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Originally published In Press as doi:10.1074/jbc.M705975200 on August 30, 2007

J. Biol. Chem., Vol. 282, Issue 43, 31789-31802, October 26, 2007
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Identification and Biochemical Characterization of Unique Secretory Nucleases of the Human Enteric Pathogen, Entamoeba histolytica*Formula

Glen C. McGugan, Jr.1, Manju B. Joshi2, and Dennis M. Dwyer3

From the Cell Biology Section, Laboratory of Parasitic Diseases, Division of Intramural Research, NIAID, National Institutes of Health, Bethesda, Maryland 20892-0425

Received for publication, July 20, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The ancient eukaryotic human pathogen, Entamoeba histolytica, is a nucleo-base auxotroph (i.e. lacks the ability to synthesize purines or pyrimidines de novo) and therefore is totally dependent upon its host for the supply of these essential nutrients. In this study, we identified two unique 28-kDa, dithiothreitol-sensitive nucleases and showed that they are constitutively released/secreted by parasites during axenic culture. Using several different molecular approaches, we identified and characterized the structure of EhNucI and EhNucII, genes that encode ribonuclease T2 family proteins. Homologous episomal expression of epitope-tagged EhNucI and EhNucII chimeric constructs was used to define the functional and biochemical properties of these released/secreted enzymes. Results of coupled immunoprecipitation-enzyme activity analyses demonstrated that these "secretory" enzymes could hydrolyze a variety of synthetic polynucleotides, as well as the natural nucleic acid substrate RNA. Furthermore, our results demonstrated that sera from acutely infected amebiasis patients recognized and immunoprecipitated these parasite secretory enzymes. Based on these observations, we hypothesize that within its host, these secretory nucleases could function, at a distance away from the parasite, to harness (i.e. hydrolyze/access) host-derived nucleic acids to satisfy the essential purine and pyrimidine requirements of these organisms. Thus, these enzymes might play an important role in facilitating the survival, growth, and development of this important human pathogen.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The ancient eukaryotic human parasite, Entamoeba histolytica, is the causative agent of invasive amebiasis in humans infecting 10% of the world population and resulting in over 100,000 deaths annually, chiefly from amebic liver abscess (1). Infection is acquired through the ingestion of the quadranucleate cyst form of the organism from fecally contaminated food or water supplies. Excystation occurs in the small intestine, releasing immature trophozoites, which move into and colonize the nutrient-poor large intestine.

As ancient eukaryotes, Entamoeba parasites contain only rudimentary endoplasmic reticulum and Golgi apparatus (2, 3). Despite this, vesicle trafficking pathways appear to be essential to the pathogenesis of these parasites as (i) uptake and digestions of nutrients by mature trophozoites in the large intestine, (ii) invasion of the intestinal epithelium, (iii) delivery of cyst wall components during encystation, and (iv) dissemination and establishment of extra-intestinal infections all rely on endocytosis and secretion. In addition, these parasites lack many traditional eukaryotic biochemical pathways, including the de novo biosynthesis of purine and pyrimidine nucleo-bases. As a result, these parasites are completely dependent upon their host for the supply of these essential preformed nutrients. In that regard, it has been reported previously that Entamoeba are capable of salvaging both purines and pyrimidines from the extracellular milieu during in vitro culture, presumably through the action of membrane transporters (4, 5). However, to date, no report exists to indicate that these organisms might be capable of producing and releasing/secreting nucleotide salvage enzymes (e.g. nucleotide hydrolases, nucleases, or other nucleic acid hydrolases) into their host environments to aid in accessing these vital nutrients.

To that end, in this study we identified, for the first time, unique nuclease activities that were released/secreted by E. histolytica parasites during their growth in vitro. Based on those observations, we used several molecular approaches to identify and characterize the structure of two genes, EhNucI and EhNucII, that encode T2 family ribonucleases from these parasites. Epitope-tagged EhNucI and EhNucII chimeric constructs were used in a homologous expression system to delineate the functional and biochemical properties of these unique parasite released/secreted enzymes.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—All reagents and chemicals used in this study were of analytical grade and were obtained from Sigma unless otherwise specified. Enzymes used for molecular studies were obtained from Roche Applied Science; DNA and RNA molecular mass standards were from Invitrogen. Protein molecular mass standards used in zymogram gels were purchased from GE Healthcare, and those used for Western blots were from Invitrogen.

Parasites—E. histolytica, strain HM-1:IMSS, were originally obtained from American Type Culture Collection (strain accession 30459, Manassas, VA). Trophozoites of this strain were maintained under axenic conditions in 15-ml glass screw cap tubes in trypticase/yeast extract/iron medium (TYI-S-33) supplemented with 15% (v/v) heat-inactivated adult bovine serum (BioSource, Rockville, MD) at 37 °C as described previously (6). Such cells were routinely assessed microscopically by trypan blue dye exclusion assay to ensure >99% viability prior to use in experiments. Culture supernatants from serum-grown cells were examined to determine whether trophozoites released/secreted any nuclease activity into their growth medium during in vitro cultivation. To that end, when cultures reached a density of ~1.5 x 105 cells ml-1 (i.e. mid-log phase), tubes were placed on ice for 10 min to release adherent parasites and were then centrifuged at ~500 x g for 10 min at 4 °C. To ensure the complete absence of cells, the supernatants from these were carefully removed and recentrifuged at 6000 x g for 10 min at 4 °C. Following this, the cell-free supernatants were carefully removed, frozen, and stored at -80 °C until assayed.

For isolation of nucleic acids and proteins, parasite cell cultures were grown to about mid-log phase and harvested as above. The resulting cell pellets were washed three times in ice-cold PBS4 by centrifugation as above and finally resuspended in buffers appropriate for the extraction of DNA, RNA, or proteins (see below).

Parasite Cell Lysates—For zymogram gel analyses, washed cell pellets of E. histolytica were solubilized in SDS-PAGE sample buffer (7) lacking any reducing agents and heated in a boiling water bath for 5 min. Following this, the solubilized samples were cooled to room temperature, frozen, and stored at -80 °C until analyzed. The protein concentration in these cell lysates was determined using the bicinchoninic acid method according to manufacturer's instructions (Micro BCA, Pierce). In preliminary experiments, to test the effects of reducing agents on nuclease activity, cell lysates were also prepared in SDS-PAGE sample buffer containing 50 mM DTT (final concentration).

In Situ Nuclease Activity/Zymogram Gels—Parasite cell lysates, cell-free culture supernatants, and samples from immunoprecipitation and affinity binding assays were separated in SDS-PAGE mini-gels and processed for in situ staining of nuclease activity essentially according to Joshi and Dwyer (8). Briefly, samples were separated by SDS-PAGE containing poly(A) (300 µg ml-1, final concentration) and a discontinuous buffer system (9). Following separation, gels were washed twice at room temperature with 100 mM HEPES, 0.1% (v/v) Triton X-100 (Protein Grade® Detergent, Calbiochem), pH 8.5, on an orbital shaker. Following this, they were washed two additional times (15 min each) in buffer containing 50 mM sodium acetate, 0.1% (v/v) Triton X-100, pH 6.0. Subsequent to washing, these gels were incubated in sodium acetate buffer above for 2 h at 37 °C with gentle agitation in a rocking-hybridization oven (Shake 'N' Bake, Boekel Scientific, Feasterville, PA). Following incubation, gels were fixed in 7.5% (v/v) aqueous acetic acid, stained with toluidine blue O (0.2% (w/v) in 10 mM HEPES, pH 8.5), and de-stained using multiple changes of deionized water (10). Nuclease activity in these gels was readily apparent as distinct, clear/colorless bands of substrate hydrolysis in an otherwise uniformly stained, dark blue background of unhydrolyzed polynucleotide substrate.

For some experiments, zymogram gels were prepared as above except that various other individual synthetic polynucleotides (i.e. poly(G), poly(I), or poly(U), all from Sigma) were substituted for the poly(A) substrate.

