015). Clearly, the diversity of these proteins is very broad within sea urchins and the diversity within the population of sea urchins must be astounding. The characteristics of the Sp185/333 immune response system in sea urchins are based on a number of attributes (Smith, 2012). 1) The differences among the genes in different individual sea urchins (Buckley and Smith, 2007), the structure of the gene family with shared sequences among genes, their tight linkage, and the association with STRs, is likely to promote swift sequence diversity among the genes (Miller et al., 2010; Oren et al., 2016). 2) mRNA editing (Buckley et al., 2008b) increases the sequence diversity of the proteins beyond the diversity encoded in the genes and generates truncated and missense proteins that appear in the CF (Dheilly et al., 2009; Smith, 2012). These truncated proteins may be functional given their propensity to be produced prior to immune challenge (Sherman et al., 2015). 3) The arrays of Sp185/333 proteins are very different among individual sea urchins, and show much greater diversity than expected (Dheilly et al., 2009; Sherman et al., 2015). The great variety of proteins may be due in part to post translational modifications that have been predicted from conserved sequence motifs for glycosylation (Terwilliger et al., 2006; Sherman et al., 2015). In 367 general, these results suggest that the diversity of the Sp185/333 proteins may be highly protective for sea urchins that are in constant contact with microbes in the marine ecosystem. The Sp185/333 proteins are expressed by the phagocyte class of coelomocytes The Sp185/333 proteins are expressed by a subset of the phagocyte class of coelomocytes including the polygonal, discoidal and small phagocytes (Brockton et al., 2008; Majeske et al., 2014). Subsets of each of these cell types have Sp185/333 proteins in small cytoplasmic vesicles that are typically positioned near the nucleus. In addition, small phagocytes have Sp185/333 proteins associated with the surface of the cell. Expression of the Sp185/333 proteins in the adult tissues before vs. after challenge with LPS shows increases in coelomocytes, axial organ, esophagus, pharynx and intestine (Majeske et al., 2013). Sp185/333-positive cells are present in all tissues and significant increases in these cells are noted for coelomocytes and for the axial organ after challenge with LPS (Brockton et al., 2008; Majeske et al., 2013). In larvae, which are free swimming, feeding members of the zooplankton, Sp185/333 gene expression is limited to a subset of differentiated blastocoelar cells called phagocytic filopodial cells (Ho et al., 2016) that have similar phagocytic functions as the discoidal and polygonal phagocytes in the adult. One of the tenets of an effective innate immune response is a quick response to the detection of pathogens before they have time to undergo significant proliferation that may overwhelm host immunity. This can be accomplished by swift up- regulation of the host immune response genes and the production of the encoded effector proteins. This has been documented for the Sp185/333 genes in both adult and larval immune cells responding to injected bacteria or microbes in the seawater (Silva, 2000; Nair et al., 2005; Terwilliger et al., 2007; Furukawa et al., 2009; Ho et al., 2016). It follows that the multiple Sp185/333 genes would be expressed simultaneously in cells to optimize the numbers of effector proteins that can be produced and secreted over time. However, evaluation of Sp185/333 gene expression in single adult phagocytes shows mRNAs of a single sequence per cell, inferring expression from a single gene (Majeske et al., 2014). This result suggests a complex mechanism for selecting the gene that is expressed and for silencing the others that is quite unexpected for an invertebrate innate immune response. It is expected that mRNA editing will result in a small set of proteins of the same element pattern but with slightly different sequence will be produced by single phagocytes. However, the wide range of Sp185/333 protein diversity observed for individual sea urchins (see below) will require that large numbers of phagocytes express the Sp185/333 genes. A recombinant Sp185/333 protein has multiple anti- pathogen activities Hundreds of different isoforms of the Sp185/333 proteins are expressed by individual sea urchins in response to immune challenge, and they Fig. 9 The diversity of the native Sp185/333 proteins are characterized by 2D Western blots. A) Native Sp185/333 proteins from the CF of S. purpuratus show the diversity, but also the preponderance of proteins in the acidic pI range. The figure is from (Dheilly et al., 2009) and is reprinted from the Journal of Immunology with permission from the American Association of Immunologists, Inc., Copyright 2009. B) When full- length, native Sp185/333 proteins with sufficient histidines are isolated by nickel affinity, these proteins are generally present in the basic pI range. This figure is republished from (Sherman et al., 2015). In both cases, the sizes of most of the proteins are larger than expected compared to sizes deduced from cDNAs and genes. may function synergistically in response to the challenge. Understanding the activities of these proteins is essential for understanding the sea urchin immune system, however this requires that individual Sp185/333 isoforms be isolated away from other versions and from other proteins in the CF. Consequently, a recombinant Sp185/333 protein called rSp0032 with an E1 element pattern (see Fig. 8A) was evaluated for predicted anti- microbial activities (Lun et al., 2016). When rSp0032 is incubated with Vibrio diazotrophicus, Bacillus subtilis, B. cereus, or Saccharomyces cerevisiae, the protein shows saturation binding to Vibrio and Saccharomyces, but no binding is observed for the Bacillus species (Fig. 10). Affinity for Vibrio and Saccharomyces is high, binding sites are specific, and once bound rSp0032 cannot be dissociated from the target cells. Mass spectrometry of protein 368 bands from gels used to analyze rSp0032 incubation with Vibrio target cells shows an association between rSp0032 and flagellin indicating that binding may be mediated through pathogen associated molecular patterns (PAMPs). Expanded PAMP binding analysis shows that rSp0032 also binds tightly with flagellin from Salmonella, LPS from E. coli, and β,1-3,glucan