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.

Reconstructing immune phylogeny: new perspectives.

Numerous studies of the mammalian immune system have begun to uncover profound interrelationships, as well as fundamental differences, between the adaptive and innate systems of immune recognition. Coincident with these investigations, the increasing experimental accessibility of non-mammalian jawed vertebrates, jawless vertebrates, protochordates and invertebrates has provided intriguing new information regarding the likely patterns of emergence of immune-related molecules during metazoan phylogeny, as well as the evolution of alternative mechanisms for receptor diversification. Such findings blur traditional distinctions between adaptive and innate immunity and emphasize that, throughout evolution, the immune system has used a remarkably extensive variety of solutions to meet fundamentally similar requirements for host protection.

Recognition of additional roles for immunoglobulin domains in immune function.

Characterization of immune receptors found in phylogenetically disparate species at the genetic, structural and functional levels has provided unique insight into the evolutionary acquisition of immune function. The roles of variable- and intermediate-type immunoglobulin (Ig) domains in direct recognition of ligands and other functions are far wider than previously anticipated. Common mechanisms of multigene family diversification and expansion as well as unique adaptations that relate to function continue to provide unique insight into the numerous patterns, processes and complex interactions that regulate the host response to infectious challenge.

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.

Associations of Microbial Diversity with Age and Other Clinical Variables among Pediatric Chronic Rhinosinusitis (CRS) Patients.

Chronic rhinosinusitis (CRS) is a heterogenous disease that causes persistent paranasal sinus inflammation in children. Microorganisms are thought to contribute to the etiology and progression of CRS. Culture-independent microbiome analysis offers deeper insights into sinonasal microbial diversity and microbe-disease associations than culture-based methods. To date, CRS-related microbiome studies have mostly focused on the adult population, and only one study has characterized the pediatric CRS microbiome. In this study, we analyzed the bacterial diversity of adenoid tissue, adenoid swab, maxillary sinus, and sinus wash samples from 45 pediatric CRS patients recruited from the Johns Hopkins All Children's Hospital (JHACH) in St. Petersburg, FL, USA. The alpha diversity in these samples was associated with baseline nasal steroid use, leukotriene receptor antagonist (LTRA) use, and total serum immunoglobulin (Ig) E (IgE) level. Streptococcus, Moraxella, and Haemophilus spp. were most frequently identified from sinus cultures and the sequenced 16S rRNA gene content. Comparative analyses combining our samples with the samples from the previous microbiome study revealed differentially abundant genera between patients with pediatric CRS and healthy controls, including Cutibacterium and Moraxella. Additionally, the abundances of Streptobacillus and Staphylococcus were consistently correlated with age in both adenoid- and sinus-derived samples. Our study uncovers new associations of alpha diversity with clinical parameters, as well as associations of specific genera with disease status and age, that can be further investigated.

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.

Reflections on the Use of an Invertebrate Chordate Model System for Studies of Gut Microbial Immune Interactions.

The functional ecology of the gastrointestinal tract impacts host physiology, and its dysregulation is at the center of various diseases. The immune system, and specifically innate immunity, plays a fundamental role in modulating the interface of host and microbes in the gut. While humans remain a primary focus of research in this field, the use of diverse model systems help inform us of the fundamental principles legislating homeostasis in the gut. Invertebrates, which lack vertebrate-style adaptive immunity, can help define conserved features of innate immunity that shape the gut ecosystem. In this context, we previously proposed the use of a marine invertebrate, the protochordate Ciona robusta, as a novel tractable model system for studies of host-microbiome interactions. Significant progress, reviewed herein, has been made to fulfill that vision. We examine and review discoveries from Ciona that include roles for a secreted immune effector interacting with elements of the microbiota, as well as chitin-rich mucus lining the gut epithelium, the gut-associated microbiome of adults, and the establishment of a large catalog of cultured isolates with which juveniles can be colonized. Also discussed is the establishment of methods to rear the animals germ-free, an essential technology for dissecting the symbiotic interactions at play. As the foundation is now set to extend these studies into the future, broadening our comprehension of how host effectors shape the ecology of these microbial communities in ways that establish and maintain homeostasis will require full utilization of "multi-omics" approaches to merge computational sciences, modeling, and experimental biology in hypothesis-driven investigations.

