Expressed sequence tags (ESTs) analysis of Acanthamoeba healyi
Article information
Abstract
Randomly selected 435 clones from Acanthamoeba healyi cDNA library were sequenced and a total of 387 expressed sequence tags (ESTs) had been generated. Based on the results of BLAST search, 130 clones (34.4%) were identified as the genes enconding surface proteins, enzymes for DNA, energy production or other metabolism, kinases and phosphatases, protease, proteins for signal transduction, structural and cytoskeletal proteins, cell cycle related proteins, transcription factors, transcription and translational machineries, and transporter proteins. Most of the genes (88.5%) are newly identified in the genus Acanthamoeba. Although 15 clones matched the genes of Acanthamoeba located in the public databases, twelve clones were actin gene which was the most frequently expressed gene in this study. These ESTs of Acanthamoeba would give valuable information to study the organism as a model system for biological investigations such as cytoskeleton or cell movement, signal transduction, transcriptional and translational regulations. These results would also provide clues to elucidate factors for pathogenesis in human granulomatous amoebic encephalitis or keratitis by Acanthamoeba.
INTRODUCTION
The genus, Acanthamoeba, a human pathogen causing granulomatous amoebic encephalitis (GAE) and amebic keratitis (Sisson et al., 1994), has been known to be a vector for pathogenic microbes such as Mycobacterium spp., Listeria spp., and Legionella spp. (Jadin, 1973; Ly & Muller, 1990; Field, 1991). In addition to these medical importances, it is also well known that Acanthamoeba is a good model system to study eukaryotic cell biology due to its relatively large size, rapid growth in culture, active motility, and well developed cytoskeleton (Byers et al., 1990).
Although the ploidy and the total DNA content of the genus Acanthamoeba are unclear at the present, Byers et al. (1990) speculated that the genome size of the amoeba would be ~1×108 bp of which the size could express more than 5,000 transcripts; however, only a few genes and proteins have been reported. Most of the genes identified are 18s rDNA sequences for the taxonomic purpose (Gast et al., 1996; Stothard et al., 1998), actin, myosins and actin binding proteins to study cytoskeleton (Nellen & Gallwitz, 1982; Cooper et al., 1986; Jung et al., 1989; Pollard & Rimm, 1991; Kelleher et al., 1995; Lee et al., 1999), and mitochondrial genome (Burger et al., 1995). Therefore most genes of Acanthamoeba have yet to be uncovered.
Characterization of the transcribed genes in a certain organism by expressed sequence tag (EST) analysis, single pass sequencing of randomly selected cDNA clones has been applied to many organisms including parasitic protozoa such as Plasmodium falciparum (Chakrabarti et al, 1994), Trypanosoma brucei rhodesiense (El-Sayed et al., 1995), Toxoplasma gondii (Wan et al., 1996; Manger et al., 1998), and Entamoeba histolytica (Azam et al., 1996; Tanaka et al., 1997). In addition to the candidate genes for pathogenic factors, many novel genes for stage specific, cell cycle regulatory, or related to cell signaling were identified in these protozoa by EST analysis.
ESTs analysis of Acanthamoeba could characterize the expression pattern of the genes, providing invaluable information to understand the genetics and the identification of many novel genes in this genus.
In the present study, we report the results of ESTs analysis of A. healyi OC-3A strain isolated from the brain of a GAE patient.
MATERIALS AND METHODS
Amoeba culture
An isolate of Acanthamoeba from the brain of a GAE patient, A. healyi OC-3A, was obtained from ATCC and cultured in Proteose peptone-Yeast extract-Glucose medium at 25℃.
RNA preparation
Trophozoites of A. healyi, washed with phosphate-buffered saline (PBS), were homogenized with β-mercaptoethanol and RNA denaturation solution (Stratagene, San Diego, CA, USA). One milliliter of 3 M sodium acetate (pH 4.0) was added to the solution and mixed by inversion. Water saturated phenol was added and mixed well with the solution. The mixture was shaken vigorously after adding chloroform/isoamyl alcohol (24:1), and then was incubated on ice for 15 min. The supernatant of the mixture after centrifugation was moved to a new tube and incubated with 1 volume of isopropanol at -20℃ for 1 hr for precipitation of the RNA. The RNA pellet was dissolved with the RNA denaturation solution and reprecipitated with equal volume of isopropanol. DEPC treated Q-water was added to the ethanol washed and vacuum dried RNA pellet. mRNA was purified from total RNA sample using Poly (A) Quick mRNA isolation kit (Stratagene, San Diego, CA, USA).
Construction of cDNA library
A unidirectional oligo (dT)-primed EcoRI/Xho I cDNA library was constructed in UNI-ZAP™ (Stratagene, San Diego, CA, USA). Briefly, first strands, synthesized by reverse transcription of mRNA of A. healyi, were used to make second strands by the action of RNase H and DNA polymerase I. cDNA with blunted termini were ligated with Eco RI adapters and digested with Xho I. After size fractionation, cDNA over the size 400 bp were packaged into UNI-ZAPTM XR vector arms by ligation. Packaged cDNAs were incubated with the host cell of XL1-blue MRF strain on agarose LB medium. More than 5 millions clones of this library were amplified and the aliquots of the library were stored at 4℃ until use.
