Use of recombinant Entamoeba histolytica cysteine proteinase 1 to identify a potent inhibitor of amebic invasion in a human colonic model.
Cysteine proteinases are key virulence factors of the protozoan parasite Entamoeba histolytica. We have shown that cysteine proteinases play a central role in tissue invasion and disruption of host defenses by digesting components of the extracellular matrix, immunoglobulins, complement, and cytokines. Analysis of the E. histolytica genome project has revealed more than 40 genes encoding cysteine proteinases. We have focused on E. histolytica cysteine proteinase 1 (EhCP1) because it is one of two cysteine proteinases unique to invasive E. histolytica and is highly expressed and released. Recombinant EhCP1 was expressed in Escherichia coli and refolded to an active enzyme with a pH optimum of 6.0. We used positional-scanning synthetic tetrapeptide combinatorial libraries to map the specificity of the P1 to P4 subsites of the active site cleft. Arginine was strongly preferred at P2, an unusual specificity among clan CA proteinases. A new vinyl sulfone inhibitor, WRR483, was synthesized based on this specificity to target EhCP1. Recombinant EhCP1 cleaved key components of the host immune system, C3, immunoglobulin G, and pro-interleukin-18, in a time- and dose-dependent manner. EhCP1 localized to large cytoplasmic vesicles, distinct from the sites of other proteinases. To gain insight into the role of secreted cysteine proteinases in amebic invasion, we tested the effect of the vinyl sulfone cysteine proteinase inhibitors K11777 and WRR483 on invasion of human colonic xenografts. The resultant dramatic inhibition of invasion by both inhibitors in this human colonic model of amebiasis strongly suggests a significant role of secreted amebic proteinases, such as EhCP1, in the pathogenesis of amebiasis.
Meléndez-López
Samuel G
SG
Department of Pathology, University of California, San Diego, San Diego, California 92103-8416, USA.
Herdman
Scott
S
Hirata
Ken
K
Choi
Min-Ho
MH
Choe
Youngchool
Y
Craik
Charles
C
Caffrey
Conor R
CR
Hansell
Elisabeth
E
Chávez-Munguía
Bibiana
B
Chen
Yen Ting
YT
Roush
William R
WR
McKerrow
James
J
Eckmann
Lars
L
Guo
Jianhua
J
Stanley
Samuel L
SL
Reed
Sharon L
SL
D000818:Animals; D003106:Colon / Q000469:parasitology* / Q000473:pathology; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000378:metabolism*; D004195:Disease Models, Animal; D004748:Entamoeba histolytica / Q000201:enzymology* / Q000472:pathogenicity* / Q000502:physiology; D004789:Enzyme Activation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D051379:Mice; D016513:Mice, SCID; D011487:Protein Conformation; D017510:Protein Folding; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013450:Sulfones / Q000737:chemistry / Q000378:metabolism; D014183:Transplantation, Heterologous; D037521:Virulence Factors / Q000235:genetics / Q000378:metabolism
D015853:Cysteine Proteinase Inhibitors; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D013450:Sulfones; D037521:Virulence Factors; C009873:divinyl sulfone; D003546:Cysteine Endopeptidases; C056657:cysteine proteinase I