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.

Alternative mechanisms of immune receptor diversity.

Our views of both innate and adaptive immunity have been significantly modified by recent studies of immune receptors and immunity in protostomes, invertebrate deuterostomes, and jawless vertebrates. Extraordinary variation in the means whereby organisms recognize pathogens has been revealed by a series of recent findings, including: novel forms of familiar immune receptors, high genetic polymorphism for new receptor types, germline rearrangement for non-Ig domain receptors, somatic variation of germline-encoded receptors, and unusually complex alternative splicing of genes with both immune and non-immune roles. Collectively, these observations underscore heretofore unrecognized pathways in the evolution of immune recognition and suggest universal processes by which immune systems co-opt and integrate existing cellular mechanisms to effect diverse recognition functions.

The development of intestinal dysbiosis in anemic preterm infants.

OBJECTIVE: Anemia and Proteobacteria-dominant intestinal dysbiosis in very low birth weight (VLBW) infants have been linked to necrotizing enterocolitis, a severe gut inflammatory disease. We hypothesize that anemia of prematurity is related to the development of intestinal dysbiosis. STUDY DESIGN: Three hundred and forty-two weekly stool samples collected prospectively from 80 VLBW infants were analyzed for bacterial microbiomes (with 16S rRNA). Linear mixed-effects model was used to determine the relationships between the onsets of anemia and intestinal dysbiosis. RESULTS: Hematocrit was associated with intestinal microbiomes, with lower Hct occurring with increased Proteobacteria and decreased Firmicutes. Infants with a hematocrit <30% had intestinal microbiomes that diverged toward Proteobacteria dominance and low diversity after the first postnatal month. The microbiome changes were also related to the severity of anemia. CONCLUSIONS: This finding supports a potential microbiological explanation for anemia as a risk factor for intestinal dysbiosis in preterm infants.

Predicted Metabolic Pathway Distributions in Stool Bacteria in Very-Low-Birth-Weight Infants: Potential Relationships with NICU Faltered Growth.

Many very-low-birth-weight (VLBW) infants experience growth faltering in early life despite adequate nutrition. Early growth patterns can affect later neurodevelopmental and anthropometric potentials. The role of the dysbiotic gut microbiome in VLBW infant growth is unknown. Eighty-four VLBW infants were followed for six weeks after birth with weekly stool collection. DNA was extracted from samples and the V4 region of the 16S rRNA gene was sequenced with Illumina MiSeq. A similar microbiota database from full-term infants was used for comparing gut microbiome and predicted metabolic pathways. The class Gammaproteobacteria increased or remained consistent over time in VLBW infants. Out of 228 metabolic pathways that were significantly different between term and VLBW infants, 133 pathways were significantly lower in VLBW infants. Major metabolic differences in their gut microbiome included pathways involved in decreased glycan biosynthesis and metabolism, reduced biosynthetic capacity, interrupted amino acid metabolism, changes that could result in increased infection susceptibility, and many other system deficiencies. Our study reveals poor postnatal growth in a VLBW cohort who had dysbiotic gut microbiota and differences in predicted metabolic pathways compared to term infants. The gut microbiota in VLBW infants likely plays an important role in postnatal growth.

Contributors to Dysbiosis in Very-Low-Birth-Weight Infants.

The objective of this commentary was to analyze the causes and outcomes of gut microbiome dysbiosis in preterm infants who are born at very low birth weight (VLBW). The intrauterine development of VLBW infants is interrupted abruptly with preterm birth and followed by extrauterine, health-threatening conditions and sequelae. These infants develop intestinal microbial dysbiosis characterized by low diversity, an overall reduction in beneficial and/or commensal bacteria, and enrichment of opportunistic pathogens of the Gammaproteobacteria class. The origin of VLBW infant dysbiosis is not well understood and is likely the result of a combination of immaturity and medical care. We propose that these factors interact to produce inflammation in the gut, which further perpetuates dysbiosis. Understanding the sources of dysbiosis could result in interventions to reduce gut inflammation, decrease enteric pathology, and improve health outcomes for these vulnerable infants.

