Variable Region-containing Chitin-binding Proteins Research Articles

Secreted immunoglobulin domain effector molecules of invertebrates and management of gut microbial ecology.

The origins of a "pass-through" gut in early bilaterians facilitated the exploration of new habitats, motivated the innovation of feeding styles and behaviors, and helped drive the evolution of more complex organisms. The gastrointestinal tract has evolved to consist of a series of interwoven exchanges between nutrients, host immunity, and an often microbe-rich environmental interface. Not surprisingly, animals have expanded their immune repertoires to include soluble effectors that can be secreted into luminal spaces, e.g., in the gut, facilitating interactions with microbes in ways that influence their settlement dynamics, virulence, and their interaction with other microbes. The immunoglobulin (Ig) domain, which is also found in some non-immune molecules, is recognized as one of the most versatile recognition domains lying at the interface of innate and adaptive immunity; among vertebrates, secreted Igs are known to play crucial roles in the management of gut microbial communities. In this mini-review, we will focus on secreted immune effectors possessing Ig-like domains in invertebrates, such as the fibrinogen-related effector proteins first described in the gastropod Biomphalaria glabrata, the Down syndrome cellular adhesion molecule first described in the arthropod, Drosophila melanogaster, and the variable region-containing chitin-binding proteins of the protochordates. We will highlight our current understanding of their function and their potential role, if not yet recognized, in the establishment and maintenance of host-microbial interfaces and argue that these Igs are likely also essential to microbiome management.

A Role for Secreted Immune Effectors in Microbial Biofilm Formation Revealed by Simple In Vitro Assays.

The formation of biofilms is critical for the successful and stable colonization of mucosal surfaces by microbes, which often build three-dimensional environments by exuding exopolysaccharides and other macromolecules such as proteins, lipids, and even DNA. It is not just bacteria, but fungi such as yeast, that form these adherent interacting communities. Historically, biofilms have been studied in the context of pathogenesis, but only recently it has been recognized that important relationships among members of host-associated microbiomes are maintained within the context of biofilms. Host immune responses impact biofilm formation in various ways; for example, it is likely that formation of stable biofilms by non-pathogens improves barrier defenses by not just filling available niche spaces but also by helping to ward off pathogens directly. Recently, it was found that soluble immune effector molecules such as immunoglobulin A (IgA) in mammals serve essential roles in modulating complex biofilm communities in ways that benefit the host. Additional lines of evidence from other secreted immune effectors, such as the variable region-containing chitin-binding proteins (VCBPs) in protochordates, now suggest that this phenomenon is much more widespread than previously recognized. The activity of these immune molecules also likely serves roles beyond those of simple defense strategies; rather, they may be improving the outcome of symbiotic interactions benefiting the host. Thus, traditional immune assays that are aimed at studying the function of secreted immune effectors, such as agglutination assays, should take into account the possibility that the first observation may not be the last if the microbes under study are not directly killed. Here, we describe a series of simple approaches to characterize biofilm formation when bacteria (or yeast) are cultured in the presence of a secreted immune effector. To model this approach, we use microbes isolated from the gut of Ciona robusta, each grown in the presence or absence of VCBPs. The approaches defined here are amenable to diverse model systems and their microbes.

Peptide receptors and immune-related proteins expressed in the digestive system of a urochordate, Ciona intestinalis.

The digestive system is responsible for nutrient intake and defense against pathogenic microbes. Thus, identification of regulatory factors for digestive functions and immune systems is a key step to the verification of the life cycle, homeostasis, survival strategy and evolutionary aspects of an organism. Over the past decade, there have been increasing reports on neuropeptides, their receptors, variable region-containing chitin-binding proteins (VCBPs) and Toll-like receptors (TLRs) in the ascidian, Ciona intestinalis. Mass spectrometry-based peptidomes and genome database-searching detected not only Ciona orthologs or prototypes of vertebrate peptides and their receptors, including cholecystokinin, gonadotropin-releasing hormones, tachykinin, calcitonin and vasopressin but also Ciona-specific neuropeptides including Ci-LFs and Ci-YFVs. The species-specific regulation of GnRHergic signaling including unique signaling control via heterodimerization among multiple GnRH receptors has also been revealed. These findings shed light on the remarkable significance of ascidians in investigations of the evolution and diversification of the peptidergic systems in chordates. In the defensive systems of C. intestinalis, VCBPs and TLRs have been shown to play major roles in the recognition of exogenous microbes in the innate immune system. These findings indicate both common and species-specific functions of the innate immunity-related molecules between C. intestinalis and vertebrates. In this review article, we present recent advances in molecular and functional features and evolutionary aspects of major neuropeptides, their receptors, VCBPs and TLRs in C. intestinalis.

