Comparative genomics of the pathogenic ciliate Ichthyophthirius multifiliis, its free-living relatives and a host species provide insights into adoption of a parasitic lifestyle and prospects for disease control

Background: Ichthyophthirius 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. Results: We 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. Conclusions: Knowledge 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

Comparative genomics of the pathogenic ciliate Ichthyophthirius multifiliis, its free-living relatives and a host species provide insights into adoption of a parasitic lifestyle and prospects for disease control

BACKGROUND: Ichthyophthirius 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. RESULTS: We 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. CONCLUSIONS: Knowledge 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

Functional analysis of the cytoplasmic dynein-2 complex in Tetrahymena thermophila

Dyneins are large molecular motor complexes containing one or more heavy chains that function as motors and several accessory subunits that bind cargoes and regulate heavy chain function. These dynein complexes perform various cellular functions within the eukaryotic cell. Axonemal outer and inner arm complexes and cytoplasmic dyneins-1 and -2 are the major types of dyneins that exist in a ciliated organism. The cytoplasmic dynein-2 complex is composed of the dynein-2 heavy chain (DYH2) and the dynein-2 light intermediate chain (D2LIC). Cytoplasmic dynein-2 heavy chain is the retrograde motor for intraflagellar transport in Chlamydomonas, C. elegans, Leishmania and mouse (Pazour et al., 1999; Porter et al., 1999; Wicks et al., 2000; May et al., 2005). Mutations of the DYH2 or D2LIC genes in these organisms result in short, stumpy flagella or cilia. In our earlier study, we knocked down the macronuclear copies of the dynein-2 heavy chain gene (DYH2) in Tetrahymena thermophila. Surprisingly, the DYH2 knockdown cells continued to form motile cilia but were misshapened and missized (Lee et al., 1999). Since our previous Tetrahymena DYH2 knockdown results were not consistent with the generally accepted model for dynein-2 in ciliogenesis, I have re-investigated the function of the Tetrahymena dynein-2 complex by a germline knockout strategy. I created knockout heterokaryon cell lines in which the DYH2 or the D2LIC gene is disrupted in the micronucleus. When two heterokaryons were mated, the somatic macronuclei of the resulting progeny were completely devoid of the targeted gene. Cells with knockout of the DYH2 gene (KO-DYH2) and the D2LIC gene (KO-D2LIC) have motile cilia but the cilia were fewer and shorter compared to wild type controls. Because of ciliary defects, the KO-DYH2 and KO-D2LIC cells swam more slowly than wild type control cells. Both KO-DYH2 and KO-D2LIC cells exhibited secondary defects including slow growth. The mutant cultures were always less dense compared to the wild type cultures. Despite the ciliary and swimming defects, both KO-DYH2 and KO-D2LIC cultures could be kept alive indefinitely. In conclusion, the cytoplasmic dynein-2 complex is important but not essential for ciliogenesis in Tetrahymena

Dynein-2 Affects the Regulation of Ciliary Length but Is Not Required for Ciliogenesis in Tetrahymena thermophila

Eukaryotic cilia and flagella are assembled and maintained by the bidirectional intraflagellar transport (IFT). Studies in alga, nematode, and mouse have shown that the heavy chain (Dyh2) and the light intermediate chain (D2LIC) of the cytoplasmic dynein-2 complex are essential for retrograde intraflagellar transport. In these organisms, disruption of either dynein-2 component results in short cilia/flagella with bulbous tips in which excess IFT particles have accumulated. In Tetrahymena, the expression of the DYH2 and D2LIC genes increases during reciliation, consistent with their roles in IFT. However, the targeted elimination of either DYH2 or D2LIC gene resulted in only a mild phenotype. Both knockout cell lines assembled motile cilia, but the cilia were of more variable lengths and less numerous than wild-type controls. Electron microscopy revealed normally shaped cilia with no swelling and no obvious accumulations of material in the distal ciliary tip. These results demonstrate that dynein-2 contributes to the regulation of ciliary length but is not required for ciliogenesis in Tetrahymena