Role of Pax genes in eye evolution: a cnidarian PaxB gene uniting Pax2 and Pax6 functions.

PaxB from Tripedalia cystophora, a cubomedusan jellyfish possessing complex eyes (ocelli), was characterized. PaxB, the only Pax gene found in this cnidarian, is expressed in the larva, retina, lens, and statocyst. PaxB contains a Pax2/5/8-type paired domain and octapeptide, but a Pax6 prd-type homeodomain. Pax2/5/8-like properties of PaxB include a DNA binding specificity of the paired domain, activation and inhibitory domains, and the ability to rescue spa(pol), a Drosophila Pax2 eye mutant. Like Pax6, PaxB activates jellyfish crystallin and Drosophila rhodopsin rh6 promoters and induces small ectopic eyes in Drosophila. Pax6 has been considered a "master" control gene for eye development. Our data suggest that the ancestor of jellyfish PaxB, a PaxB-like protein, was the primordial Pax protein in eye evolution and that Pax6-like genes evolved in triploblasts after separation from Cnidaria, raising the possibility that cnidarian and sophisticated triploblastic eyes arose independently.

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.

Longitudinal Microbiome Composition and Stability Correlate with Increased Weight and Length of Very-Low-Birth-Weight Infants.

The microbiomes of 83 preterm very-low-birth-weight (VLBW) infants and clinical covariates were analyzed weekly over the course of their initial neonatal intensive care unit (NICU) stay, with infant growth as the primary clinical outcome. Birth weight significantly correlated with increased rate of weight gain in the first 6 weeks of life, while no significant relationship was observed between rate of weight gain and feeding type. Microbial diversity increased with age and was significantly correlated with weight gain and percentage of the mother's own milk. As expected, infants who received antibiotics during their NICU stay had significantly lower alpha diversity than those who did not. Of those in the cohort, 25 were followed into childhood. Alpha diversity significantly increased between NICU discharge and age 2 years and between age 2 years and age 4 years, but the microbial alpha diversity of 4-year-old children was not significantly different from that of mothers. Infants who showed improved length over the course of their NICU stay had significantly more volatile microbial beta diversity results than and a significantly decreased microbial maturity index compared with infants who did not; interestingly, all infants who showed improved length during the NICU stay were delivered by Caesarean section. Microbial beta diversity results were significantly different between the time of the NICU stay and all other time points (for children who were 2 or 4 years old and mothers when their children were 2 or 4 years old). IMPORTANCE Preterm infants are at greater risk of microbial insult than full-term infants, including reduced exposure to maternal vaginal and enteric microbes, higher rates of formula feeding, invasive procedures, and administration of antibiotics and medications that alter gastrointestinal pH. This investigation of the VLBW infant microbiome over the course of the neonatal intensive care unit (NICU) stay, and at ages 2 and 4 years, showed that the only clinical variables associated with significant differences in taxon abundance were weight gain during NICU stay (Klebsiella and Staphylococcus) and antibiotic administration (Streptococcus and Bifidobacterium). At 2 and 4 years of age, the microbiota of these VLBW infants became similar to the mothers' microbiota. The number of microbial taxa shared between the infant or toddler and the mother varied, with least the overlap between infants and mothers. Overall, there was a significant association between the diversity and structure of the microbial community and infant weight and length gain in an at-risk childhood population.

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

BACKGROUND: The 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. RESULTS: VCBP 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. CONCLUSION: The 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.

Unprecedented Diversity of ssDNA Phages from the Family Microviridae Detected within the Gut of a Protochordate Model Organism (Ciona robusta).