A Soluble Immune Effector Binds Both Fungi and Bacteria via Separate Functional Domains.

The gut microbiome of animals consists of diverse microorganisms that include both prokaryotes and eukaryotes. Complex interactions occur among these inhabitants, as well as with the immune system of the host, and profoundly influence the overall health of both the host and its microbial symbionts. Despite the enormous importance for the host to regulate its gut microbiome, the extent to which animals generate immune-related molecules with the capacity to directly influence polymicrobial interactions remains unclear. The urochordate, Ciona robusta, is a model organism that has been adapted to experimental studies of host/microbiome interactions. Ciona variable-region containing chitin-binding proteins (VCBPs) are innate immune effectors, composed of immunoglobulin (Ig) variable regions and a chitin-binding domain (CBD) and are expressed in high abundance in the gut. It was previously shown that VCBP-C binds bacteria and influences both phagocytosis by granular amoebocytes and biofilm formation via its Ig domains. We show here that the CBD of VCBP-C independently recognizes chitin molecules present in the cell walls, sporangia (spore-forming bodies), and spores of a diverse set of filamentous fungi isolated from the gut of Ciona. To our knowledge, this is the first description of a secreted Ig-containing immune molecule with the capacity to directly promote transkingdom interactions through simultaneous binding by independent structural domains and could have broad implications in modulating the establishment, succession, and homeostasis of gut microbiomes.

Immunity in Protochordates: The Tunicate Perspective.

Tunicates are the closest relatives of vertebrates, and their peculiar phylogenetic position explains the increasing interest toward tunicate immunobiology. They are filter-feeding organisms, and this greatly influences their defense strategies. The majority of the studies on tunicate immunity were carried out in ascidians. The tunic acts as a first barrier against pathogens and parasites. In addition, the oral siphon and the pharynx represent two major, highly vascularized, immune organs, where circulating hemocytes can sense non-self material and trigger immune responses that, usually, lead to inflammation and phagocytosis. Inflammation involves the recruitment of circulating cytotoxic, phenoloxidase (PO)-containing cells in the infected area, where they degranulate as a consequence of non-self recognition and release cytokines, complement factors, and the enzyme PO. The latter, acting on polyphenol substrata, produces cytotoxic quinones, which polymerize to melanin, and reactive oxygen species, which induce oxidative stress. Both the alternative and the lectin pathways of complement activation converge to activate C3: C3a and C3b are involved in the recruitment of hemocytes and in the opsonization of foreign materials, respectively. The interaction of circulating professional phagocytes with potentially pathogenic foreign material can be direct or mediated by opsonins, either complement dependent or complement independent. Together with cytotoxic cells, phagocytes are active in the encapsulation of large materials. Cells involved in immune responses, collectively called immunocytes, represent a large fraction of hemocytes, and the presence of a cross talk between cytotoxic cells and phagocytes, mediated by secreted humoral factors, was reported. Lectins play a pivotal role as pattern-recognition receptors and opsonizing agents. In addition, variable region-containing chitin-binding proteins, identified in the solitary ascidian Ciona intestinalis, control the settlement and colonization of bacteria in the gut.

Gut immunity in a protochordate involves a secreted immunoglobulin-type mediator binding host chitin and bacteria.

