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proteinases have been generated. Null
mutants for the gene encoding CPa cysteine
proteinase exhibit a wild-type phenotype,
suggesting that the enzyme is not essential for
viability. Null mutants for CPb cysteine tandem
arrays also are able to grow and differentiate,
although their macrophage infectivity
in vitro is considerably reduced, and lesions
caused in mice are much smaller than those
caused by the wild type. Re-incorporation of
one of the Type I genes to the null mutant
restores full infectivity. Double-null mutants
for CPa and CPb have a phenotype similar to
that of the CPb null mutants. Mutants null for
CPc also are considerably less infective to
macrophages in vitro, but only a little less
infective than wild type parasites to BALB/c
mice. Since the low infectivity CPa/CPb
mutants did not produce an established disease,
but were able to elicit a protective
immune response, these mutants have been
proposed as candidates for live attenuated
vaccines against leishmaniasis.
E. histolytica pathogenesis requires penetration
of the trophozoite through the intestinal
mucosal cells and the basal membrane. The
amoebal cysteine proteinases are highly active
with collagen types I, IV and V, fibronectin
and laminin, which is consistent with the participation
of these enzymes in penetration.
Although several of the cysteine proteinase
genes present in pathogenic E. histolytica are
also present in the non-pathogenic Entamoeba
dispar, there seems to be a clear correlation
between the secretion of cysteine proteinases
and virulence. The gene encoding CP5, an
E. histolytica cysteine proteinase able to interact
with membranes but still retain its proteolytic
activity, is missing in E. dispar. Neutral
cysteine proteinases are able to degrade human
IgGs, in addition to their participation in tissue
invasion, thus protecting the parasite against
the immune response of the host.
The role of the cysteine proteinase activities
present in T. vaginalis is still not well understood,
but some membrane-bound forms have
been linked to adhesion to epithelial cells and
cytotoxicity. Some of the secreted proteinases
seem to participate in the degradation of
immunoglobulins (serum and secreted IgA and
IgG), and may be involved in defensive mechanisms
of the parasite against the immune system
of the host. A G. lamblia cathepsin B-like
cysteine proteinase seems to be involved in the
excystation of the trophozoite.
Parasite proteinases as targets for
chemotherapy
Over the last ten years, a number of studies have
suggested that some parasite proteinases may
be excellent targets for the development of new
drugs for malaria, leishmaniasis, and Chagas
disease. Cruzipain inhibitors are effective in
killing the parasite in vitro, and also in effectively
treating the disease in experimental
animals. McKerrow and his co-workers have
tested two vinyl sulfone inhibitors, designed to
increase selectivity towards cruzipain, increase
their half-life in vivo, provide oral bioavailability
and reduce toxicity, as compared with the
previously used dipeptide-based inhibitors, on
T. cruzi-infected mice. These compounds rescued
all animals from lethal acute infection,
and those exhibiting chronic infections were
cured by a second treatment. It is noteworthy
that these inhibitors also selectively arrest
replication of other protozoan pathogens, like
L. major and P. falciparum, without untoward
toxicity to the host.
Similarly, inhibitors of both falcipains and
plasmepsins kill the intraerythrocytic stages of
P. falciparum, by inhibiting the intravacuolar
proteolysis of globin. Recent studies have
suggested that the use of both cysteine and
aspartic proteinase inhibitors has a synergistic
effect, both in vitro, on cultured parasites, and
BIOCHEMISTRY AND CELL BIOLOGY: PROTOZOA