A novel chemotherapy in development for Chagas' disease targets cruzain, the major cysteine protease of Trypanosoma cruzi. Peptidomimetic inhibitors disrupt the intracellular cycle of the parasite and rescue animals from a lethal infection. Inhibitor killing of parasites results from interruption of autocatalytic cruzain processing and transport to lysosomes, and massive accumulation of precursor protein in the Golgi complex. To further understand the mechanisms of protease processing and transport in this primitive eukaryote, and uncover potential mechanisms for resistance to these drugs, we generated cysteine-protease inhibitor (CPI)-resistant epimastigotes in vitro and investigated the mechanisms involved at the biochemical and structural levels. Resistance to 20-fold the lethal CPI concentration, achieved after a year of gradual drug increase, was accompanied by a modest decrease in growth rate. A marked increase in the number of vesicles trafficking from the Golgi complex to the flagellar pocket occurs in resistant cells. No mature protease reaches lysosomes though accumulation of endocytosed gold particles in lysosomes appears to be normal. Higher molecular mass cruzain species, consistent with complexes of cruzain precursors and inhibitor, are secreted by CPI-resistant parasites into the culture supernatant. Release of these cruzain precursors may be facilitated by an enhanced acidification of trans-Golgi cisternae in resistant parasites. The pH within Golgi cisternae is higher in control epimastigotes and most mature cruzain is lysosomal. Cruzain activity is negligible in CPI-resistant epimastigote extracts compared to the parental clone. Activity is restored following withdrawal of the inhibitor. No cross-resistance to the therapeutic drugs nifurtimox and benznidazole occurred and, conversely, parasites resistant to these drugs were sensitive to CPI. Protease inhibitors are thus potential therapeutical alternatives in cases of nifurtimox/benznidazole resistance. Cumulatively, these results suggest that CPI-resistance induces upregulation of Golgi complex function and post-Golgi secretory pathway, and release of precursors before the enzyme reaches its site of biologic activity.

REFERENCES

Andrade
S. G.
,
Stocker-Guerret
S.
,
Pimentel
A. S.
,
Grimaud
J. A.
(
1991
).
Reversibility of cardiac fibrosis in mice chronically infected with Trypanosoma cruzi under specific chemotherapy.
Mem. Ins. Osw. Cruz
86
,
187
200
Apt
W.
,
Aguilera
X.
,
Arribada
A.
,
Perez
C.
,
Miranda
C.
,
Sanchez
G.
,
Zulantay
I.
,
Cortes
P.
,
Rodriguez
J.
,
Juri
D.
(
1998
).
Treatment of chronic Chagas' disease with itraconazole and allopurinol.
Am. J. Trop. Med. Hyg
59
,
133
138
Borst
P.
,
Ouellette
M.
(
1995
).
New mechanisms of drug resistance in parasitic protozoa.
Annu. Rev. Microbiol
49
,
426
460
Brener
Z.
(
1979
).
Present status of chemotherapy and chemoprofilaxis of human trypanosomiasis in the western hemisphere.
Pharmacol. Ther
7
,
71
90
Burleigh
B. A.
,
Caler
E. V.
,
Webster
P.
,
Andrews
N. W.
(
1997
).
A cytosolic serine endopeptidase from Trypanosoma cruzi is required for the generation of Ca2+signaling in mammalian cells.
J. Cell Biol
136
,
609
620
Cazzulo
J. J.
,
Hellman
U.
,
Couso
R.
,
Parodi
A. J.
(
1990
).
Amino acid and carbohydrate composition of a lysosomal cysteine proteinase from Trypanosoma cruzi. Absence of phosphorylated mannose residues.
Mol. Biochem. Parasitol
38
,
41
48
Chan
V. J.
,
Selzer
P. M.
,
McKerrow
J. H.
,
Sakanari
J.
(
1999
).
Expression and alteration of the S2 subsite of the Leishmania major cathepsin B-like cysteine protease.
Biochem. J
340
,
113
117
Demaurex
N.
,
Furuya
W.
,
D'Souza
S.
,
Bonifacino
J. S.
,
Grinstein
S.
(
1998
).
Mechanism of acidification of the trans-Golgi network (TGN). In situ measurements of pH using retrieval of TGN38 and furin from the cell surface.
J. Biol. Chem
273
,
2044
2051
Docampo
R.
(
1990
).
Sensitivity of parasites to free radical damage by antiparasitic drugs.
Chem. Biol. Interact
73
,
1
27
Doyle
P. S.
,
Dwyer
D. M.
(
1993
).