Protochordate variable region-containing chitin-binding proteins (VCBPs) consist of immunoglobulin-type V domains and a chitin-binding domain (CBD). VCBP V domains facilitate phagocytosis of bacteria by granulocytic amoebocytes; the function of the CBD is not understood. Here we show that the gut mucosa of Ciona intestinalis contains an extensive matrix of chitin fibrils to which VCBPs bind early in gut development, before feeding. Later in development, VCBPs and bacteria colocalize to chitin-rich mucus along the intestinal wall. VCBP-C influences biofilm formation in vitro and, collectively, the findings of this study suggest that VCBP-C may influence the overall settlement and colonization of bacteria in the Ciona gut. Basic relationships between soluble immunoglobulin-type molecules, endogenous chitin and bacteria arose early in chordate evolution and are integral to the overall function of the gut barrier.

An Immune Effector System in the Protochordate Gut Sheds Light on Fundamental Aspects of Vertebrate Immunity.

A variety of germline and somatic immune mechanisms have evolved in vertebrate and invertebrate species to detect a wide array of pathogenic invaders. The gut is a particularly significant site in terms of distinguishing pathogens from potentially beneficial microbes. Ciona intestinalis, a filter-feeding marine protochordate that is ancestral to the vertebrate form, possesses variable region-containing chitin-binding proteins (VCBPs), a family of innate immune receptors, which recognize bacteria through an immunoglobulin-type variable region. The manner in which VCBPs mediate immune recognition appears to be related to the development and bacterial colonization of the gut, and it is likely that these molecules are critical elements in achieving overall immune and physiological homeostasis.

Expression of Ciona intestinalis variable region-containing chitin-binding proteins during development of the gastrointestinal tract and their role in host-microbe interactions.

Variable region-containing chitin-binding proteins (VCBPs) are secreted, immune-type molecules that have been described in both amphioxus, a cephalochordate, and sea squirt, Ciona intestinalis, a urochordate. In adult Ciona, VCBP-A, -B and -C are expressed in hemocytes and the cells of the gastrointestinal tract. VCBP-C binds bacteria in the stomach lumen and functions as an opsonin in vitro. In the present paper the expression of VCBPs has been characterized during development using in situ hybridization, immunohistochemical staining and quantitative polymerase chain reaction (qPCR) technologies. The expression of VCBP-A and -C is detected first in discrete areas of larva endoderm and becomes progressively localized during differentiation in the stomach and intestine, marking the development of gut tracts. In “small adults” (1–2 cm juveniles) expression of VCBP-C persists and VCBP-A gradually diminishes, ultimately replaced by expression of VCBP-B. The expression of VCBP-A and -C in stage 7–8 juveniles, at which point animals have already started feeding, is influenced significantly by challenge with either Gram-positive or -negative bacteria. A potential role for VCBPs in gut-microbiota interactions and homeostasis is indicated.

The amphioxus genome provides unique insight into the evolution of immunity

Immune systems evolve as essential strategies to maintain homeostasis with the environment, prevent microbial assault and recycle damaged host tissues. The immune system is composed of two components, innate and adaptive immunity. The former is common to all animals while the latter consists of a vertebrate-specific system that relies on somatically derived lymphocytes and is associated with near limitless genetic diversity as well as long-term memory. Deuterostome invertebrates provide a view of immune repertoires in phyla that immediately predate the origins of vertebrates. Genomic studies in amphioxus, a cephalochordate, have revealed homologs of genes encoding most innate immune receptors found in vertebrates; however, many of the gene families have undergone dramatic expansions, greatly increasing the innate immune repertoire. In addition, domain-swapping accounts for the innovation of new predicted pathways of receptor function. In both amphioxus and Ciona, a urochordate, the VCBPs (variable region containing chitin-binding proteins), which consist of immunoglobulin V (variable) and chitin binding domains, mediate recognition through the V domains. The V domains of VCBPs in amphioxus exhibit high levels of allelic complexity that presumably relate to functional specificity. Various features of the amphioxus immune repertoire reflect novel selective pressures, which likely have resulted in innovative strategies. Functional genomic studies underscore the value of amphioxus as a model for studying innate immunity and may help reveal how unique relationships between innate immune receptors and both pathogens and symbionts factored in the evolution of adaptive immune systems.

