A novel malaria vaccine candidate antigen expressed in Tetrahymena thermophila

Development of effective malaria vaccines is hampered by the problem of producing correctly folded Plasmodium proteins for use as vaccine components. We have investigated the use of a novel ciliate expression system, Tetrahymena thermophila, as a P. falciparum vaccine antigen platform. A synthetic vaccine antigen composed of N-terminal and C-terminal regions of merozoite surface protein-1 (MSP-1) was expressed in Tetrahymena thermophila. The recombinant antigen was secreted into the culture medium and purified by monoclonal antibody (mAb) affinity chromatography. The vaccine was immunogenic in MF1 mice, eliciting high antibody titers against both N- and C-terminal components. Sera from immunized animals reacted strongly with P. falciparum parasites from three antigenically different strains by immunofluorescence assays, confirming that the antibodies produced are able to recognize parasite antigens in their native form. Epitope mapping of serum reactivity with a peptide library derived from all three MSP-1 Block 2 serotypes confirmed that the MSP-1 Block 2 hybrid component of the vaccine had effectively targeted all three serotypes of this polymorphic region of MSP-1. This study has successfully demonstrated the use of Tetrahymena thermophila as a recombinant protein expression platform for the production of malaria vaccine antigens

Distinct Functional Roles of β-Tubulin Isotypes in Microtubule Arrays of Tetrahymena thermophila, a Model Single-Celled Organism

<div><h3>Background</h3><p>The multi-tubulin hypothesis proposes that each tubulin isotype performs a unique role, or subset of roles, in the universe of microtubule function(s). To test this hypothesis, we are investigating the functions of the recently discovered, noncanonical β-like tubulins (BLTs) of the ciliate, <em>Tetrahymena thermophila</em>. <em>Tetrahymena</em> forms 17 distinct microtubular structures whose assembly had been thought to be based on single α- and β-isotypes. However, completion of the macronuclear genome sequence of <em>Tetrahymena</em> demonstrated that this ciliate possessed a β-tubulin multigene family: two synonymous genes (<em>BTU1</em> and <em>BTU2</em>) encode the canonical β-tubulin, BTU2, and six genes (<em>BLT1-6</em>) yield five divergent β-tubulin isotypes. In this report, we examine the structural features and functions of two of the BLTs (BLT1 and BLT4) and compare them to those of BTU2.</p> <h3>Methodology/Principal Findings</h3><p>With respect to BTU2, BLT1 and BLT4 had multiple sequence substitutions in their GTP-binding sites, in their interaction surfaces, and in their microtubule-targeting motifs, which together suggest that they have specialized functions. To assess the roles of these tubulins <em>in vivo</em>, we transformed <em>Tetrahymena</em> with expression vectors that direct the synthesis of GFP-tagged versions of the isotypes. We show that GFP-BLT1 and GFP-BLT4 were not detectable in somatic cilia and basal bodies, whereas GFP-BTU2 strongly labeled these structures. During cell division, GFP-BLT1 and GFP-BLT4, but not GFP-BTU2, were incorporated into the microtubule arrays of the macronucleus and into the mitotic apparatus of the micronucleus. GFP-BLT1 also participated in formation of the microtubules of the meiotic apparatus of the micronucleus during conjugation. Partitioning of the isotypes between nuclear and ciliary microtubules was confirmed biochemically.</p> <h3>Conclusion/Significance</h3><p>We conclude that <em>Tetrahymena</em> uses a family of distinct β-tubulin isotypes to construct subsets of functionally different microtubules, a result that provides strong support for the multi-tubulin hypothesis.</p> </div

Kin5 Knockdown in Tetrahymena thermophila Using RNAi Blocks Cargo Transport of Gef1

A critical process that builds and maintains the eukaryotic cilium is intraflagellar transport (IFT). This process utilizes members of the kinesin-2 superfamily to transport cargo into the cilium (anterograde transport) and a dynein motor for the retrograde traffic. Using a novel RNAi knockdown method, we have analyzed the function of the homodimeric IFT kinesin-2, Kin5, in Tetrahymena ciliary transport. In RNAi transformants, Kin5 was severely downregulated and disappeared from the cilia, but cilia did not resorb, although tip structure was affected. After deciliation of the knockdown cell, cilia regrew and cells swam, which suggested that Kin5 is not responsible for the trafficking of axonemal precursors to build the cilium, but could be transporting molecules that act in ciliary signal transduction, such as guanine nucleotide exchange proteins (GEFs). Gef1 is a Tetrahymena ciliary protein, and current coimmunoprecipitation and immunofluorescence studies showed that it is absent in regrowing cilia of the knockdown cells lacking ciliary Kin5. We suggest that one important cargo of Kin5 is Gef1 and knockdown of Kin5 results in cell lethality