from Saccharomyces, but does not bind to peptidoglycan (PGN) from Bacillus. This suggests that there are multiple types of foreign cells to which rSp0032 can bind, that it does not bind indiscriminately to all foreign cells, and that it uses several PAMPs as specific binding targets that have very different molecular characteristics. Despite the diversity that has been documented for the full-length Sp185/333 proteins, they have a standard structure with an N-terminal glycine rich (gly-rich) region, a central region that is the C- terminal end of the gly-rich (C-gly) region that has an arginine-glycine-aspartic acid motif (which has not been tested for integrin binding), a more C- terminal histidine rich (his-rich) region, and a C- terminal region (Fig. 8C). Based on the significant differences in the amino acid content of the gly-rich and his-rich regions, in addition to observations that edited mRNAs (Buckley et al., 2008b) encoding truncated proteins missing the his-rich region are more prevalent prior to immune challenge (Terwilliger et al., 2007; Sherman et al., 2015), these two regions of the proteins are expected to have different activities or functions. Consequently, when tested, the recombinant fragments of the full- length rSp0032 (rGly-rich, rC-Gly, rHis-rich fragments; Fig. 8C) show differences in activities compared to rSp0032 (Lun et al., 2016). All recombinants bind to all foreign target cells including Bacillus species to which rSp0032 does not bind indicating a broadening of binding characteristics (Fig. 9). Furthermore, the rC-Gly fragment multimerizes in the presence or absence of binding targets in agreement with the rGly-rich and rHis-rich fragments, which do not include the C- gly region and do not multimerize. The binding specificity of the rHis-rich fragment for yeast is identical to that of the full-length protein, however, the rGly-rich fragment shows expanded binding compared to either the full-length protein or the rHis- rich fragment. These results indicate several noteworthy aspects of the binding activities that include 1) rSp0032 binds specifically and with high affinity to quite different foreign cell targets, 2) rSp0032 binds to multiple PAMPs that have very different characteristics, 3) the central C-gly region mediates multimerization, 4) the gly-rich and his-rich regions of the protein have different activities, and 5) when all regions of the full-length protein function together, they show increased specificity for binding targets. The binding characteristics of the rGly-rich fragment predict broadened binding activities of truncated Sp185/333 proteins that are not characteristic of either the his-rich region or the full- length protein. This suggests interesting possibilities for truncated proteins, which are more likely to be present prior to challenge, and may be important for immune surveillance. Fig. 10 Multitasking binding characteristics of rSp0032 and the recombinant fragments to microbes and PAMPs. rSp0032 (to the left) is composed of a gly-rich region (orange) including the C-terminal end of the gly-rich region (red), a his-rich region (blue), and a C-terminal region (gray). The recombinant fragments that correspond to the regions of the full-length protein are shown to the right in corresponding colors. The binding targets against which rSp0032 and the recombinant fragments were tested are shown in the middle. Colored arrows indicate binding, whereas black blocked lines indicate no binding. The major differences in binding is to Bacillus and to peptidoglycan (PGN) from Bacillus, which are highlighted with the red box. The recombinant fragments were not tested against the PAMPs. The rC-Gly fragment also mediates dimerization and multimerization of the full-length protein. The figure was modified from (Lun et al., 2016; Smith and Lun, 2016). 369 When the multitasking activities of rSp0032 are applied to the hundreds of native Sp185/333 proteins that are expressed by coelomocytes, this has vast implications for the sea urchin immune response and its activities in host protection. If all or most of the Sp185/333 proteins have multitasking binding activities with slightly different but perhaps overlapping targets to which they bind, and many or all of the isoforms function synergistically, the outcome is a highly diverse and flexible innate immune effector response directed at controlling attack and infection from foreign microbial cells. If the expressed arrays of Sp185/333 proteins have broad binding capabilities, it is highly unlikely that microbes will be able to circumvent or defeat the Sp185/333 system because it would likely require simultaneous changes to multiple characteristics of the microbes and their PAMPs. The Sp185/333 system, including the sequence diversity noted in the members of the gene family, the (putative) directed editing of mRNAs that are translated to truncated and missense proteins, and the extraordinarily diverse arrays of proteins that are expressed among individual sea urchins strongly suggests that this system represents the central immune diversification system for echinoids. Conclusion The common theme reviewed here describes host immune responses to microbial pathogens and the abilities to drive diversity at one or more levels to produce broad sequence variations among immune proteins that function in detection of foreign cells and effector proteins that act to remove and/or kill the invaders. There are commonalities among animals for detecting foreign molecules including the TLR and NOD proteins among others, however, the wide variety of effector proteins and their modes of action make different immune systems appear to function quite differently. The underlying commonality is that although mechanisms to generate immune diversity are vastly different, ranging from RAG-induced somatic recombination and AID-mediated gene assembly in the vertebrate Ig and VLR systems respectively (Tonegawa, 1983; Davis et al., 1984; Nagawa et al., 2007; Rogozin et al., 2007), to extensive alternative splicing of DSCAM genes in arthropods (Schmucker and Chen, 2009; Ng et al., 2014; Brites and du Pasquier, 2015), to clusters of duplicated genes with shared sequences that drive gene recombination, deletion, duplication (Oren et al., 2016), the outcome for effective immunity and survival of organisms is to stay ahead of the pathogens. 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