Oligonucleotide Primers, PCR, and Cloning—In preliminary experiments, we found that during their growth in vitro, E. histolytica cells released/secreted two nuclease activities of ~24 and 25 kDa into their culture medium. Based on these observations, we searched the E. histolytica Gene Data Base for potential "secretory" nucleases having the following properties: 1) a predicted signal peptide sequence defined using the SignalP 3.0 Server; 2) lack of any apparent membrane-anchoring motifs, and 3) a predicted molecular mass of ~20-30 kDa. Among those meeting these criteria, two were chosen for further analyses. The deduced proteins encoded by these two open reading frames (ORFs) were annotated in the data base as putative ribonucleases (Gene Data Base identifiers 177.m00126 and 100.m00129) and designated here as EhNucI and EhNucII, respectively. Comparisons of these two E. histolytica sequences were done using the Gap-Global Alignment program of Genetic Computer Group (GCG) via NIH helix (molbio.info.nih.gov/molbio/gcglite/compare.html).

Primers were designed against each of these sequences toward amplifying them using PCR. To that end, to amplify EhNucI, a forward primer (Fwd 5'-ATGATTTCATTGTGTGTATTAC-3') with a methionine initiation codon (boldface type) corresponding to aa 1-7 and a reverse primer (Rev 5'-TTAATACAAACATCCACC-3') corresponding to aa 247-251 were synthesized by beta-cyanoethylphosphoramidite chemistry using an ExpediteTM nucleic acid synthesis system (Applied Biosystems). To amplify EhNucII, a forward primer (Fwd 5'-ATGATTTCATTGTGTTTATTAC-3') with a methionine initiation codon (boldface type) corresponding to aa 1-7 and a reverse primer (Rev 5'-TTAATACAAACATCCACC-3') corresponding to aa 247-251 were synthesized as above.

For PCR amplifications, these primers were used with 500 ng of E. histolytica HM-1 strain genomic (g) DNA as template and a high fidelity polymerase mix (High Fidelity PCR Master Mix, Roche Applied Science). After an initial "hot start" at 94 °C for 2 min, the conditions used for amplification were as follows: 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min (35 cycles) and a final step at 72 °C for 5 min. The resulting ~750-bp amplified products were cloned into the pCR®2.1-TOPO vector (Invitrogen). The resulting plasmid clones of EhNucI and EhNucII were subjected to nucleotide sequencing using vector-encoded M13 forward and reverse primers. Results of those analyses demonstrated that their nucleotide sequences were identical to those annotated in the E. histolytica Gene Data Base.

Nucleotide Sequencing and Analyses—DNA was sequenced using the fluorescent di-deoxy chain terminator cycle sequencing method (11) at The Johns Hopkins University DNA Analysis Facility (Baltimore, MD). Sequence data obtained from both strands were analyzed using the GCG software package (12) running on a National Institutes of Health Unix System and SequencherTM3.0 software (Gene Codes Corp., Ann Arbor, MI). Furthermore, such sequences were also subjected to BLAST-N and BLAST-P analyses using the NCBI BLAST-link (www.ncbi.nlm.nih.gov/BLAST/). Signal peptide sequence and protease cleavage sites were predicted using the SignalP link available at the worldwide ExPASy (Expert Protein Analysis System) proteomics server. Protein domain analysis was done using the Simple Modular Architecture Research Tool (SMART) available via EMBL-EBI, European Bioinformatics Institute). Protein multiple sequence alignments were done using the ClustalW program (13) using a MacVector® 7.0 software package (Accelrys).

Isolation of Genomic DNA—Total gDNA was isolated from mid-log phase E. histolytica trophozoites using the Fast CTAB DNA isolation method as described (14, 15).

Isolation of RNA and RT-PCR—Total RNA was isolated from trophozoites of E. histolytica using RNA STAT-60 according to the manufacturer's recommendations (Tel-Test, Inc., Friendswood, TX). To verify that the isolated parasite RNA was free of DNA contamination, it was tested in a control reaction that lacked reverse transcriptase. Reverse transcription of E. histolytica total RNA was done using the GeneAmp kit (Applied Biosystems) with dT16 to generate cDNA according to manufacturer's instructions. PCR amplification was carried out using conditions as above but with the following oligonucleotide primer pairs: for EhNucI, Fwd 5'-GGCTCTTAAATCAGGATT-3' and Rev 5'-GAAAGAGAAGCCCAGTTG-3', and for EhNucII, Fwd 5'-GGCTACTAAACCAGGATT-3' and Rev 5'-GTTAATGAAGCCCAATTG-3'. In addition, a set of forward and reverse oligonucleotide primers corresponding to a portion of an E. histolytica actin gene (16) were synthesized and used as a positive control in these reactions. The PCR-amplified products resulting from these reactions were separated and analyzed using 1.2% E-Gels® (Invitrogen). These PCR fragments were cloned into the pCR®2.1-TOPO vector (Invitrogen) and subjected to nucleotide sequencing using vector encoded M13-forward and -reverse primers.

Generation of Epitope-tagged Expression Constructs—A homologous episomal system was used to express epitope-tagged EhNucI-HA and EhNucII-HA chimeric proteins in E. histolytica trophozoites. To that end, individual constructs were designed that contained the complete open reading frame of the EhNucI and EhNucII genes (including 5'-ends encoding putative signal peptides) joined, at the 3'-ends, with a nine amino acid sequence encoding the influenza virus hemagglutinin (HA) epitope (Roche Applied Science). These constructs were generated by PCR using E. histolytica gDNA as template with the following forward primers: for EhNucI, Fwd 5'-GGTACCATGATTTCATTGTGTGTATTAC-3' (containing a KpnI restriction site shown in boldface), and for EhNucII, Fwd 5'-GGTACCATGATTTCATTGTGTTTATTAC-3'. A common reverse primer was used for both EhNucI and EhNucII in these reactions, i.e. Rev 5'-GGATCCTTAAGCGTAATCTGGAACATCGTATGGGTAATACAAACATCCACC-3' (containing a BamHI restriction site shown in boldface; stop codon in boldface italics; and the HA epitope sequence (underlined)). The conditions used for these PCR were as follows: a hot start at 94 °C for 5 min, followed by 10 cycles of amplification: 94 °C for 30 s, 50 °C for 30 s, 72 °C for 30 s, followed by 25 cycles of amplification: 94 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s, and a final step at 72 °C for 5 min. The resulting ~800-bp amplified fragments (EhNucI-HA and EhNucII-HA) were gel-purified and cloned into the pCR®2.1-TOPO vector (Invitrogen) to generate pCR2.1::EhNucI-HA and pCR2.1:: EhNucII-HA plasmids. The inserts were excised from these plasmids using KpnI and BamHI. Subsequently, the excised inserts were ligated into the KpnI and BamHI sites of the E. histolytica expression vector ExEhNeo (17) (kindly provided by Dr. Egbert Tannich, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany) to generate ExEhNeo:: EhNucI-HA and ExEhNeo::EhNucII-HA plasmid constructs. The nucleotide sequences of these constructs were verified by DNA sequence analysis.

Homologous Episomal Expression of EhNucI and EhNucII—The ExEhNeo::EhNucI-HA and ExEhNeo::EhNucII-HA plasmid constructs as well as the [ExEhNeo] control plasmid were transfected into E. histolytica trophozoites using electroporation conditions modified from those described by Nickel and Tannich (18). Briefly, mid-log phase trophozoites (~1.5 x 105 cells ml-1) were detached by chilling on ice for 10 min, pelleted by centrifugation at 500 x g at 4 °C for 10 min, washed twice in ice-cold PBS and once in electroporation buffer (120 mM KCl, 0.15 mM CaCl2, 25 mM HEPES, 2 mM EGTA, and 5 mM MgCl2, 10 mM K2HPO4/KH2PO4, pH 7.6). Subsequently, 2.4 x 106 cells were resuspended in 0.8 ml of electroporation buffer supplemented immediately before use with 3 µg/ml (final concentration) DEAE-dextran (ProFection® kit, Promega) and transferred to 4-mm gap, electroporation cuvettes (BTX, Harvard Apparatus) in the presence or absence of 100 µg of plasmid DNA. Cells were pulsed once using a BTX-ECM600 electroporator under conditions of 500 V/cm charging voltage, 800-microfarad capacitance, and 129-ohm resistance. Subsequently cells were placed on ice for 5 min and then subjected to a second electroporation pulse. Following this, they were placed in complete culture medium as above and allowed to recover for 48 h at 37 °C before being selected for their growth in the same medium containing 5 µgml-1 Geneticin® (G418, Invitrogen). Once drug-resistant parasites emerged, they were further selected for growth in increasing concentrations of G418 up to 10 µgml-1. For routine purposes, these transfectants were maintained and grown at 37 °C in complete growth medium containing 10 µgml-1 of G418. Both cell lysates and cell-free culture supernatants were prepared from such transfectants as described above.

