N-glycosylation as a biochemical basis for virulence in Leishmania mexicana amazonensis.

Promastigotes of Leishmania mexicana amazonensis grown in vitro under different conditions showed variable degrees of virulence, as determined quantitatively by the size of the lesions and the number of intracellular parasites produced in mice and in cultured macrophages, respectively. Promastigotes newly transformed from amastigotes gave the highest degree of virulence, which decreased progressively with periods of their continuous in vitro cultivation. This loss of virulence was prevented by making virulent wildtype promastigotes resistant to tunicamycin, an inhibitor of N-acetylglucosamine-1-phosphate transferase in the dolichol pathway of protein glycosylation. In the wildtype cells, the progressive loss of virulence during a period of two years was marked by a gradual decrease in the activity of the glycosyltransferase, incorporation of 2-D-[3H]mannose and the expression of a surface glycoprotein (gp63). Virulent and avirulent wildtype cells differed in these activities by 3-4 fold. In contrast, during equivalent periods of in vitro cultivation, tunicamycin-resistant cells were found to consistently maintain the biochemical phenotypes of the virulent wildtype, except for a disproportional elevation of the glycosyltransferase activity. Thus, only a portion of the over-produced enzyme is relevant to leishmanial virulence in the drug-resistant variants. The bulk of its activity in these cells serves to overcome the inhibitory effect of tunicamycin responsible for their resistance to this drug, as shown previously. A role of N-glycosylation in leishmanial virulence is now indicated by studying cells of this phenotype selected by two independent methods. It is inferred that leishmanial virulence may be regulated by the level of the glycosyltransferases for N-glycosylation of proteins, e.g. gp63 in relation to their expression as virulent determinants.