A Basal Chordate Model for Studies of Gut Microbial Immune Interactions

Complex symbiotic interactions at the surface of host epithelia govern most encounters between host and microbe. The epithelium of the gut is a physiologically ancient structure that is comprised of a single layer of cells and is thought to possess fully developed immunological capabilities. Ciona intestinalis (sea squirt), which is a descendant of the last common ancestor of all vertebrates, is a potentially valuable model for studying barrier defenses and gut microbial immune interactions. A variety of innate immunological phenomena have been well characterized in Ciona, of which many are active in the gut tissues. Interactions with gut microbiota likely involve surface epithelium, secreted immune molecules including variable region-containing chitin-binding proteins, and hemocytes from a densely populated laminar tissue space. The microbial composition of representative gut luminal contents has been characterized by molecular screening and a potentially relevant, reproducible, dysbiosis can be induced via starvation. The dialog between host and microbe in the gut can be investigated in Ciona against the background of a competent innate immune system and in the absence of the integral elements and processes that are characteristic of vertebrate adaptive immunity.

A role for variable region-containing chitin-binding proteins (VCBPs) in host gut–bacteria interactions

A number of different classes of molecules function as structural matrices for effecting innate and adaptive immunity. The most extensively characterized mediators of adaptive immunity are the immunoglobulins and T-cell antigen receptors found in jawed vertebrates. In both classes of molecules, unique receptor specificity is effected through somatic variation in the variable (V) structural domain. V region-containing chitin-binding proteins (VCBPs) consist of two tandem Ig V domains as well as a chitin-binding domain. VCBPs are encoded at four loci (i.e., VCBPA-VCBPD) in Ciona, a urochordate, and are expressed by distinct epithelial cells of the stomach and intestine, as well as by granular amoebocytes present in the lamina propria of the gut and in circulating blood. VCBPs are secreted into the gut lumen, and direct binding to bacterial surfaces can be detected by immunogold analysis. Affinity-purified native and recombinant VCBP-C, as well as a construct consisting only of the tandem V domains, enhance bacterial phagocytosis by granular amoebocytes in vitro. Various aspects of VCBP expression and function suggest an early origin for the key elements that are central to the dialogue between the immune system of the host and gut microflora.

The basis for haplotype complexity in VCBPs, an immune-type receptor in amphioxus

Innate immune gene repertoires are restricted primarily to germline variation. Adaptive immunity, by comparison, relies on somatic variation of germline-encoded genes to generate extraordinarily large numbers of non-heritable antigen recognition motifs. Invertebrates lack the key features of vertebrate adaptive immunity, but have evolved a variety of alternative mechanisms to successfully protect the integrity of "self"; in many cases, these appear to be taxon-specific innovations. In the protochordate Branchiostoma floridae (amphioxus), the variable region-containing chitin-binding proteins (VCBPs) constitute a multigene family (comprised of VCBPs 1-5), which possesses features that are consistent with innate immune-type function. A large number of VCBP alleles and haplotypes are shown to exhibit levels of polymorphism exceeding the elevated overall levels determined for the whole amphioxus genome (JGI). VCBP genes of the 2 and 5 types are distinguished further by a highly polymorphic segment (exon 2) in the N-terminal immunoglobulin domain, defined previously as a "hypervariable region" or a "hotspot." Genomic deoxyribonucleic acid (DNA) and complementary DNA (cDNA) sequences from large numbers of animals representing different populations reveal further significant differences in sequence complexity within and across VCBP2/5 haplotypes that arise through overlapping mechanisms of genetic exchange, gene copy number variation as well as mutation and give rise to distinct allelic lineages. The collective observations suggest that mechanisms were in place at the time of divergence of the cephalochordates that could selectively hyperdiversify immune-type receptors within a multigene family.