Absence of Positive Selection on Centromeric Histones in Tetrahymena Suggests Unsuppressed Centromere-Drive in Lineages Lacking Male Meiosis

Centromere-drive is a process where centromeres compete for transmission through asymmetric "female" meiosis for inclusion into the oocyte. In symmetric "male" meiosis, all meiotic products form viable germ cells. Therefore, the primary incentive for centromere-drive, a potential transmission bias, is believed to be missing from male meiosis. In this article, we consider whether male meiosis also bears the primary cost of centromere-drive. Because different taxa carry out different combinations of meiotic programs (symmetric + asymmetric, symmetric only, asymmetric only), it is possible to consider the evolutionary consequences of centromere-drive in the context of these differing systems. Groups with both types of meiosis have large, rapidly evolving centromeric regions, and their centromeric histones (CenH3s) have been shown to evolve under positive selection, suggesting roles as suppressors of centromere-drive. In contrast, taxa with only symmetric male meiosis have shown no evidence of positive selection in their centromeric histones. In this article, we present the first evolutionary analysis of centromeric histones in ciliated protozoans, a group that only undergoes asymmetric "female" meiosis. We find no evidence of positive selection acting on CNA1, the CenH3 of Tetrahymena species. Cytological observations of a panel of Tetrahymena species are consistent with dynamic karyotype evolution in this lineage. Our findings suggest that defects in male meiosis, and not mitosis or female meiosis, are the primary selective force behind centromere-drive suppression. Our study raises the possibility that taxa like ciliates, with only female meiosis, may therefore undergo unsuppressed centromere drive

Tetrahymena Metallothioneins Fall into Two Discrete Subfamilies

BACKGROUND: Metallothioneins are ubiquitous small, cysteine-rich, multifunctional proteins which can bind heavy metals. METHODOLOGY/PRINCIPAL FINDINGS: We report the results of phylogenetic and gene expression analyses that include two new Tetrahymena thermophila metallothionein genes (MTT3 and MTT5). Sequence alignments of all known Tetrahymena metallothioneins have allowed us to rationalize the structure of these proteins. We now formally subdivide the known metallothioneins from the ciliate genus Tetrahymena into two well defined subfamilies, 7a and 7b, based on phylogenetic analysis, on the pattern of clustering of Cys residues, and on the pattern of inducibility by the heavy metals Cd and Cu. Sequence alignment also reveals a remarkably regular, conserved and hierarchical modular structure of all five subfamily 7a MTs, which include MTT3 and MTT5. The former has three modules, while the latter has only two. Induction levels of the three T. thermophila genes were determined using quantitative real time RT-PCR. Various stressors (including heavy metals) brought about dramatically different fold-inductions for each gene; MTT5 showed the highest fold-induction. Conserved DNA motifs with potential regulatory significance were identified, in an unbiased way, upstream of the start codons of subfamily 7a MTs. EST evidence for alternative splicing in the 3′ UTR of the MTT5 mRNA with potential regulatory activity is reported. CONCLUSION/SIGNIFICANCE: The small number and remarkably regular structure of Tetrahymena MTs, coupled with the experimental tractability of this model organism for studies of in vivo function, make it an attractive system for the experimental dissection of the roles, structure/function relationships, regulation of gene expression, and adaptive evolution of these proteins, as well as for the development of biotechnological applications for the environmental monitoring of toxic substances

CEP41 is mutated in Joubert syndrome and is required for tubulin glutamylation at the cilium

Tubulin glutamylation is a post-translational modification that occurs predominantly in the ciliary axoneme and has been suggested to be important for ciliary function. However, its relationship to disorders of the primary cilium, termed ciliopathies, has not been explored. Here we mapped a new locus for Joubert syndrome (JBTS), which we have designated as JBTS15, and identified causative mutations in CEP41, which encodes a 41-kDa centrosomal protein. We show that CEP41 is localized to the basal body and primary cilia, and regulates ciliary entry of TTLL6, an evolutionarily conserved polyglutamylase enzyme. Depletion of CEP41 causes ciliopathy-related phenotypes in zebrafish and mice and results in glutamylation defects in the ciliary axoneme. Our data identify CEP41 mutations as a cause of JBTS and implicate tubulin post-translational modification in the pathogenesis of human ciliary dysfunction