Growth Kinetics of Transfected Parasites—ExEhNeo control, ExEhNeo::EhNucI-HA, and ExEhNeo::EhNucII-HA transfected parasites were monitored at regular intervals during the course of their growth in vitro. Parasite cultures used for growth kinetic studies were initiated at 3 x 103 cells ml-1 from stock cultures in their exponential phase of growth. Aliquots of the resulting cultures were taken at regular intervals, diluted appropriately, and counted using disposable cell counting chambers (CellometerTM, Nexcelom Bioscience, Lawrence, MA).

Temporal Release Assays—Temporal release assays were used to determine whether transfected trophozoites constitutively released/secreted any episomally expressed proteins into their extracellular environment over time. For these experiments, mid-log phase transfected trophozoites were harvested by centrifugation at ~500 x g as above, resuspended, washed twice with PBS (10 mM sodium phosphate, 145 mM NaCl, pH 7.4) by centrifugation, and finally resuspended at 3 x 105 cells ml-1 in fresh complete growth medium. Following incubation at 37 °C for various periods (i.e. 30, 60, 90, and 180 min), aliquots were removed and centrifuged as above to produce cell-free supernatants for Western blot analyses.

Western Blots—Lysates of E. histolytica episomally transfected parasites, as well as aliquots of their cell-free culture supernatants (all in 1x LDS sample buffer; Invitrogen) were separated in SDS-polyacrylamide gels (10%, pre-cast, BisTris polyacrylamide, NuPAGE® gels; Invitrogen). These gels were trans-blotted onto polyvinylidene difluoride membranes (Invitrogen), and the membranes were blocked and washed as described previously (19). Such blots were probed with a rabbit anti-HA polyclonal antibody (Sigma) as described (8). Subsequent to incubation and washing, the blots were reacted with a donkey anti-rabbit-horseradish peroxidase-conjugated secondary antibody (GE Healthcare) as described previously (19). Immunodetection was carried out using ECL Western blot kit reagents according to manufacturer's recommendations (GE Healthcare), and images were captured using BIOMAXTM-MR x-ray film (Eastman Kodak Co.).

Subcellular Fractionation—Trophozoites of E. histolytica transfected with EhNucI-HA and EhNucII-HA were subjected to subcellular fractionation using Dounce homogenization and differential centrifugation essentially as described by Sato et al. (20).

Light Microscopy—The subcellular localization of EhNucI-HA and EhNucII-HA was investigated using indirect immunofluorescence microscopy. For these assays, episomally transfected trophozoites were chilled on ice for 10 min, pelleted by centrifugation at 500 x g for 10 min, and then washed three times in ice-cold PBS. These cells were resuspended in fresh culture medium lacking bovine serum, and aliquots were placed onto glass coverslips and allowed to adhere for 30 min at 37 °C. For some experiments, the medium was supplemented with either 100 µgml-1 Texas Red dextran (Mr 10,000; Invitrogen) to label pinosomes or with 6 x 108 Texas Red-labeled Escherichia coli BioParticles® (Invitrogen) to label phagosomes, and cells were incubated for up to 3 h at 37 °C. Subsequently, the adherent cells were washed three times with pre-warmed PBS, fixed in pre-warmed 2% paraformaldehyde (Polysciences, Inc., Warrington, PA) in PBS, incubated for 30 min at room temperature, and then post-fixed for 5 min in -20 °C methanol. For immunofluorescence, fixed cells were blocked with 0.5% (w/v) bovine serum albumin (MP BioMedicals), 0.045% (w/v) fish gelatin (Sigma) in PBS, pH 7.2, at room temperature for 30 min. These fixed and blocked cells were then reacted with a rabbit anti-HA polyclonal antibody (Sigma) appropriately diluted in the blocking buffer as above for 1 h at room temperature. Following washing with PBS, these cells were reacted for 30 min at room temperature with a fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (H +L) secondary antibody (Jackson ImmunoResearch Laboratories) diluted in blocking buffer. Subsequently, cells were washed three times with PBS and once with distilled water. Stained, washed coverslips were mounted on glass slides with VECTASHIELD® mounting medium containing 15 µg/ml 4',6-diamidino-2-phenylindole (Vector Laboratories, Inc., Burlingame, CA) and sealed with nail polish.

Confocal and z-series images were obtained using a Leica model TCS-AOBS/SP2 confocal microscope with a 63x, 1.4 NA objective. Images were adjusted for contrast in Adobe Photoshop CS2 (Adobe Systems). Some z-series were processed for deconvolution with Huygens®Essential, version 2.9.1p0 (Scientific Volume Imaging BV, The Netherlands). Three-dimensional reconstructions and isosurface models were assembled with Imaris®, version 5.5.2 (Bitplane AG, Zurich, Switzerland).

Immunoprecipitation and Detection of Parasite Nuclease Activity—To test whether our episomally expressed EhNucI-HA and EhNucII-HA actually possessed functional nuclease activity, we immunoprecipitated these proteins from whole cell lysates of E. histolytica trophozoites. Immunoprecipitations were performed using the Catch and Release® version 2.0 system (Upstate-Cell Signaling Systems, Millipore) using 1 mg of total cellular protein and a rabbit anti-HA antibody (Sigma) according to the manufacturer's instructions. Following binding to this bead matrix, the immunoprecipitated proteins were washed and eluted using buffers according to the manufacturer's recommendations. Similarly, cell-free culture supernatants from these transfectants were subjected to immunoprecipitations as above to determine whether the expressed EhNucI-HA and EhNucII-HA released/secreted by these parasites also possessed functional nuclease activity. For these assays, parasite cell-free culture supernatants were made to 1x immunoprecipitation (IP) buffer (i.e. 100 mM Tris-HCl, 150 mM NaCl, 0.1% (v/v) Nonidet P-40 (Sigma), pH 7.4, final concentration) and reacted with a rabbit anti-HA antibody (Sigma) and protein A/G-agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA) for 12 h on a platform rocker at 4 °C. Subsequently, the bead-bound immunoprecipitates were washed three times with IP buffer, twice with 50 mM Tris-HCl, pH 7.4, and eluted by boiling in SDS-PAGE sample buffer. Aliquots of immunoprecipitates obtained from both E. histolytica cell lysates and their cell-free culture supernatants were analyzed for their nuclease activity using SDS-PAGE-poly(A) zymogram gels in the presence or absence of DTT as described above.

For some experiments, aliquots of cell-free culture supernatants were also subjected to immunoprecipitations with either sera obtained from patients suffering from acute amebic disease or sera from normal, uninfected volunteers in conjunction with a protein A/G affinity bead matrix, washed, and eluted as above. The resulting immune complexes were subjected to SDS-PAGE and subsequent Western blotting using a rabbit anti-HA polyclonal antibody.

Nuclease Assays Using Nucleic Acid Substrates—Total RNA was isolated from Leishmania donovani, as described previously (8), and it was used as substrate to evaluate the ribonuclease activity of the E. histolytica EhNucI-HA and EhNucII-HA episomally expressed nucleases. Aliquots of this RNA were incubated with immunoprecipitates of EhNucI-HA and EhNucII-HA obtained from whole cell lysates as described above. Such reactions were carried out in 100 mM MES buffer, pH 6.0, or 100 mM HEPES buffer, pH 8.0, in a total assay volume of 20 µl. Following incubation at 37 °C for 30 min, the reaction products were mixed with RNA-loading buffer containing ethidium bromide (Sigma), boiled for 5 min, and then resolved in 1.2% agarose gels containing 0.7% formaldehyde (v/v) in MOPS buffer (Quality Biosciences, Inc.). Subsequently, the hydrolysis products in these gels were visualized using an ultraviolet trans-illuminator with an UVP-Biochemi Imaging System (UVP-Bioimaging Systems, UVP, Inc., Upland, CA), and images were processed using Adobe Photoshop CS2.


