Acanthamoeba healyi, originally isolated from the brain of a GAE patient, was obtained from ATCC (ATCC # 30866), and grown axenically in peptone-yeast extract-glucose (PYG) medium at 25℃. Amoebae of approx. 4.0×10⁶ cells/ml were centrifuged at 1100 rpm for 10 min at room temperature. The cell pellet was subjected to extraction of RNA and DNA for further study.

Acanthamoeba healyi mRNA was prepared from trophozoites using the mRNA isolation kit (Qiagen, Germany) following the manufacturer's instruction. cDNA was synthesized using a ZAP-cDNA synthesis kit (Stratagene, California, U.S.A.). Following EcoR I adaptor ligation to the 5' terminus and Xho I digestion, the cDNA was inserted into the EcoR I-Xho I site of lambda ZAP (Stratagene, California, U.S.A.). Degenerate oligonucleotide primers for reverse transcription-PCR of A. healyi gene encoding cysteine proteinase were CPP5'(5'-ACAGAATTCCARTGYGGITCITGG-3') and CPP 3'(5'-TTAAAGCTTCCATTYTTIACRATCCARTA-3'), based on the amino acid sequences of the active sites conserved in C1 family of cysteine proteinases. Amplification reaction was 30 cycles of denaturation at 94℃ for 1min, annealing at 55℃ for 1 min, and extension at 72℃ for 1min, and the final extension step was 5 min at 72℃ in a DNA Thermal Cycler (Model 2400, Perkin-Elmer Cetus). The amplified DNA fragment was gel-purified and cloned into the Sma I-digested pBluescript SK(-) vector (Stratagene, California, U.S.A.). Recombinant phages were spread on agar plates and lifted to nylon membranes in duplicate. Membranes were hybridized to the cDNA insert of AhCP1, which had been labeled with [α-³²P]dCTP using random primed DNA labeling kit (Boehringer Mannheim, Germany). The library was screened by standard methods. Co-infection with Exassist helper phage was used to rescue pBluescript phagemid from plaque-purified phage according to the manufacturer's instructions. All DNA sequencing was performed by the dideoxynucleotide method using custom synthesized primers.

Homology search was performed with the Basic Local Alignment Search Tool (BLAST) program of the National Center for Biotechnology Information, National Institutes for Health (Altschul et al., 1990; Altschul et al., 1994). The cleavage site of the AhCP1 signal peptide was predicted following Nielson et al. (1997). Phylogeny was obtained from the alignment of 26 cysteine proteinases using CLUSTALW 1.7 program with a low gap penalty using the European Bioinformatics Institute server (Pearson and Lipman, 1988). The sequence alignment of catalytic domains of cysteine proteinases was performed with the corresponding regions of various organisms (Siezen and Leunissen, 1997).

Total RNA from A. healyi OC-3A, Acanthamoeba sp. KA/E1 and KA/E2, two corneal isolates, KA/L5 and KA/S8, two environmental isolates, A. castellanii Castellani, A. polyphaga, A. castellanii Ma, and A. mauritaniensis was extracted. Poly(A)+ RNA was denatured and separated on an 1.1% agarose gel containing 6.7% formaldehyde, and transferred to a Hybond-N+ nylon membrane (Amersham, U.S.A.). Oligonucleotide primers for PCR amplification of the catalytic domain of cysteine proteinase were CYS (5'-CAGGGTCAGTGCGGCTCGTGCTGGTCGTTCTCGACC-3') and HIS (5'-GCCCCAGCCGACGACGAGCACACCGTGGTCGAG-3'), based on the amino acid sequences Gln¹³² to Thr¹⁴³ and Leu²⁷⁵ to Gly²⁸⁵ of AhCP1. The 462 bp PCR product of AhCP1 labeled by the random prime labeling method (Boehringer Mannheim, Germany) was used as a probe in standard Northern hybridization analysis (Sambrook and Russell, 2001)