Sequencing of randomly selected cDNA clones
cDNA library mixture were incubated with XL1-Blue MRF cell to allow in vivo excision using ExAssist helper phage, transfected into Escherichia coli SOLR strain, and plated on LB-ampicillin agar plates. Each randomly selected colony from the plates was inoculated into LB-ampicillin broth and incubated at 37℃ overnight. Plasmid with the cDNA insert was extracted with plasmid DNA purification system (Wizard® Plus Minipreps, Promega, USA). The size of cDNA inserts obtained by digestion of the plasmid DNA with EcoR I and Xho I were estimated by electrophoresis on agarose gel with Hind III digested λ phage DNA, a DNA size standard. Alkaline denaturation of the plasmid DNA and the dideoxy chain termination method using DNA sequencing kit (T7 Sequenase version 2.0, Amersham, USA) and 35S dATP were applied to elucidate the sequence data of the randomly selected cDNA clones. After electrophoresis of the reaction samples for sequencing, the vacuum dried acrylamide gel was exposed on X-ray film. Sequence data were edited to remove vector and ambiguous sequences, and less than 100 bases were also rejected.
Basic Local Alignment Search Tool (BLAST) search
The sequence data of cDNA clones by random partial sequencing were subjected to examine similarities in the nucleic acid and protein databases using the BLAST on the National Center for Biotechnology Information (NCBI). The cDNA sequences were compared against nucleotide data by the program BLASTN, and the conceptual translation products of query sequences against translated nucleic acid and protein databases by BLASTX. Matches were considered to be significant only when the probability (P) was less than 0.0001 and scores were >160 for BLASTN and >80 for BLASTX.
RESULTS
Among 435 cDNA clones sequenced, the sequence data of 378 clones were submitted for blast search (Table 1). It was 130 clones (34.4%) identified by high homology with the DNA sequence of Acanthamoeba or other organisms in the public data base. Although 15 clones were matched with Acanthamoeba genes already studied, they were just 3 kinds of genes and twelve of them were the actin gene, the most commonly found gene in this study. The other 115 clones (88.5%, 94 different genes) are reported in the present paper for the first time in this genus.
Acanthamoeba healyi cDNA library sequencing
Based on the results of BLAST search, ESTs with predicted or known functions were classified into putative cellular roles (Table 2). They were 4 clones for surface protein, 2 clones for DNA metabolism, 12 clones for energy metabolism, 10 clones for kinase and phosphatase, 17 clones for other metabolism, 1 clone for protease, 10 clones for signal transduction, 18 clones for structure and cytoskeleton, 3 clones for cell cycle related proteins, 5 clones for transcription factor, 32 clones for transcription and translational machinery, 4 clones for transporters, and 12 clones for not classified.
Significant matches of Acanthamoeba healyi ESTs with database sequences from other organisms
DISCUSSION
It is the first time to analyze ESTs of Acanthamoeba known as an human pathogen and a good model system for biological studies. The most frequently presented gene was actin gene which appeared 12 times. This was as expected because Acanthamoeba has well developed cytoskeleton and move very actively (Pollard, 1982). The biggest class among identified clones was genes for protein synthesis with the number of 32 from 130 clones. Similar results were reported in the EST analysis of Entamoeba histolytica, Typanosoma cruzi, and E. dispar (Tanaka et al., 1997; Verdun et al., 1998; Sharma et al., 1999).
Among four clones of genes for surface protein, two were identified as non-integrin type laminin binding protein. Adhesion would be the very first and important step to infect host by tissue invading parasites. Laminin binding proteins has been reported from several parasitic organisms including Trichomonas vaginalis (Silva Filho et al., 1988), Trypanosoma brucei rhodesiense (gene bank, W99296), Leishmania donovani (Ghosh et al., 1996) and Echinococcus granulosus (Zhang et al., 1997). The ability to recognize extracellular matrix proteins such as laminin or fibronectin has been known to correlate with invasiveness (Silva Filho et al., 1988; Ghosh et al., 1999). Further characterization of laminin binding protein in A. healyi would help to discover the mechanisms of the invasion by the amoeba.
A lot of genes for proteins involved in various metabolism were found in Acanthamoeba for the first time in this study except for the ubiquitin (Ub) gene (Hu & Henney, 1997). Six clones were identified to be associated with Ub-proteasome protein destruction system. Ub-proteasome pathway of intracellular proteolysis has been shown to be involved in various biologically important processes, such as the cell cycle, cellular metabolism, apoptosis, signal transduction, immune response, and protein quality control (Hilt and Wolf, 1996; Ciechanover, 1998; Tanaka, 1998).
Little information has been reported for signal transduction in genus Acanthamoeba. In this study, many kinds of cell signaling molecules, including Rabs, 14-3-3 protein and rac, were identified. Rabs regulate the flux through individual steps of the intracellular membrane trafficking pathway. The small GTPase Rab2 is a resident of pre-Golgi intermediates and required for protein transport from the endoplasmic reticulum to the Golgi complex (Tisdale et al., 1992). The GTP binding motif, GDTGVGKS, was conserved in the sequence of the clone Ahc040 identified as Rab2. A Rab protein isolated by EST analysis had been characterized in Trypanosoma brucei by Field et al. (1999).
In addition, gene for prohibitin known to negatively regulate cell proliferation in mammals was identified in Acanthamoeba. Further studies on this gene would give information in the regulation of Acanthamoeba proliferation and development. Three clones were identified as gene for vault protein showing high homology with that of Dictyostelium discoideum. Although vault proteins are found in nearly all eukaryotic cells, the function of the protein has yet to be elucidated.
Lots of the genetic information of Acanthamoeba obtained in this study would be very helpful to figure out pathogenetic mechanisms of GAE or keratitis by Acanthamoeba and to develop therapeutic reagents specific to the amoeba.
Notes
This study was in part supported by a grant from Kyungpook National University.
Note: EST sequence data reported in this paper is available in GenBankTM database under the accession number from AT001240 to AT001616.