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Toll-like receptors (TLRs) are essential components of innate immunity, mediating the recognition of pathogen-associated molecular patterns (PAMPs) through extracellular leucine-rich repeat (LRR) domains and initiating signaling via intracellular Toll/interleukin-1 receptor (TIR) domains. In the ascidianCiona robusta, two canonical TLRs (CiTLR1 and CiTLR2) and several putative TLR-like genes (TLR3, -4, -6, -7, -13) have been annotated; however, their authenticity has remained uncertain due to limited structural and functional validation. Methodswe systematically reanalyzed theCionagenome using the latest nearly complete assembly (HT genome) in combination with domain prediction, three-dimensional structural modeling, and transcriptomic expression profiling. ResultsGenomic mapping and sequence comparison demonstrated that TLR13 is identical to CiTLR1, while TLR3, -6, and -7 lack a complete TIR domain, indicating that these are not canonical TLRs. We further identified a novel TLR gene, CiTLRs1, located approximately 42 kb from CiTLR1 on chromosome 14, which encodes all essential structural features including LRR and TIR domains. AlphaFold3 structural predictions confirmed that CiTLR1, CiTLR2, and CiTLRs1 possess canonical solenoid LRR folds and typical TIR domain architectures. In addition, we found no convincing evidence that CiTLR3, CiTLR6, or CiTLR7 function as soluble TLRs. Transcriptomic analyses revealed distinct tissue-specific expression profiles of these genes, suggesting nonredundant immune functions. ConclusionsOur findings revise the repertoire of bona fide TLRs inCionato three (CiTLR1, CiTLR2, CiTLRs1) and emphasize the risk of overestimating TLR diversity based solely on sequence homology without domain and functional validation. This work refines the structural and functional landscape of ascidian TLRs. 

ABSTRACT We isolatedPseudovibrio ascidiaceicolastrain 5337, a gut bacterium of the ascidianCiona robusta,from Mission Bay, San Diego. The genomic assembly is 6.94 Mb and 99.99% complete, comprising 22 contigs and 6,613 protein-coding genes. Unicycler identified seven circular contigs, and PHASTEST identified 11 prophage regions, including two gene transfer agents. 

Lysogens, bacteria with one or more viruses (prophages) integrated into their genomes, are abundant in the gut of animals. Prophages often influence bacterial traits; however, the influence of prophages on the gut microbiota–host immune axis in animals remains poorly understood. Here, we investigate the influence of the prophage SfPat onShewanella fidelis3313, a persistent member of the gut microbiome of the model marine tunicate,Ciona robusta. Establishment of a SfPat deletion mutant (ΔSfPat) reveals the influence of this prophage on bacterial physiology in vitro and during colonization of theCionagut. In vitro, deletion of SfPat reducesS. fidelis3313 motility and swimming while increasing biofilm formation. To understand the in vivo impact of these prophage-induced changes in bacterial traits, we exposed metamorphic stage 4Cionajuveniles to wildtype (WT) and ΔSfPat strains. During colonization, ΔSfPat localizes to overlapping and distinct areas of the gut compared to the WT strain. We examined the differential expression of various regulators of cyclic-di-GMP, a secondary signaling molecule that mediates biofilm formation and motility. ThepdeBgene, which encodes a bacterial phosphodiesterase known to influence biofilm formation and motility by degrading cyclic-di-GMP, is upregulated in the WT strain but not in ΔSfPat when examined in vivo. Expression of theCionagut immune effector, VCBP-C, is enhanced during colonization by ΔSfPat compared to the WT strain; however, VCBP-C binding to the WT strain does not promote the excision of SfPat in an SOS-dependent pathway. Instead, VCBP-C binding significantly reduces the expression of a phage major capsid protein. Our findings suggest that SfPat influences host perception of this important colonizing commensal and highlights the significance of investigating tripartite dynamics between prophages, bacteria, and their animal hosts to better understand the gut microbiota-host immune axis. 

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. 

ABSTRACT 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.