Episomally Driven Antisense mRNA Abrogates the Hyperinducible Expression and Function of a Unique Cell Surface Class I Nuclease in the Primitive Trypanosomatid Parasite, Crithidia luciliae

Here, we show that Crithidia luciliae, a primitive trypanosomatid, purine auxotroph, up-expressed its unique, bi-functional, surface membrane 3′-nucleotidase/nuclease (Cl 3′NT/NU) activity by ∼ 1000-fold in response to purine starvation. A second surface membrane phospho-monoesterase, i.e. a tartrate-resistant acid phosphatase (Cl MAcP) was also found to be up-expressed in such purine-starved cells. Here, we used homologous episomal-expression of an antisense construct of the Cl3′NT/NU to dissect the functional expression of these two surface membrane enzymes. In antisense transfected cells, a large excess of the antisense transcript was produced and no trace of any endogenous Cl3′NT/NU sense message was detected. Further, the purine-starvation hyper-induced levels of 3′NT/NU enzyme activity were completely abrogated in these transfected cells versus controls. Moreover, such antisense transcription completely abolished the ability of these transfectants to grow in poly(A)-containing medium demonstrating the essential nature of the 3′NT/NU for the growth/survival of this parasite. In contrast, antisense transcription had no apparent deleterious effects on either endogenous or purine-starvation-induced levels of MAcP enzyme activity, its steady-state mRNA levels, or the constitutive expression of house-keeping genes (e.g. Cl α-tubulin) in these transfectants.Cumulatively, results of our antisense experiments demonstrated that the functional nuclease activity of the surface membrane Cl 3′NT/NU was, in fact, critical/essential for the growth and development of these primitive parasites.