Ciliates display promise for foreign gene expression

The study of Tetrahymena thermophila, the simple ciliate of introductorybiology laboratory exercises, has led to a remarkable series of discoveriesin biology, many of which have found important applications in biotechnology.These range from the identification of ribozymes and telomerase to the firstexample of a noncanonical nuclear genetic code and the role of histone acetyltransferaseA in gene activation^1,2,3,4. Now this remarkable ciliatehas made the leap from mere model organism in basic biology to a new roleas a potentially useful expression system in biotechnology. As Tetrahymena is nonpathogenic, has a very short generation time, and can be grownto high cell density in inexpensive media, it is an ideal candidate systemfor production of pharmaceuticals and vaccines.

When transformation of an expression system with a foreign gene does notproduce a selectable phenotype, identification of transformed cells and themaintenance of the transformed state can be tricky, if not impossible. Inthis issue, Gaertig et al. describe the engineering of a Tetrahymenaexpression system that gets around this problem by exploiting the drug sensitivityof a particular mutant strain⁵. A mutation in either one ofthe two Tetrahymena β-tubulin genes—a substitution of Lys350by methionine—produces sensitivity to the microtubule drug, paclitaxel⁷, and knocking out the mutant β-tubulin gene restores resistance(see Fig. 1). Thus, if a foreign gene is targeted tothe mutant β-tubulin gene by homologous recombination, transformed cellscan be selected by their resistance to paclitaxel and the foreign gene isexpressed under the control of the β-tubulin promoter. In one fell swoop,an introduced foreign gene is expressed and produces a selectable phenotype.This approach is not unique to Tetrahymena and should be applicableto other systems.

Figure 1: Tetrahymena expressing a mutant copy of the β-tubulin gene(btu1-1K350M) are paclitaxel sensitive.

When transformed with an Ichthyophthirius multifiliis i-antigenflanked by the 5´ and 3´ regions of the β-tubulin gene, themutant gene is disrupted, the cells are paclitaxel resistant, and the i-antigenis expressed and displayed on the cell surface.

This system has been used to express a surface immobilization antigen (i-antigen)from the parasitic cousin of Tetrahymena, the ciliate Ichthyophthiriusmultifiliis, which causes "Ich" or "white spot" disease in economicallyimportant freshwater fish⁶. The i-antigen of I. multifiliis is apparently responsible for protective immunity and is thus a primecandidate for vaccine development⁸. Unfortunately, however, I. multifiliis can only be grown in association with its host, makingit difficult to obtain sufficient quantities of i-antigen protein⁹.Moreover, like most ciliates, I. multifiliis employs a noncanonicalnuclear genetic code, in which codons UAA and UAG specify glutamine ratherthan chain termination¹⁰. This means that any attempt to translate I. multifiliis genes in a conventional expression system employing a canonicalnuclear genetic code will generally produce premature termination and resultin nonfunctional truncated proteins. Tetrahymena conveniently sharesthe same noncanonical nuclear genetic code used by I. multifiliis,allowing accurate translation of the parasite's proteins in Tetrahymena. Of course, the noncanonical system used by Tetrahymena may createproblems when it is used to translate genes from organisms using the canonicalnuclear genetic code that terminates with a UAA or UAG codon. That said, amongmammalian genes found in GenBank, UGA appears more frequently than UAA andUAG termination codons combined¹¹. When a foreign gene terminatingwith either UAA or UAG is translated in Tetrahymena, the resultingprotein is extended at the C terminus by a polypeptide corresponding to thesequence between the normal termination codon and the next in-frame UGA. Theseadditional C-terminal amino acids are unlikely to interfere with the functionof the protein, however. Thus, not only is the Tetrahymena system capableof translating foreign genes using the noncanonical nuclear genetic code,it should also produce active proteins from many foreign genes using the canonicalnuclear genetic code.

Importantly, the i-antigen is expressed in Tetrahymena at the cellsurface. Probing Tetrahymena with a fluorescently labeled antiserumdirected against the i-antigen reveals strong labeling of both oral and somaticcilia in a pattern very similar to that observed in I. multifiliis.Moreover, antiserum directed against I. multifiliis i-antigen causesimmediate immobilization of transformed Tetrahymena (by cross-linkingcilia). The surface localization of the i-antigen suggests that its signalpeptide is functioning appropriately in Tetrahymena. It remains tobe seen, however, whether proteins from more distantly related organisms willalso be appropriately localized when expressed in Tetrahymena.

The expression of I. multifiliis i-antigen in Tetrahymenamakes possible the large-scale production of this protein as a potential vaccineagainst I. multifiliis. The high cell density to which Tetrahymena can be grown suggests that the i-antigen may be produced on the orderof milligrams per liter. Furthermore, its display on the cell membrane opensthe possibility that transformed Tetrahymena organisms could be usedas a live vaccine against I. multifiliis. Since Tetrahymenaare normally present in freshwater ecosystems, the introduction of quantitiesof transformed cells displaying the I. multifiliis i-antigen in theircell membranes may immunize fish against the parasitic I. Multifiliis. Whether or not the use of Tetrahymena as a living vaccine against"white spot" disease becomes a reality, the innovative system described byGaertig et al. for the identification and maintenance of transformedcells expressing foreign proteins that do not confer a selectable phenotypehas real merit and is not for ciliates only.