Targeted reduction in expression of Trypanosoma cruzi surface glycoproteingp90 increases parasite infectivity

A previous study had shown that the expression of gp90, a stage-specific su rface glycoprotein of Trypanosoma cruzi metacyclic trypomastigotes, is inve rsely correlated with the parasite's ability to invade mammalian cells. By using antisense oligonucleotides complementary to a region of the gp90 gene implicated in host cell adhesion, we investigated whether the selective in hibition of gp90 synthesis affected the capacity of metacyclic forms to ent er target cells, Parasites were incubated for 24 h with 20 muM PS1, a phosp horothioate oligonucleotide based on a sequence of the gp90 coding strand; PS2, the antisense counterpart of PS1; or PO2, the unmodified version of PS 2 containing phosphodiester linkages, and the expression of surface molecul es was analyzed by flow cytometry and immunoblotting using specific monoclo nal antibodies. PS2 but not PS1 or PO2 inhibited the expression of gp90. In hibition by PS2 was dose dependent. Northern blot analysis revealed that st eady-state gp90 mRNA levels were diminished in PS2-treated parasites compar ed to untreated controls. Treatment with PS2 did not affect the expression of other metacyclic stage surface glycoproteins involved in parasite-host c ell interaction, such as gp82 and the mucin-like gp35/50. Expression of gp9 0 was also inhibited by other phosphorothioate oligonucleotides targeted to the gp90 gene (PS4, PS5, PS6, and PS7) but not by PS3, with the same base composition as PS2 but a mismatched sequence. Parasites treated with PS2, P S4, or PS5 entered HeLa cells in significantly higher numbers than untreate d controls, whereas the invasive capacity of PS1- and PS3-treated parasites was unchanged, confirming the inverse association between infectivity and gp90 expression.