Phages (viruses that infect bacteria) play important roles in the gut ecosystem through infection of bacterial hosts, yet the gut virome remains poorly characterized. Mammalian gut viromes are dominated by double-stranded DNA (dsDNA) phages belonging to the order Caudovirales and single-stranded DNA (ssDNA) phages belonging to the family Microviridae. Since the relative proportion of each of these phage groups appears to correlate with age and health status in humans, it is critical to understand both ssDNA and dsDNA phages in the gut. Building upon prior research describing dsDNA viruses in the gut of Ciona robusta, a marine invertebrate model system used to study gut microbial interactions, this study investigated ssDNA phages found in the Ciona gut. We identified 258 Microviridae genomes, which were dominated by novel members of the Gokushovirinae subfamily, but also represented several proposed phylogenetic groups (Alpavirinae, Aravirinae, Group D, Parabacteroides prophages, and Pequeñovirus) and a novel group. Comparative analyses between Ciona specimens with full and cleared guts, as well as the surrounding water, indicated that Ciona retains a distinct and highly diverse community of ssDNA phages. This study significantly expands the known diversity within the Microviridae family and demonstrates the promise of Ciona as a model system for investigating their role in animal health.

The gut virome of the protochordate model organism, Ciona intestinalis subtype A.

The identification of host-specific bacterial and viral communities associated with diverse animals has led to the concept of the metaorganism, which defines the animal and all of its associated microbes as a single unit. Here we sequence the viruses found in the gut (i.e., the gut virome) of the marine invertebrate model system, Ciona intestinalis subtype A, in samples collected one year apart. We present evidence for a host-associated virome that is distinct from the surrounding seawater and contains some temporally-stable members. Comparison of gut tissues before and after starvation in virus-free water enabled the differentiation between the Ciona-specific virome and transient viral communities associated with dietary sources. The Ciona gut viromes were dominated by double-stranded DNA tailed phages (Order Caudovirales) and sequence assembly yielded a number of complete circular phage genomes, most of which were highly divergent from known genomes. Unique viral communities were found in distinct gut niches (stomach, midgut and hindgut), paralleling the compartmentalization of bacterial communities. Additionally, integrase and excisionase genes, including many that are similar to prophage sequences within the genomes of bacterial genera belonging to the Ciona core microbiome, were prevalent in the viromes, indicating the active induction of prophages within the gut ecosystem. Knowledge of the gut virome of this model organism lays the foundation for studying the interactions between viruses, bacteria, and host immunity.

Genome Sequence of PM2-Like Phage Cr39582, Induced from a Pseudoalteromonas sp. Isolated from the Gut of Ciona robusta.

Phage Cr39582 was induced by mitomycin C from Pseudoalteromonas sp. strain Cr6751, isolated from a marine invertebrate gut. Pseudoalteromonas phage Cr39582 has 85% pairwise nucleotide identity with phage PM2 but lacks sequence homology in the spike protein. This report supports previous bioinformatic identification of corticoviral sequences within aquatic bacterial genomes.

Chitin protects the gut epithelial barrier in a protochordate model of DSS-induced colitis.

The gastrointestinal tract of Ciona intestinalis, a solitary tunicate that siphon-filters water, shares similarities with its mammalian counterpart. The Ciona gut exhibits other features that are unique to protochordates, including certain immune molecules, and other characteristics, e.g. chitin-rich mucus, which appears to be more widespread than considered previously. Exposure of Ciona to dextran sulphate sodium (DSS) induces a colitis-like phenotype similar to that seen in other systems, and is characterized by alteration of epithelial morphology and infiltration of blood cells into lamina propria-like regions. DSS treatment also influences the production and localization of a secreted immune molecule shown previously to co-localize to chitin-rich mucus in the gut. Resistance to DSS is enhanced by exposure to exogenous chitin microparticles, suggesting that endogenous chitin is critical to barrier integrity. Protochordates, such as Ciona, retain basic characteristics found in other more advanced chordates and can inform us of uniquely conserved signals shaping host-microbiota interactions in the absence of adaptive immunity. These simpler model systems may also reveal factors and processes that modulate recovery from colitis, the role gut microbiota play in the onset of the disease, and the rules that help govern the reestablishment and maintenance of gut homeostasis.