The intestinal protozoan parasite Entamoeba histolytica contains 20 cysteine protease genes, of which only a small subset is expressed during in vitro cultivation

Abstract

Cysteine proteases are known to be important pathogenicity factors of the protozoan parasite Entamoeba histolytica. So far, a total of eight genes coding for cysteine proteases have been identified in E. histolytica, two of which are absent in the closely related nonpathogenic species E. dispar. However, present knowledge is restricted to enzymes expressed during in vitro cultivation of the parasite, which might represent only a subset of the entire repertoire. Taking advantage of the current E. histolytica genome-sequencing efforts, we analyzed databases containing more than 99% of all ameba gene sequences for the presence of cysteine protease genes. A total of 20 full-length genes was identified (including all eight genes previously reported), which show 10 to 86% sequence identity. The various genes obviously originated from two separate ancestors since they form two distinct clades. Despite cathepsin B-like substrate specificities, all of the ameba polypeptides are structurally related to cathepsin L-like enzymes. None of the previously described enzymes but 7 of the 12 newly identified proteins are unique compared to cathepsins of higher eukaryotes in that they are predicted to have transmembrane or glycosylphosphatidylinositol anchor attachment domains. Southern blot analysis revealed that orthologous sequences for all of the newly identified proteases are present in E. dispar. Interestingly, the majority of the various cysteine protease genes are not expressed in E. histolytica or E. dispar trophozoites during in vitro cultivation. Therefore, it is likely that at least some of these enzymes are required for infection of the human host and/or for completion of the parasite life cycle.

FIG. 1.

Structural organization of 20 E. histolytica cysteine proteases. Numbers indicate the number of amino acid residues forming the predomains, prodomains, or catalytic domains.

FIG. 2.

Phylogenetic tree of all 20 E. histolytica cysteine proteases and representative family members of other organisms. The tree was generated using sequence alignments of the region between the active-site Cys and the active-site His as described in Materials and Methods. Human cathepsin B was used as an outgroup. Two distinct groups are clustered within the E. histolytica protease family (EhCP-A and EhCP-B). The accession numbers for the various proteases are as follows: EhCP1, Q01957; EhCP2, Q01958; EhCP3, CAA60673; EhCP4, CAA62833; EhCP5, CAA62835; EhCP6, CAA62835; EhCP7, CAC34069; EhCP8, AY156066; EhCP9, AY156067; EhCP10, AY156068; EhCP11, AY156096; EhCP12, AY156070; EhCP13, AY156071; EhCP14, AY156072; EhCP15, AY156073; EhCP16, Ay156074; EhCP17, AY156075; EhCP18, AY156076; EhCP19, AY156077; EhCP112, AAF04255; Brugia malayi, AAK16513; human cathepsin B, KHHUB; Dictyostelium discoideum 1, KHDO; Dictyostelium discoideum 2, ACC47482; Drosophila melanogaster, Q95029; Giardia lamblia cathepsin C (CatC), AAK97078; human cathepsin L, KHHUL; Leishmania mexicana, CAA44094; Litomosoides sigmodontis, AAK16515; Mus musculus, NP034114; Naegleria fowleri, AAB01769; Onchocerca volvulus, AAK16514; papain, P00784, Plasmodium falciparum, A45624; Schistosoma mansoni, CAA83538; Trypanosoma brucei, S07051; Trypanosoma congolense, S37048; Trypanosoma cruzi, P25779; and Trichomonas vaginalis, S41427.

FIG. 3.

Comparison between the N termini of the various E. histolytica cysteine protease catalytic domains. Conserved amino acid residues (>50% identity) are printed in bold type, and the active-site residues Gln and Cys are marked by asterisks. For optimal alignment, gaps were introduced into the sequences.

FIG. 4.

Alignment of the ERFNIN motifs located within the prodomains of the various E. histolytica cysteine proteases. Conserved amino acid residues are printed in bold type.

FIG. 5.

Comparison of the E. histolytica cysteine protease sequences spanning the region around active-site residues His and Asn (both marked by asterisks). Conserved amino acid residues are printed in bold type. For optimal alignment, gaps were introduced into the sequences.

FIG. 6.

Southern blot analysis of E. histolytica and E. dispar genomic DNA. Total genomic DNAs of either the E. histolytica isolate HM-1:IMSS or the E. dispar isolates SAW 760 were digested using HincII, HindIII, and NdeI, separated on agarose gels, and blotted onto nylon membranes. The blots were sequentially hybridized with the coding regions of all 20 E. histolytica cysteine protease genes. Hybridization and washing were performed as described in Materials and Methods. Size markers are indicated on the left. They represent 10, 8, 6, 5, 4, 3.5, 3, 2, 1, and 0.5 kb.

FIG. 7.

Expression of the various cysteine protease genes in the E. histolytica isolate HM-1: IMSS and the E. dispar isolate SAW 760. (A) Total cellular RNA of each isolate were separated on formaldehyde-agarose gels, blotted onto nylon membranes, and hybridized with the coding region of the various E. histolytica cysteine protease genes as indicated. (B and C) As controls, the blots were stripped and sequentially hybridized with ehcp9 (B) and an E. histolytica actin gene probe (C).