Figure 1
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  		FIGURE 1.
Detection of E. histolytica released/secreted nuclease activities in SDS-polyacrylamide zymogram gels. A, cell-free culture supernatants (Cult Sup,10 µl, lane 1;20 µl, lane 2) obtained from cells grown in complete medium, prepared in SDS-PAGE sample buffer in the absence of reducing agent (-DTT), and separated in a poly(A)-containing SDS-polyacrylamide substrate gel. Following renaturation and staining with toluidine blue O, nuclease activity was visualized in these gels as a clear/colorless zone of substrate hydrolysis in an otherwise uniformly stained, dark blue background of unhydrolyzed poly(A) substrate. Arrow indicates the two zones ~24 and ~25 kDa of nuclease activity observed in these samples. B, samples as in A (lanes 1 and 2) prepared in SDS-PAGE sample buffer but containing reducing agent (+DTT, 50 mM, final concentration) and analyzed as above. Note the absence of any activity in the presence of the reducing agent. Protein molecular mass standards in kDa are shown on the left of each panel.

 
To further evaluate the nuclease activities of the EhNucI-HA- and EhNucII-HA-expressed proteins, they were reacted with both an ~7,249-nt, ssDNA (M13mp18 phage, Sigma) and a circular 7,249-bp, dsDNA (M13mp18 RF I, Sigma) as substrates. For experiments, aliquots of such ssDNA (0.5 µg) or dsDNA (0.5 µg) were incubated with anti-HA immunoprecipitates of EhNucI-HA and EhNucII-HA obtained from cell lysates (as above) in a total reaction volume of 25 µl containing 100 mM MES, pH 6.0. Following incubation (30 min at 37 °C), the hydrolysis products were resolved in 0.8% E-Gels® (Invitrogen) according to the manufacturer's instructions. Subsequently, images were captured using an ultraviolet trans-illuminator and an UVP-Biochemi Imaging System (UVP-Bioimaging Systems, UVP, Inc., Upland, CA) and processed using Adobe Photoshop CS2, as above.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of E. histolytica Released/Secreted Nuclease Activity—Previously, it has been shown experimentally that E. histolytica parasites are incapable of the de novo biosynthesis of purine nucleo-bases (i.e. purine auxotrophs) (21). Furthermore, recent analysis of the E. histolytica genome has revealed that these parasites also lack the essential enzymes for pyrimidine biosynthesis (22, 23). Thus, these organisms are totally dependent upon their human hosts to provide these essential nutrients for their growth and survival. In that regard, although it has been shown that axenically cultured E. histolytica parasites are capable of taking up such nutrients from their growth medium in vitro (5, 24, 25), the means by which these parasites might access these essential compounds in vivo within their human host are unknown. One potential mechanism by which Entamoeba might access exogenous nucleic acids present in their host environment is via the release/secretion of nucleic acid hydrolases (e.g. nucleases). To examine this concept further, experiments were designed to evaluate whether E. histolytica trophozoites released any nuclease activity into their culture medium during their growth in vitro. This was accomplished by analyzing cell-free culture supernatants from parasites for their nuclease activities using SDS-PAGE, poly(A)-containing substrate zymogram gels. For these assays, samples were prepared either in the presence of DTT (50 mM final concentration (19, 26)) or absence of this reducing agent (27).

Results obtained from these in situ activity gel assays indicated that under nonreducing conditions, cell-free culture supernatants of E. histolytica contained two distinct zones of nuclease activity of ~24 and ~25 kDa (Fig. 1A, lanes 1 and 2). However, no nuclease activity was observed when these samples were first treated with the reducing agent DTT (Fig. 1B, lanes 1 and 2). In addition, no nuclease activity was observed from similarly treated (-/+ DTT) control samples of fresh parasite culture medium (data not shown). Taken together, results of these activity gel assays demonstrated that E. histolytica trophozoites possess at least two DTT-sensitive nuclease activities. Furthermore, these nucleases appear to be constitutively released by these parasites into their culture medium during their growth in vitro.

Identification and Characterization of EhNucI and EhNucII Genes—In preliminary experiments we used a variety of different affinity-based bead matrices (e.g. binding to concanavalin A and the nucleotide dye mimetic, Reactive Red) in attempts to isolate sufficient quantities of these nucleases for functional characterization, including direct amino acid sequencing (data not shown). However, we were unable to obtain sufficient quantities of these proteins for such purposes. In that regard, it is important to point out that although the functional activity of the parasite secreted nucleases could be readily detected in the in situ activity gels above using cell-free culture supernatants, the actual amount of protein present in these samples was far below the level of detection. These observations suggested that the parasite-secreted nucleases might exhibit high levels of specific activity. Therefore, we adopted a molecular approach to determine the possible identity of these secreted nucleases. To accomplish this, an annotated protein data base of E. histolytica (Gene DB) was examined for putative secretory nucleases using the follow search criteria: (i) a predicted molecular mass of ~20-30 kDa; (ii) possession of a predicted signal peptide; and (iii) the lack of any apparent membrane-anchoring motif(s). Among the 51 putative nucleases present in the E. histolytica data base, 5 met our search criteria, and 2 of these were selected for further investigation. These two deduced proteins were annotated in Gene DB as 177.m00126 and 100.m00129, and both were designated as putative ribonucleases (i.e. no demonstrated functional activities). To amplify genes encoding these two putative E. histolytica nucleases, oligonucleotide primers were synthesized complementary to the sequences of their 5'- and 3'-ends and used in PCR with E. histolytica genomic DNA as template. The resulting ~750-bp products obtained from these amplification reactions were gel-purified, cloned, subjected to nucleotide sequencing, and designated as EhNucI and EhNucII. These sequences were subjected to both BLAST-N and BLAST-P analyses. Those analyses showed that the PCR clones contained ORFs that had complete nucleotide and deduced amino acid sequence identity with the putative E. histolytica 177.m00126 and 100.m00129 ribonucleases above.


Figure 2
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  		FIGURE 2.
The deduced amino acid sequence and structural features of the genes encoding EhNucI and EhNucII. The E. histolytica EhNucI and EhNucII genes encode deduced polypeptides of 251 aa residues each. The shaded boxed sequences delineate the putative 14-aa signal peptide (SP, Met1-Ala14), and the predicted cleavage sites are indicated by the arrow for each deduced protein. The unshaded boxes delineate the two conserved amino acid sequence domains (CAS I and CAS II) present in all members of the T2-RNase family. The histidine residues (asterisks) within these sequences have been shown to be necessary for the catalytic activity of various members of this enzyme family. In addition, several conserved cysteine residues have been shown to be responsible for the formation of disulfide bridges in all T2-RNase family members. The predicted disulfide bridges in EhNucI and EhNucII involve Cys81-Cys128, black-filled flags, and Cys195-Cys232, white flags.

 
The deduced amino acid sequences encoded by the open reading frames of EhNucI and EhNucII were compared with all available, nonredundant data bases using BLAST-P. Results of these comparisons showed that they had homologies to nucleases from diverse sources (28-31). Furthermore, analyses of the EhNucI- and EhNucII-deduced proteins using the Pfam data base showed that they had high homology with members of the T2 family of ribonucleases. Members of the T2 RNase family have been isolated from a variety of bacteria, fungi, plants, and animals and typically show an acid pH preference (reviewed in Ref. 32). Although Aspergillus oryzae, the prototypical T2 RNase family member, shows a preference for adenylic acid (33), most members of this family show no specific base preference (reviewed in Ref. 32).

Based on the von Heijne algorithm (34, 35), the hydrophobic, N-terminal 14 amino acids of both the EhNucI- and EhNucII- deduced proteins constitute putative signal peptides (Fig. 2). Cleavage at these sites, presumably within the parasite endoplasmic reticulum-like structure(s), would result in mature proteins with Lys15 as the N-terminal amino acid residue. Such cleavages in EhNucI and EhNucII would result in mature proteins consisting of 237 amino acids with calculated molecular masses of 26,340 and 26,339 Da, respectively. Both proteins have a calculated pI of 6.56. The deduced proteins were also analyzed using various other structural algorithms. Those analyses indicated that both of these enzymes lacked any apparent hydrophobic trans-membrane domains or glycosylphosphatidylinositol phosphate anchor signature sequences (36). Similarly, no KDEL or analogous endoplasmic reticulum retention sequences (37) or any other intracellular organelle-specific targeting sequences were identified in either deduced protein. The overall hydrophilicity, the presence of N-terminal signal peptides, and the absence of both membrane anchors and endoplasmic reticulum retention motifs suggest that both EhNucI and EhNucII represent soluble, released/secreted proteins. Interestingly, many microbial members of the T2 ribonuclease family have been shown to function as extracellular enzymes (38-40).