Leishmania: Immunochemical comparison of the secretory (extracellular) acid phosphatases from various species.
Exp. Parasitol
77
,
435
444
Doty
S. B.
,
Smith
C. E.
,
Hand
A. R.
,
Oliver
C.
(
1977
).
Inorganic trimetaphosphate as a histochemical marker for lysosomes in light and electron microscopy.
J. Histochem. Cytochem
25
,
1381
1384
Eakin
A. E.
,
Mills
A. A.
,
Harth
G.
,
McKerrow
J. H.
,
Craik
C. S.
(
1993
).
The sequence, organization, and expression of the major cysteine protease (cruzain) from Trypanosoma cruzi.
J. Biol. Chem
267
,
7411
7420
Engel
J. C.
,
Segura
E. L.
,
Dvorak
J. A.
,
Crane
M. S. J.
(
1982
).
Trypanosoma cruzi: biological characterization of 19 clones derived from two chronic chagasic patients. I. Growth kinetics in liquid medium.
J. Protozool
29
,
555
560
Engel
J. C.
,
Franke de Cazzulo
B. M.
,
Stoppani
A. O.
,
Cannata
J. J. B.
,
Cazzulo
J. J.
(
1987
).
Aerobic glucose fermentation by Trypanosoma cruzi axenic culture amastigote-like forms during growth and differentiation to epimastigotes.
Mol. Biochem. Parasitol
26
,
1
10
Engel
J. C.
,
Doyle
P. S.
,
Dvorak
J. A.
(
1990
).
Isolate dependent differences in the oxidative metabolism of Trypanosoma cruzi epimastigotes.
Mol. Biochem. Parasitol
39
,
69
76
Engel
J. C.
,
Doyle
P. S.
,
Palmer
J.
,
Hsieh
I.
,
Bainton
D. F.
,
McKerrow
J. H.
(
1998
).
Growth arrest of T. cruzi by cysteine protease inhibitors is accompanied by alterations in Golgi complex and ER ultrastructure.
J. Cell Sci
111
,
597
606
Engel
J. C.
,
Doyle
P. S.
,
Hsieh
I.
,
McKerrow
J. H.
(
1998
).
Cysteine protease inhibitors curean experimental Trypanosoma cruzi infection.
J. Exp. Med
188
,
725
734
Gardiner
P.
,
Dwyer
D. M.
(
1983
).
Radioiodination and identification of externally disposed membrane components of Leishmania tropica.
Mol. Biochem. Parasitol
8
,
283
295
Filardi
L. S.
,
Brener
Z.
(
1987
).
Susceptibility and natural resistance of Trypanosoma cruzi strains to drugs used clinically in Chagas' disease.
Trans. Royal Soc. Trop. Med. Hyg
81
,
755
759
Gorla
N. B.
,
Ledesma
O. S.
,
Barbieri
G. P.
,
Larripa
I. B.
(
1988
).
Assessment of cytogenetic damage in chagasic children treated with benznidazole.
Mutat. Res
206
,
217
220
Harth
G.
,
Andrews
N.
,
Miles
A. A.
,
Engel
J. C.
,
Smith
R.
,
McKerrow
J. H.
(
1993
).
Peptide-fluoromethyl ketones arrest intracellularreplication and intracellular transformation of Trypanosoma cruzi.
Mol. Biochem. Parasitol
58
,
17
24
Huete-Perez
J. A.
,
Engel
J. C.
,
Brinen
L. S.
,
Mottram
J. C.
,
McKerrow
J. H.
(
1999
).
Protease trafficking in two primitive eukaryotes is mediated by a prodomain protein motif.
J. Biol. Chem
274
,
16249
16256
Kirchhoff
L. V.
(
1993
).
American trypanosomiasis (Chagas' disease)- A tropical disease now in the United States.
New Eng. J. Med
329
,
639
644
Lowndes
C. M.
,
Bonaldo
M. C.
,
Thomaz
N.
,
Goldenberg
S.
(
1996
).
Heterogeneity of metalloprotease expression in Trypanosoma cruzi.
Parasitology
112
,
393
399
Matsudaira
P.
(
1987
).
Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes.
J. Biol. Chem
262
,
10035
10038
McGrath
M.
,
Eakin
A. E.
,
Engel
J. C.
,
McKerrow
J. H.
,
Craik
C. S.
,
Fletterick
R.
(
1995
).
The crystal structure of cruzain: A therapeutic target for Chagas' disease.
