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.