Figure 3
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  		FIGURE 3.
RT-PCR of EhNucI and EhNucII. A, diagrammatic representation of the complete 753-nt open reading frames of EhNucI and EhNucII. The black box (nt 1-42) represents the sequence encoding the predicted signal peptide (SP) of both EhNucI and EhNucII. The unshaded boxes indicate the relative positions of the sequences encoding the CAS I and CAS II domains of both EhNucI and EhNucII (see Fig. 2 above). Arrows represent the annealing positions of the specific EhNucI and EhNucII forward and reverse primers (RT-F and RT-R, respectively) used for RT-PCR experiments. These specific primers were designed to amplify the most variable region between EhNuc1 and EhNucII. B, ethidium bromide-stained agarose gel showing the amplification products of RT-PCR. Total RNA extracted from E. histolytica trophozoites was reverse-transcribed using reverse transcriptase and dT16 as primer. Aliquots (100 ng) of the resulting cDNAs were used as templates in PCR with EhNuc1- and EhNucII- specific RT-F and RT-R primers, respectively. Products of these reactions were separated and visualized in 1.2% agarose E-gels® containing ethidium bromide. No reaction products were observed from controls in which reverse transcriptase was omitted (lane 1). A single amplified product of ~160 bp was obtained for EhNucI with its specific set of primers (lane 2), and a similar 160-bp product was obtained for EhNucII with its specific set of primers (lane 3). The specific identity of each of these PCR products was independently verified by nucleotide sequence analyses. In addition, a single ~250-bp amplified product was obtained using primers specific for an E. histolytica actin gene (lane 4), which served as a positive control in these RT-PCR experiments. DNA standards in base pairs are shown on the left.

 
Previously, it has been reported that members of the T2 family of ribonucleases possess two conserved amino acid sequences (CAS I and II), each containing a single histidine residue essential for catalytic activity (41, 42). Analyses of the EhNucI- and EhNucII-deduced proteins showed that they both possess the CAS I and II domains, including the two critical histidine residues (Fig. 2, boxed aa), further solidifying the identity of these Entamoeba enzymes as T2 ribonucleases. Additionally, all members of the T2 ribonuclease family possess at least four structurally conserved cysteine residues involved in intra-chain disulfide bridges, and such bridges are essential for the enzymatic functions of these nucleases (43). In that context, our analyses showed that the mature EhNucI- and EhNucII- deduced proteins also possess four similarly conserved cysteine residues that could be involved in forming disulfide bridges (Fig. 2, Cys81, Cys128, Cys195, and Cys232). Such disulfide bonds appear to be essential for the function of the endogenous E. histolytica wild-type released/secreted enzymes, as treatment with the reducing agent, DTT, abolished all parasite nuclease activity (cf. Fig. 1, A and B).

Expression of EhNucI and EhNucII—RT-PCR was used to determine whether EhNucI and EhNucII were indeed transcribed by E. histolytica parasites. To accomplish this, total RNA was extracted from E. histolytica trophozoites and reverse-transcribed using reverse transcriptase and dT16. Aliquots (100 ng) of the resulting cDNA were used as template in PCR with primers (RT-F and RT-R) specific to EhNucI and EhNuc II, respectively (Fig. 3A). A control reaction that lacked reverse transcriptase was used to verify that the isolated parasite RNA was free of DNA contamination. In addition, a set of oligonucleotide primers corresponding to a portion of an E. histolytica actin gene (16) was used as a positive control in these reactions. Products from these reactions were separated and visualized in 1.2% agarose E-Gels®. Results of these analyses showed that no PCR product was generated from a control reaction that lacked reverse transcriptase verifying that the isolated parasite RNA was free of DNA contamination (Fig. 3B, lane 1). As anticipated, a single amplified product of ~220 bp was obtained from the E. histolytica actin-positive control (Fig. 3B, lane 4). More importantly, a single amplified product of ~180 bp was generated for both EhNucI and EhNucII (Fig. 3B, lanes 2 and 3, respectively). These results indicated that E. histolytica trophozoites were actively synthesizing message for each of these genes. To analyze this further, the products of these reactions were gel-purified, cloned, and sequenced. Results of such sequence analyses verified that mRNAs for each of these nucleases (i.e. EhNucI and EhNucII) were in fact readily transcribed by these parasites during their growth in vitro.

Transfection of E. histolytica Trophozoites with Epitope-tagged Gene Constructs—We used a homologous episomal expression system to functionally characterize the proteins encoded by the EhNucI and EhNucII genes. To accomplish this, individual chimeric constructs were generated that contained the complete ORF of either EhNucI or EhNucII, in-frame, at the 3'-end with a sequence encoding a 9-aa HA epitope of the influenza virus (designated as EhNucI-HA and EhNucII-HA, respectively). Following ligation into the ExEhNeo Entamoeba expression vector, these constructs were used to transfect E. histolytica trophozoites (Fig. 4A). Cells transfected with the ExEhNeo vector alone served as controls in all transfection experiments. Transfected parasites were selected for their growth in complete medium containing increasing concentrations of G418 (i.e. up to 10 µgml-1). Following drug selection, the growth kinetics of these transfected parasites were compared. For experiments, triplicate cultures of EhNucI-HA, EhNucII-HA, and ExEhNeo control transfectants were initiated at 4 x 103 cells ml-1 from stock cultures in their exponential phase of growth. Aliquots from such cultures were taken at 24-h intervals, diluted appropriately, and counted using CellometerTM disposable cell counting chambers. Results of these in vitro assays showed that cells transfected with either the EhNucI-HA, EhNucII-HA chimeric constructs or the ExEhNeo control plasmid had very similar growth kinetics (Fig. 4B). These observations indicated that these episomal transfections did not appear to alter the characteristic growth kinetics of the parental E. histolytica cell line. Subsequently, such transfected cells were analyzed for the expression of the EhNucI-HA and EhNucII-HA chimeric proteins using Western blots, in situ activity gel assays, and immunofluorescence confocal microscopy.


Figure 4
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  		FIGURE 4.
Episomal expression constructs and growth kinetics of transfected parasites. A, map of the EhNucI-HA and EhNucII-HA chimeric constructs. A diagrammatic representation of the complete open reading frame (i.e. nt 1-753, minus the terminal TAA stop codon) of the EhNuc1 and EhNucII genes fused at their 3'-ends with a 27-nt sequence encoding the influenza virus HA epitope (light gray box). The black box at the 5'-end represents nt 1-42 encoding the putative 14-aa signal peptide (SP) of the EhNucI and EhNucII proteins. The thick black line represents the ExEhNeo expression plasmid vector backbone and the KpnI and BamHI restriction endonuclease sites used for cloning the chimeric inserts. B, growth kinetics of transfected parasites. E. histolytica trophozoites transfected with the EhNucI-HA (bullet), EhNucII-HA ({circ}), or the ExEhNeo control plasmid ({square}) were monitored at regular intervals during the course of their growth in vitro. Triplicate cultures of parasites were initiated at 4 x 103 cells ml-1 from stock cultures in their exponential phase of growth. Aliquots of the resulting cultures were taken at regular intervals as indicated, diluted appropriately, and counted using a hemocytometer. Values shown represent the results obtained from a typical experiment.

 


