Summary The conserved transmembrane protein, HAP2/GCS1, has been linked to fertility in a wide range of taxa and is hypothesized to be an ancient gamete fusogen. Using template-based homology modeling, we now show that the ectodomain of HAP2 orthologs from Tetrahymena thermophila and other species adopt a protein fold remarkably similar to the Dengue Virus E glycoprotein and related class II viral fusogens. To test the functional significance of this predicted structure, we developed a flow cytometry-based assay that measures cytosolic exchange across the conjugation junction to rapidly probe the effects of HAP2 mutations in the Tetrahymena system. Using this assay, alterations to a region in and around a predicted “fusion loop” in T. thermophila HAP2 were found to abrogate membrane pore formation in mating cells. Consistent with this, a synthetic peptide corresponding to the HAP2 “fusion loop” was found to interact directly with model membranes in a variety of biophysical assays. These results raise interesting questions regarding the evolutionary relationships of class II membrane fusogens, and harken back to a long-held argument that eukaryotic sex arose as the by-product of selection for the horizontal transfer of a “selfish” genetic element from cell-to-cell via membrane fusion.

BackgroundIchthyophthirius multifiliis, commonly known as Ich, is a highly pathogenic ciliate responsible for 'white spot', a disease causing significant economic losses to the global aquaculture industry. Options for disease control are extremely limited, and Ich's obligate parasitic lifestyle makes experimental studies challenging. Unlike most well-studied protozoan parasites, Ich belongs to a phylum composed primarily of free-living members. Indeed, it is closely related to the model organism Tetrahymena thermophila. Genomic studies represent a promising strategy to reduce the impact of this disease and to understand the evolutionary transition to parasitism.ResultsWe report the sequencing, assembly and annotation of the Ich macronuclear genome. Compared with its free-living relative T. thermophila, the Ich genome is reduced approximately two-fold in length and gene density and three-fold in gene content. We analyzed in detail several gene classes with diverse functions in behavior, cellular function and host immunogenicity, including protein kinases, membrane transporters, proteases, surface antigens and cytoskeletal components and regulators. We also mapped by orthology Ich's metabolic pathways in comparison with other ciliates and a potential host organism, the zebrafish Danio rerio.ConclusionsKnowledge of the complete protein-coding and metabolic potential of Ich opens avenues for rational testing of therapeutic drugs that target functions essential to this parasite but not to its fish hosts. Also, a catalog of surface protein-encoding genes will facilitate development of more effective vaccines. The potential to use T. thermophila as a surrogate model offers promise toward controlling 'white spot' disease and understanding the adaptation to a parasitic lifestyle.

Dendritic cells are specialized antigen presenting cells that bridge innate and adaptive immunity in mammals. This link between the ancient innate immune system and the more evolutionarily recent adaptive immune system is of particular interest in fish, the oldest vertebrates to have both innate and adaptive immunity. It is unknown whether dendritic cells co-evolved with the adaptive response, or if the connection between innate and adaptive immunity relied on a fundamentally different cell type early in evolution. We approached this question using the teleost model organism, rainbow trout ( Oncorhynchus mykiss ), with the aim of identifying dendritic cells based on their ability to stimulate naïve T cells. Adapting mammalian protocols for the generation of dendritic cells, we established a method of culturing highly motile, non-adherent cells from trout hematopoietic tissue that had irregular membrane processes and expressed surface MHCII. When side-by-side mixed leukocyte reactions were performed, these cells stimulated greater proliferation than B cells or macrophages, demonstrating their specialized ability to present antigen and therefore their functional homology to mammalian dendritic cells. Trout dendritic cells were then further analyzed to determine if they exhibited other features of mammalian dendritic cells. Trout dendritic cells were found to have many of the hallmarks of mammalian DCs including tree-like morphology, the expression of dendritic cell markers, the ability to phagocytose small particles, activation by toll-like receptor-ligands, and the ability to migrate in vivo . As in mammals, trout dendritic cells could be isolated directly from the spleen, or larger numbers could be derived from hematopoietic tissue and peripheral blood mononuclear cells in vitro .

No abstract