To generate a cDNA fragment of Acanthamoeba cysteine proteinase, RT-PCR using degenerate primers specific for cysteine proteinases was carried out with mRNA of A. healyi. The oligonucleotide primers were designed based on the amino acid sequences of cysteine proteinase. PCR primers, CPP5' (sense) and CPP3'(antisense) were based on the conserved motifs flanking the catalytic cysteine and asparagine residues Gln¹⁹ to Trp²⁶ and Tyr¹⁷⁰ to Trp¹⁷⁷ respectively of the papain superfamily (C1) of cysteine proteinase. The PCR reaction using degenerate primers yields a cDNA fragment of approximately 500 bp (Fig. 1). The cDNA was sequenced and a comparison of the deduced amino acid sequence with the protein databases indicated strong homology with a number of cathepsin L like cysteine proteinases. To generate the full length cDNA sequence of cysteine proteinase, the cDNA fragment was used as probe for screening the A. healyi cDNA library. After three rounds of stringent hybridization, six potentially positive clones were selected. When the insert DNAs were hybridized with the cDNA fragment by Southern blot analysis, all cDNA clones hybridized to the probe and were confirmed to be the products of a single gene by sequence analysis. The largest insert had a single open reading frame of 990 bp, predicting a protein of 330 amino acid residues (Fig. 2).

Based on homology searches using BLASTP, the deduced amino acid sequence of the cloned gene clearly belongs to the C1 peptidase (papain superfamily) proteinase clan as defined by Rawlings and Barrett (1994). Thus, the cloned gene was termed as Acanthamoeba healyi cysteine proteinase (AhCP1). When the amino acid sequence was aligned with C1 peptidase previously reported from eukaryotes or prokaryotes, AhCP1 was homologous at the core catalytic triad residues, Cys²⁵, His¹⁵⁹, and Asn¹⁷⁵, cysteine proteinase defining sequence domains (Fig. 3). The active site of AhCP1 has great homology with those cysteine proteinase; 73% similarity with cysteine proteinase of Sarcophaga. Among mammalian cathepsin L and S, it displays 70% similarity with boar cathepsin L, 69% similarity with cattle, 73% with mouse and 71% with human cathepsin L, and 70% with cathepsin S. Phylogenetic analysis in this study suggested that AhCP1 belongs to the cathepsin L subgroup of the papain superfamily (Fig. 4). Sequence comparisons with other cathepsin L proteinase also suggested that Ahcp1, like majority of cysteine proteinase, could be formed as a pre-pro-protein precursor. The pre-region cleavage site of the signal sequence was predicted to be between Ala²⁰ and Ala²¹ (Fig. 2, one downward arrow) by weight matrix analysis (Nielson et al., 1997). The pro-region cleavage site was predicted between Asn⁹² and Phe⁹³ (Fig. 2, double downward arrows). Cleavage of pre-pro-region would reduce the molecular mass of Ahcp1 from a predicted 330 amino acid protein with a molecular mass of 36 kDa to a 216 amino acid mature protein with a molecular mass of 24 kDa. As in the other proteinases of C1 family, there was a motif GCNGG (residues 64-68) which is suggested to have an important structural role. The enzyme has neither N-terminal nor C-terminal extension observed in Plasmodium falciparum and Leishmania major cysteine proteinases. AhCP1 had a highly conserved interspersed amino acid motif, the ERFNIN motif (EX3RX2(V/I) FX2NX3IX3N) in the propeptide region (Fig. 2 & 3).

By Northern blot analysis of A. healyi poly (A+) mRNA, a single band of approximately 1.8 kb transcript was detected, thus supporting that the cloned cDNA should be a full-length (Fig. 5). To compare the level of mRNA expressions in pathogenic and nonpathogenic Acanthamoeba spp., total RNA from 9 different isolates were probed with the PCR product of AhCP1. As a control, the blot was reprobed with a fragment of rRNA cDNA of A. healyi, and intensities of the hybridization signals showed that comparable quantities of total RNA were present in all samples. There was no significant differences among isolates from infected cornea (KA/E1, KA/E2), contact lens storage cases (KA/L5) or infected brain (A. healyi OC-3A). Interestingly, two isolates from soil (KA/S8 and KA/CS) showed higher expression than other isolates (Fig. 5).