Figure 5
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  		FIGURE 5.
Episomal expression of EhNucI-HA and EhNucII-HA chimeras in E. histolytica. A, expression of the EhNucI-HA and EhNucII-HA chimeric proteins in E. histolytica-transfected cells. E. histolytica cells transfected with the EhNucI-HA, EhNucII-HA constructs, or the control ExEhNeo plasmid were grown in complete medium. Whole cell lysates (Cell Lys., 10 µg of total protein, lanes 1-3) of these transfectants were subjected to SDS-PAGE and reacted in Western blots with a rabbit anti-HA polyclonal (primary) antibody and a horseradish peroxidase-conjugated, goat anti-rabbit IgG (secondary) antibody. The anti-HA antibody showed no reactivity with any protein in ExEhNeo controls (lane 1) but showed strong reactivity with a single ~28-kDa chimeric protein expressed in both the EhNucI-HA- and EhNucII-HA transfected cells (arrow, lanes 2 and 3). Protein molecular mass standards (in kDa) are shown on the left. B, detection of EhNucI-HA and EhNucII-HA chimeric proteins released into the culture supernatants of E. histolytica-transfected cells. Cell-free culture supernatants (Cult. Sup, 20 µl, lanes 1-3) obtained from E. histolytica cells transfected with the EhNucI-HA, EhNucII-HA constructs, or the control ExEhNeo plasmid were subjected to SDS-PAGE and Western blotting. Blots were probed with a rabbit anti-HA polyclonal (primary) antibody and a horseradish peroxidase-conjugated, goat anti-rabbit IgG (secondary) antibody, as above. The anti-HA antibody showed no reactivity with any protein in supernatants of the ExEhNeo controls (lane 1) but showed strong reactivity with a single ~28-kDa chimeric protein present in the culture supernatants of both the EhNucI-HA- and EhNucII-HA-transfected cells (arrow, lanes 2 and 3). The additional proteins of lower apparent molecular mass (<17 kDa) observed in lanes 2 and 3 presumably represent proteolytic degradation products of the released/secreted EhNucI-HA- and EhNucII-HA-expressed proteins. Protein molecular mass standards (in kDa) are shown on the left. C, release of EhNucI-HA and EhNucII-HA chimeric proteins over the course of a temporal in vitro incubation assay. Log-phase transfected parasites were harvested, washed, and resuspended to 3 x 105 cells ml-1 in fresh culture medium and incubated for 30, 60, 90, and 180 min at 37 °C. Cell-free aliquots (20 µl) of such supernatants were subjected to SDS-PAGE and Western blotting as above. The anti-HA antibody reacted with a single ~28-kDa chimeric protein present in the supernatants of both the EhNucI-HA- and EhNucII-HA-transfected cells. Furthermore, the ~28-kDa chimeric proteins were detectable in as little as 30 min postincubation and appeared to accumulate in the supernatants of each of these transfected cell lines over time.

 
Expression of EhNucI-HA and EhNucII-HA Chimeric Proteins in Transfected Parasites—Western blot analyses were performed to determine whether transfected parasites synthesized and released/secreted the chimeric proteins during their growth in vitro. For these experiments, cells transfected with either the EhNucI-HA or EhNucII-HA constructs or ExEhNeo control plasmid were grown in complete medium containing 10 µgml-1 of G418. Lysates and cell-free culture supernatants from such cells were subjected to SDS-PAGE and subsequent Western blotting using a rabbit anti-HA polyclonal antibody. Results of such assays showed that the anti-HA antibody reacted with only a single ~28-kDa protein present in lysates of both EhNucI-HA- and EhNucII-HA-transfected cells (Fig. 5A, lanes 2 and 3). In parallel blots, the anti-HA antibody reacted most strongly with an ~28-kDa protein present in the cell-free culture supernatants of both EhNucI-HA- and EhNucII-HA- transfected cells (Fig. 5B, lanes 2 and 3). This antibody also reacted to a lesser extent with several lower apparent molecular weight proteins present in the cell-free culture supernatants of these transfectants. Presumably, the latter represent proteolytic degradation products of the released/secreted EhNucI-HA- and EhNucII-HA-expressed proteins. In contrast, the anti-HA antibody showed no reactivity with either lysates (Fig. 5A, lane 1) or cell-free culture supernatants (Fig. 5B, lane 1) obtained from control ExEhNeo-transfected cells.


Figure 6
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  		FIGURE 6.
Localization of chimeric EhNucI-HA and EhNucII-HA by fluorescence microscopy. Log phase E. histolytica transfected with either the EhNucI-HA or the EhNucII-HA construct were allowed to adhere to glass coverslips, fixed, permeabilized, and reacted with a primary anti-HA rabbit polyclonal antibody ({alpha}-HA) followed by a FITC-labeled goat anti-rabbit secondary antibody. To visualize the nuclei in these cells, coverslips were mounted on glass slides using mounting medium containing 4',6-diamidino-2-phenylindole (Vector Laboratories). Differential interference contrast (DIC) images show the overall loboid morphology of these transfected parasites as well as some of their intracellular compartments (e.g. nuclei and various sized vesicles). The fluorescence images shown are projected confocal z-series processed using deconvolution software. Both the EhNucI-HA- (A) and the EhNucII-HA (B)-expressed proteins showed very similar patterns of vesicular distribution throughout the cytoplasm of these transfected cells. In the merged images, 4',6-diamidino-2-phenylindole (DAPI) stain is blue and {alpha}-HA staining is green. Bar = 10 µm.

 
By having demonstrated that the chimeric proteins were in fact synthesized and released by transfected parasites into their culture supernatants, it was of interest to investigate the temporal release of both EhNucI-HA and EhNucII-HA. To that end, 3 x 10-5 cells transfected with either the EhNucI-HA or EhNucII-HA construct were incubated for up to 3 h in complete medium. At defined intervals, cells were harvested, and their resulting cell-free culture supernatants were subjected to SDS-PAGE and subsequent Western blotting as above. Results from such blots showed that the transfected parasites released/secreted detectable quantities of both EhNucI-HA and EhNucII-HA into their culture medium in as little as 30 min postincubation (Fig. 5C, 30 min). In addition, both of these expressed proteins appear to accumulate in the culture medium over time (Fig. 5C, 60-180 min).

Taken together, results of these Western blot experiments demonstrated the following. 1) The EhNucI-HA and EhNucII-HA chimeric-gene constructs were readily transcribed and translated into single ~28-kDa chimeric proteins by transfected E. histolytica cells. 2) Both proteins were released/secreted by these transfectants during their growth in vitro.3) Both proteins appeared to accumulate in the culture medium of these transfectants over time.

Intracellular Distribution of EhNucI-HA and EhNucII-HA—In preliminary experiments, subcellular fractionations were carried out to determine the intracellular localization of the EhNucI-HA- and EhNucII-HA-expressed proteins. For such experiments, transfected parasites were Dounce-homogenized, subjected to differential centrifugation, and the resulting pellets and supernatants analyzed by SDS-PAGE and Western blotting with an anti-HA antibody as above. Results obtained from these experiments demonstrated that the EhNucI-HA- and EhNucII-HA-expressed proteins were pelletable and appeared to fractionate with vesicular compartments (data not shown). Similar results have been obtained with other compartmentalized proteins in E. histolytica (20).

Subsequently, we used indirect immunofluorescence microscopy to visualize the subcellular distribution of EhNucI-HA and EhNucII-HA in E. histolytica-transfected parasites. For these experiments, EhNucI-HA-, EhNucII-HA-, and ExEhNeo control-transfected cells were fixed, permeabilized, and reacted with a rabbit anti-HA polyclonal antibody. Subsequently, cells were reacted with a FITC-conjugated secondary antibody and examined by confocal fluorescence microscopy. Results obtained from microscopic evaluation revealed that the EhNucI-HA- and EhNucII-HA-expressed proteins were present in vesicular compartments dispersed throughout the cytoplasm of EhNucI-HA and EhNucII-HA transfectant cells (Fig. 6, A and B). Such staining is consistent with processing of the EhNucI-HA and EhNucII-HA proteins through the rudimentary endoplasmic reticulum present in these parasites for entry into the secretory system. Importantly, very similar immunofluorescence staining patterns have been reported for other secretory proteins in these parasites (44, 45). In contrast to the above, no fluorescence was detected in cells transfected with the ExEhNeo control plasmid (data not shown). Results of these experiments showed that the EhNucI-HA- and EhNucII-HA-expressed proteins were readily synthesized and localized in vesicular compartments in cultured E. histolytica trophozoites.

To further characterize the nature of the vesicular compartments seen in the above microscopic experiments, both phagosomes and pinosomes were labeled in the EhNucI-HA- and EhNucII-HA-transfected cells. To label pinosomes, cells were allowed to ingest 10-kDa Texas Red-labeled dextran for 60 min. To label phagosomes, cells were allowed to ingest Texas Red-labeled E. coli for 60 min. Following such incubations, cells were fixed, permeabilized, and reacted with a rabbit anti-HA polyclonal antibody as above, and then reacted with a FITC-conjugated secondary antibody and examined by confocal-fluorescence microscopy. Results obtained from deconvolved z-series images showed that the EhNucI-HA- and EhNucII-HA-expressed proteins did not co-localize with either compartments containing labeled dextran (i.e. pinosomes, Fig. 7, A and B) or those containing labeled bacteria (i.e. phagosomes, Fig. 7, C and D). In addition, some of these images were subjected to three-dimensional projections and isosurface modeling. Videos of these were assembled using Imaris®, version 5.5.2 (Bitplane AG, Zurich, Switzerland). Results obtained from analysis of the latter further confirmed that the EhNucI-HA- and EhNucII-HA-expressed proteins did not colocalize with compartments containing endosomal markers. A representative video illustrating the three-dimensional intracellular distribution of EhNucI-HA and Texas-red labeled pinosomes is shown in supplemental Fig. S1.