J. Mol. Biol
247
,
251
259
Murta
S. M. F.
,
Romanha
A. J.
(
1998
).
In vivo selection of a population of Trypanosoma cruzi and clones resistant to benznidazole.
Parasitology
116
,
265
271
Murta
S. M. F.
,
Gazzinelli
R. T.
,
Brener
Z.
,
Romanha
A. J.
(
1998
).
Molecular characterization of susceptible and naturally resistant strains of Trypanosoma cruzi to benznidazole and nifurtimox.
Mol. Biochem. Parasitol
93
,
204
214
Neal
R. A.
,
van Bueren
J.
(
1988
).
Comparative studies of drug susceptibility of five strains of Trypanosoma cruzi in vitro and in vivo.
Trans. R. Soc. Trop. Med. & Hyg
82
,
709
714
Nobrega
O. T.
,
Santos Silva
M. A.
,
Texeira
A. R. L.
,
Santana
J. M.
(
1998
).
Cloning and sequencing of tccb, a gene encoding a Trypanosoma cruzi cathepsin B-like protease.
Mol. Biochem. Parasitol
97
,
235
240
Parodi
A. J.
,
Labriola
C.
,
Cazzulo
J. J.
(
1995
).
The presence of complex-type oligosaccharides at the C-terminal domain glycosylation site of some molecules of cruzipain.
Mol. Biochem. Parasitol
69
,
247
255
Poore
T. E.
,
Barrett
S. G.
,
Kadin
M. E.
,
Bainton
D. F.
(
1981
).
Ultrastructural localization of acid phosphatase in rosetted T and B lympocytes of normal human blood.
Am. J. Pathol
102
,
72
83
Prescianotto-Baschong
C.
,
Riezman
H.
(
1998
).
Morphology of the yeast endocytic pathway.
Mol. Biol. Cell
9
,
173
189
Quan
S. G.
,
Poosawat
S. S.
,
Golde
D. W.
(
1980
).
Ultrastructure and tartrate-resistant acid phosphatase localization in a T-cell hairy-cell leukemia cell line.
J. Histochem. Cytochem
28
,
434
440
Roush
W. R.
,
Gwaltney
S. L.
II
,
Chebg
J.
,
Scheidt
K. A.
,
McKerrow
J. H.
,
Hansell
E.
(
1999
).
Vinyl sulfonate esters and vinylsulfonamides: Potent, irreversible inhibitors of cysteine proteases.
J. Am. Chem. Soc
120
,
10994
10995
Serveau
C.
,
Lalmanach
G.
,
Juliano
M. A.
,
Scharfstein
J.
,
Juliano
L.
,
Gautheir
F.
(
1996
).
Investigation of the substrate specificity of cruzipain, the major cysteine proteinase of Trypanosoma cruzi, through the use of cystatin-derived substrates and inhibitors.
Biochem. J
313
,
951
956
Sosa Estani
A.
,
Segura
E. L.
,
Ruiz
A. M.
,
Velazquez
E.
,
Porcel
B. M.
,
Yampotis
C.
(
1998
).
Efficacy of chemotherapy with benznidazole in children in the indeterminate phase of Chagas' disease.
Am. J. Trop. Med. Hyg
59
,
526
529
Ullman
B.
(
1995
).
Multi drug resistance and P-glycoproteins in parasitic protozoa.
J. Bioenerg. Biomem
27
,
77
84
Urbina
J. A.
,
Payares
G.
,
Molina
J.
,
Sonoja
C.
,
Liendo
A.
,
Lazardi
K.
,
Piras
M. M.
,
Piras
R.
,
Perez
N.
,
Wincker
P.
,
Ryley
J. F.
(
1996
).
Cure of short-and long-term experimental Chagas' disease using D0870.
Science
273
,
969
971
Viotti
R.
,
Vigliano
C.
,
Armenti
H.
,
Segura
E. L.
(
1994
).
Treatment of chronic Chagas' disease with benznidazole: Clinical and serological evolution of patients with long-term follow up.
Am. Heart J
127
,
151
162
Westen
H.
,
Bainton
D. F.
(
1979
).
Association of alkaline phosphatase positive reticulum cells in bone marrow with granulocytic precursors.
J. Exp. Med
150
,
919
937
Wiser
M. F.
,
Lonsdale-Eccles
J. D.
,
D'Alessandro
A.
,
Grab
D. J.
(
1997
).
A cryptic protease activity from Trypanosoma cruzi revealed by preincubation with kininogen.
Bioch. Bioph. Res. Commun
240
,
540
544
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