TARGETED GENE DELETION OF LEISHMANIA-MAJOR GENES ENCODING DEVELOPMENTAL STAGE-SPECIFIC LEISHMANOLYSIN (GP63)

The major surface glycoprotein of Leishmania major is a zinc metallopr oteinase of 63 kDa referred to as leishmanolysin or GP63, which is enc oded by a family of seven genes, Targeted gene replacement was used to delete gp63 genes 1-6 encoding the highly expressed promastigote and constitutively expressed GP63, In the L. major homozygous mutants defi cient in gp63 genes 1-6, there was no expression of GP63 as detected b y reverse transcription-polymerase chain reaction (RT-PCR) or fluoresc ent staining in promastigotes from the procyclic stage (logarithmic gr owth phase), The remaining L. major gP63 gene 7 was shown to be develo pmentally regulated, as it was expressed exclusively in infectious met acyclic stage (late stationary growth phase) promastigotes and in lesi on amastigotes, The gp63 genes 1-6-deficient mutants showed increased sensitivity to complement-mediated lysis, The sensitivity to lysis was greater in procyclics than in metacyclics when compared with the equi valent wild-type stages, Increased resistance of the mutant metacyclic promastigotes correlated with the expression of gp63 gene 7 and was r estored to the same levels as wild-type promastigotes by transfection with gp63 gene 1, Thus, expression of GP63 is clearly involved in conf erring resistance to complement-mediated lysis, The L. major GP63 1-6 mutants were capable of infecting mouse macrophages and differentiatin g into amastigotes, Similar levels of infection and subsequent intrace llular survival were observed when mouse macrophages were infected in vitro with wild type, GP63 1-6 mutants and mutants transfected with gp 63 gene 1,The GP63 1-6 mutants were capable of lesion formation in BAL B/c mice and, thus, gp63 genes 1-6 do not play a role in the survival of the parasite within mouse macrophages. The role of gp63 genes 1-6 i n parasite development within the sandfly vector was studied. GP63 1-6 mutants grew normally in the blood-engorged midgut of both Phlebotomu s argentipes and P. papatasi, However, both wild-type and mutant proma stigotes were lost after 2 days' growth in P. papatasi. The complete d evelopmental pathway in P. argentipes was observed for wild-type proma stigotes, GP63 1-6 mutants and mutants transfected with gp63 gene 1, N ormal stage differentiation from amastigotes to procyclics, to nectomo nads, to haptomonads and to infectious metacyclics was observed, Thus, the highly expressed promastigote forms of GP63, encoded by gp63 gene s 1-6, do not appear to be required for nutrient utilization in the bl oodmeal during the early stages of development in the sandfly or for m idgut attachment and further development, gp63 1-6 genes do, however, play a major protective role against complement-mediated lysis when pr omastigotes are introduced into the mammalian host.