Figure 7
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  		FIGURE 7.
Intracellular distribution of the chimeric EhNucI-HA- and EhNucII-HA-expressed proteins in relation to pinosomes and phagosomes by fluorescence microscopy. E. histolytica transfected with the EhNucI-HA or the EhNucII-HA construct were allowed to endocytose Texas Red-labeled ~10-kDa dextran to visualize pinosomes (A and B) or Texas Red-labeled E. coli to visualize phagosomes (C and D). Cells were fixed, permeabilized, and reacted with a primary rabbit anti-HA polyclonal antibody ({alpha}-HA) followed by a FITC-labeled goat anti-rabbit secondary antibody. All images are projected confocal z-series processed using deconvolution software. Results of such image analyses indicated that both the EhNucI-HA- and EhNucII-HA-expressed proteins occupied compartments distinctly separate from either pinosomes containing dextran or phagosomes containing E. coli. In the merged images, the endosomal markers are red and {alpha}-HA staining is green. Bar = 10 µm.

 


Figure 8
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  		FIGURE 8.
Zymogram analyses of EhNucI-HA and EhNucII-HA released/secreted by transfected parasites. A, cell-free culture supernatants (Sup) from parasites transfected with either the ExEhNeo control plasmid, the EhNucI-HA construct, or the EhNucII-HA construct were subjected to immunoprecipitation with an {alpha}-HA antibody. The resulting immunoprecipitates (IP) were solubilized in SDS-PAGE sample buffer in the absence of reducing agent (-DTT) and analyzed for their nuclease activity in a poly(A) containing SDS-polyacrylamide zymogram gels. Arrow indicates the major ~25-kDa nuclease activity immunoprecipitated from the supernatants of EhNucI-HA (lane 2) and EhNucII-HA (lane 3). The additional nuclease bands of higher apparent molecular mass seen in lanes 2 and 3 presumably reflect aggregates of the 25-kDa immunoprecipitated nucleases. Note the absence of any nuclease activity in the immunoprecipitates obtained from the ExEhNeo transfected controls (lane 1). B, samples as in A (lanes 1-3) prepared in SDS-PAGE sample buffer containing reducing agent (+DTT, 50 mM, final concentration) and analyzed as above. Note the absence of any activity in the presence of the reducing agent. Protein molecular mass standards in kDa are shown on the left of each panel.

 
Similarly, EhNucI-HA- and EhNucII-HA-expressed proteins did not co-localize with compartments containing various other endosomal markers (i.e. latex beads, human red blood cells, and Chinese hamster ovary cells (data not shown)). Results of these immunofluorescence experiments showed the following: 1) the EhNucI-HA- and EhNucII-HA-expressed proteins were synthesized and processed by the transfectants, and 2) the expressed proteins were localized in putative secretory vesicles (i.e. vesicular compartments distinct from either phagosomes or pinosomes).

Functional Enzyme Activity Analyses of the EhNucI-HA- and EhNucII-HA-expressed Proteins—Results of our Western blots and subcellular localization studies above demonstrated that both EhNucI-HA and EhNucII-HA chimeric proteins were synthesized and released/secreted by transfected cells. It was important, however, to also demonstrate that these expressed proteins actually possessed functional nuclease activity. To accomplish this, parasites transfected with the EhNucI-HA construct, the EhNucII-HA construct, or the ExEhNeo control plasmid were grown in complete medium, and their cell-free culture supernatants were harvested. Aliquots of these supernatants were subjected to immunoprecipitations using a rabbit anti-HA polyclonal antibody in conjunction with an affinity matrix, and the resulting immune complexes were analyzed for their nuclease activity using poly(A)-containing SDS-polyacrylamide zymogram gels. Results of these assays demonstrated that the anti-HA antibody specifically immunoprecipitated a major ~25-kDa band of nuclease activity from the cell-free culture supernatants of both EhNucI-HA and EhNucII-HA transfectants (Fig. 8A, lanes 2 and 3, respectively). These immunoprecipitates also contained several additional bands of nuclease activity of higher apparent molecular mass, which presumably reflect aggregates of these 25-kDa nucleases. As expected, however, no activity was detected in anti-HA immunoprecipitates obtained from culture supernatants of ExEhNeo control transfectants (Fig. 8A, lane 1). Furthermore, no detectable nuclease activity was observed with immunoprecipitates obtained from parallel samples incubated with the reducing agent DTT (50 mM final concentration) and then analyzed using a poly(A)-containing SDS-polyacrylamide zymogram gel (Fig. 8B).


Figure 9
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  		FIGURE 9.
Detection of EhNucI-HA- and EhNucII-HA-expressed nuclease activity with various synthetic polynucleotide substrates. Whole cells lysates (1 mg of total cellular protein) of parasites transfected with either the ExEhNeo control plasmid, the EhNucI-HA construct, or the EhNucII-HA construct were subjected to immunoprecipitation with an {alpha}-HA antibody. The resulting immunoprecipitates were solubilized in SDS-PAGE sample buffer in the absence of reducing agent (-DTT) and analyzed for their nuclease activity in SDS-polyacrylamide zymogram gels containing various synthetic polynucleotide substrates: A, poly(A); B, poly(U); and C, poly(I). Arrows indicate that the major ~25-kDa nuclease immunoprecipitated from cell lysates of EhNucI-HA had activity against poly(A), poly(U), and poly(I) (lane 2 in A-C). The immunoprecipitates from EhNucII-HA showed similar nuclease activity with each of these polynucleotide substrates (lane 3 in A-C). The additional nuclease bands of higher apparent molecular mass seen in lanes 2 and 3 presumably reflect aggregates of the 25-kDa immunoprecipitated nucleases. Note the absence of any nuclease activity in the immunoprecipitates obtained from the ExEhNeo-transfected controls (lane 1 in A-C). Molecular mass standards (in kDa) are shown on the left.

 
It is important to mention that the nuclease activities immunoprecipitated above appeared to have relative molecular masses different from those observed in our Western blots. To reconcile these apparent differences, parallel immunoprecipitated samples were separated in poly(A)-containing SDS-polyacrylamide gels, transferred to polyvinylidene difluoride, and subsequently analyzed by Western blot as above. Results of these analyses showed that inclusion of poly(A) in these SDS-polyacrylamide zymogram gels altered the relative mobility of both the EhNucI-HA- and EhNucII-HA-expressed proteins (data not shown). Taken together, results of these coupled immunoprecipitation/zymogram analyses demonstrated that the EhNucI-HA- and EhNucII-HA-expressed proteins released/secreted by transfected parasites did in fact possess functional nuclease activities that, like the endogenous wild-type parasite enzymes, were completely inhibited by treatment with DTT.

Activity of the EhNucI-HA- and EhNucII-HA-expressed Proteins Using Synthetic Polynucleotide Substrates—As shown above, the wild-type parasite released/secreted nucleases and the EhNucI-HA- and EhNucII-HA-expressed enzymes readily hydrolyzed the poly(A) substrate in zymogram gels. In preliminary experiments, we found that the nuclease activities released/secreted by wild-type parasites were also capable of hydrolyzing various other polynucleotide substrates. Therefore, it was of interest to determine whether the EhNucI-HA- and EhNucII-HA-expressed nucleases were also capable of hydrolyzing such substrates. To that end, anti-HA immunoprecipitates obtained from lysates of EhNucI-HA-, EhNucII-HA-, and ExEhNeo control-transfected parasites were analyzed for their nuclease activity using SDS-polyacrylamide zymogram gels containing various individual polynucleotide substrates (poly(A), poly(U), poly(I), or poly(G)). Results of such analyses demonstrated that the anti-HA immunoprecipitates obtained from EhNucI-HA and EhNucII-HA transfectants each possessed a predominant ~25-kDa nuclease activity, capable of hydrolyzing three of the polynucleotide substrates tested: poly(U) > poly(A) > poly(I) (Fig. 9, A-C, lanes 2 and 3, respectively). In contrast, such immunoprecipitates failed to show any detectable nuclease activity with poly(G)-containing zymogram gels (data not shown). As expected, the anti-HA immunoprecipitates obtained from ExEhNeo control transfectants showed no nuclease activity with any of the polynucleotide substrates tested in these assays (Fig. 9, lane 1 in A-C). Results of these coupled anti-HA immunoprecipitation-zymogram assays demonstrated that the EhNucI-HA- and EhNucII-HA-expressed proteins, similar to the native wild-type released/secreted nucleases, were capable of hydrolyzing various different polynucleotide substrates.