Plasticity of the immunoglobulin domain in the evolution of immunity

Immune receptors are omnipresent in multicellular organisms and comprise a vast array of molecular structures that serve to detect and eliminate pathogenic threats. The immunoglobulin (Ig) domain, a central structural feature of the antigen binding receptors that mediate adaptive immunity in jawed vertebrates, appears to play a particularly widespread role in metazoan immunity. Recent reports also have implicated Ig domains in the immune responses of protostomes such as flies and snails. Our research has focused on understanding the utilization of the Ig domain in the immunity of chordates and has identified numerous multigene families of Ig domain-containing receptors that appear to serve roles distinct from the adaptive antigen-binding receptors. Three families have received particular focus: novel immune-type receptors (NITRs) of bony fish, modular domain immune-type receptors (MDIRs) of cartilaginous fish and variable region-containing chitin-binding proteins (VCBPs) of amphioxus. NITRs and MDIRs are encoded in large multigene families of highly diversified forms and exhibit a striking dichotomy of an apparently ubiquitous presence but extensive diversification of sequence both within and among the particular taxonomic groups in which they are found. Crystal structures of VCBPs and NITRs demonstrate significant similarity to those of antigen-binding receptors but at the same time exhibit key differences that imply acquisition of separate and distinct ligand-binding functions. The tremendous plasticity of the Ig domain makes it a strong focus for studies of evolutionary events that have shaped modern integrated immune systems. Current data are consistent with a model of extremely rapid emergence and divergence of immune receptors, perhaps specific to individual species, as organisms contend with environments in which pathogens are continually selected for variation of their own molecular signatures.

Genomic complexity of the variable region-containing chitin-binding proteins in amphioxus.

BackgroundThe variable region-containing chitin-binding proteins (VCBPs) are found in protochordates and consist of two tandem immunoglobulin variable (V)-type domains and a chitin-binding domain. We previously have shown that these polymorphic genes, which primarily are expressed in the gut, exhibit characteristics of immune genes. In this report, we describe VCBP genomic organization and characterize adjacent and intervening genetic features which may influence both their polymorphism and complex transcriptional repertoire.ResultsVCBP genes 1, 2, 4, and 5 are encoded in a single contiguous gene-rich chromosomal region and VCBP3 is encoded in a separate locus. The VCBPs exhibit extensive haplotype variation, including copy number variation (CNV), indel polymorphism and a markedly elevated variation in repeat type and density. In at least one haplotype, inverted repeats occur more frequently than elsewhere in the genome. Multi-animal cDNA screening, as well as transcriptional profilingusing a novel transfection system, suggests that haplotype-specific transcriptional variants may contribute to VCBP genetic diversity.ConclusionThe availability of the Branchiostoma floridae genome (Joint Genome Institute, Brafl1), along with BAC and PAC screening and sequencing described here, reveal that the relatively limited number of VCBP genes present in the amphioxus genome exhibit exceptionally high haplotype variation. These VCBP haplotypes contribute a diverse pool of allelic variants, which includes gene copy number variation, pseudogenes, and other polymorphisms, while contributing secondary effects on gene transcription as well.

Immunoglobulin variable regions in molecules exhibiting characteristics of innate and adaptive immune receptors

The antigen combining sites of immunoglobulin (Ig) and T cell antigen receptors (TCRs), which are present in all jawed vertebrates, consist of a paired variable (V) domain heterodimer that exhibits varying degrees of germline- and extraordinarily high levels of somatically-derived variation. The near limitless variation in receptor specificity on the surface of individual lymphocytes is the basis for clonal selection in the adaptive immune response. A basic question arises as to whether or not there are other forms of immune-type receptors in vertebrates as well as in invertebrates that derive immune specificity through sequence differences in V domains. Our laboratory has discovered two such families of molecules, the novel immune-type receptors and the variable region-containing chitin-binding proteins. Both families of molecules encode V domains that share some characteristics of adaptive immune receptors but likely mediate innate functions.

Ancient evolutionary origin of diversified variable regions demonstrated by crystal structures of an immune-type receptor in amphioxus

Although the origins of genes encoding the rearranging binding receptors remain obscure, it is predicted that their ancestral forms were nonrearranging immunoglobulin-type domains. Variable region-containing chitin-binding proteins (VCBPs) are diversified immune-type molecules found in amphioxus (Branchiostoma floridae), an invertebrate that diverged early in deuterostome phylogeny. To study the potential evolutionary relationships between VCBPs and vertebrate adaptive immune receptors, we solved the structures of both a single V-type domain (to 1.15 A) and a pair of V-type domains (to 1.85 A) from VCBP3. The deduced structures show integral features of the ancestral variable-region fold as well as unique features of variable-region pairing in molecules that may reflect characteristics of ancestral forms of diversified immune receptors found in modern-day vertebrates.