Activity of the EhNucI-HA- and EhNucII-HA-expressed Enzymes with Nucleic Acid Substrates—The activities of the Entamoeba expressed nucleases were also evaluated using several different nucleic acid substrates. For these assays, RNase activity was assessed using total RNA isolated from an unrelated eukaryotic organism as substrate, whereas DNase activities were evaluated using both a single-stranded M13mp18 phage DNA and double-stranded M13mp18 RF I DNA as substrates. To determine whether EhNucI-HA and EhNucII-HA possessed RNase or DNase activities, these expressed proteins were immunoprecipitated from cell lysates using an anti-HA antibody as above. Aliquots of the eluted immune complexes were reacted with the RNA, ssDNA, and dsDNA substrates above in either a pH 6.5 or pH 8.0 buffer for 30 min at 37 °C. Following incubation, RNA containing samples were separated in 1.2% agarose gels containing formaldehyde and ethidium bromide, and DNA-containing samples were separated in 0.8% E-Gels®. Subsequently, all samples were visualized and evaluated using UV trans-illumination. Results of these experiments demonstrated that immunoprecipitates obtained from lysates of both the EhNucI-HA and EhNucII-HA transfectants readily hydrolyzed the RNA substrate (Fig. 10A, lanes 2 and 3, respectively). In contrast such immunoprecipitates showed no activity with either the ssDNA or the dsDNA substrates (Fig. 10, B and C, lanes 2 and 3, respectively). Interestingly, the RNA substrate seemed to be more efficiently hydrolyzed at acidic pH (pH 6.5) than under alkaline conditions (pH 8.5, data not shown). As expected, anti-HA immunoprecipitates obtained from control ExEhNeo-transfected parasites failed to hydrolyze any of these RNA, ssDNA, or dsDNA substrates (lanes 1 in Fig. 10, A-C, respectively). Results of these coupled anti-HA immunoprecipitation-nucleic acid hydrolysis assays demonstrated that the EhNucI-HA- and EhNucII-HA-expressed proteins possessed functional RNA hydrolase activity consistent with them being members of the T2 family of ribonucleases.


Figure 10
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  		FIGURE 10.
Activity of EhNucI-HA- and EhNucII-HA-expressed nucleases with various nucleic acid substrates. Whole cells lysates (1 mg of total cellular protein) of parasites transfected with either the ExEhNeo control plasmid, the EhNucI-HA construct, or the EhNucII-HA construct were subjected to immunoprecipitation with an {alpha}-HA antibody. The resulting immunoprecipitates were analyzed for their nuclease activity with several different nucleic acid substrates. A, immunoprecipitates were incubated at pH 6.0 with total RNA as substrate, and the resulting reaction products were separated and visualized in EtBr-stained formaldehyde-agarose gel. Note that the immunoprecipitates obtained from parasites transfected with the ExEhNeo control plasmid (lane 1) showed no nuclease activity with the RNA substrate (arrow). In contrast, immunoprecipitates obtained from EhNucI-HA (lane 2) and EhNucII-HA (lane 3) transfectants readily hydrolyzed the RNA substrate. B, immunoprecipitates as in A were incubated at pH 6.0 with a single-stranded circular DNA substrate and the reaction products separated in a 0.8% agarose, EtBr-stained, E-Gel®. Neither immunoprecipitates from ExEhNeo control transfectants (lane 1) nor those obtained from the EhNucI-HA (lane 2) and EhNucII-HA (lane 3) transfectants showed any nuclease activity with the ssDNA substrate (arrow). C, immunoprecipitates as in A were incubated at pH 6.0 with a dsDNA substrate, and the reaction products were separated and visualized in a 0.8% agarose, EtBr-stained, E-Gel®. None of the immunoprecipitates obtained from the control, ExEhNeo (lane 1), EhNucI-HA (lane 2), or EhNucII-HA (lane 3) transfectants showed any nuclease activity with the dsDNA substrate. Arrow indicates the unhydrolyzed dsDNA substrate.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
According to World Health Organization estimates, E. histolytica, a major enteric protozoan pathogen of humans, is the etiologic agent responsible for causing up to 50 million cases of amebic dysentery and more than 100,000 deaths per year worldwide (1, 46). These parasites typically colonize, and some cases invade, the large intestine of their human hosts. Although the human small intestine represents an environment relatively rich in nutrients and enzymatic activities, the large intestine produces no digestive enzymes and is generally nutrient-poor (47). E. histolytica parasites are purine auxotrophs (i.e. they are incapable of synthesizing the purine ring de novo) (21). In addition, it has been recently suggested, based on the cumulative data from the completed E. histolytica genome project, that these parasites are also incapable of de novo pyrimidine biosynthesis (22, 23). Thus, these parasites are completely reliant on salvaging these essential compounds from their host environment for their own survival, growth, and development. We hypothesized that one mechanism by which Entamoeba might access such nucleo-bases from their host environment is through the release/secretion of enzymes capable of hydrolyzing nucleic acids (i.e. nucleases). It is of interest to note that E. histolytica is unique among eukaryotes in that it contains only rudimentary endoplasmic reticulum and Golgi (2, 3). However, homologues to important ER-associated proteins such as BiP, calreticulin, and protein-disulfide isomerase and Golgi-associated proteins such as ERD2 have been identified in this pathogen (48-51). Furthermore, several Entamoeba cell surface and secreted proteins (i.e. Gal/GalNac lectin, amebapores, and cysteine proteases) contain typical signal sequences similar to higher eukaryotes for targeting through the ER and Golgi (52-54). In fact, the secretion of cysteine proteases is essential for the pathogenicity of E. histolytica (55-57). Thus, it appears that Entamoeba is quite capable of secretion despite the apparent lack of traditional eukaryotic secretory machinery. In that regard, in preliminary experiments, using SDS-PAGE, poly(A)-zymogram gels, we demonstrated that E. histolytica did, in fact, constitutively synthesize and release/secrete soluble, DTT-sensitive, nuclease activities into their culture supernatants during their growth in vitro.

Having found these nuclease activities, it was of interest to further characterize their properties. Consequently, in preliminary experiments, multiple approaches were used in attempts to purify these secretory nucleases from parasite culture supernatants. Results of those experiments showed, however, that the actual amount of protein obtained from such samples was far below the level needed for these analyses. Therefore, we adopted an alternative, molecular approach to identify E. histolytica genes encoding putative secretory nucleases and subsequently expressed them to examine some of their biochemical and functional properties. To that end, using a PCR-based approach, we isolated and characterized two highly similar open reading frames (EhNucI and EhNucII) encoding 251-aa deduced proteins, both with calculated molecular masses of ~28 kDa. Analyses of the EhNucI- and EhNucII-deduced proteins, using various algorithms, suggested that they both possessed features typical of soluble, secreted proteins (i.e. the presence of putative N-terminal signal peptides, overall hydrophilicity, and the absence of membrane anchoring domains or organelle retention signals). In addition, BLAST-P analyses of the deduced amino acid sequences showed that both EhNucI and EhNucII had homologies with a variety of nucleases from diverse sources (28-31). Significantly, results of Pfam data base comparisons reveled that both EhNucI- and EhNucII-deduced proteins belong to the T2 family of ribonucleases (reviewed in Ref. 32). It has been reported that all T2 ribonuclease family members possess two conserved blocks of amino acid residues designated as CAS I and CAS II, each containing a histidine residue involved in the catalytic activity of the enzymes (41, 42). Results of our sequence analyses showed that these domains were also present in both EhNucI- and EhNucII-deduced proteins. Additionally, all T2 ribonuclease family members possess at least two intra-chain disulfide bonds, which may be critical for the enzymatic activity of these proteins (43). In that regard, structural analyses of the mature EhNucI- and EhNucII-deduced proteins showed they both possess four cysteine residues, which could be involved in intra-chain disulfide bonding. Interestingly, disulfide bonds were found to be essential for the function of the native released parasite enzymes because treatment with reducing agents, such as DTT, completely abolished parasite nuclease activity. In addition, many members of the T2 family are soluble, secreted enzymes, which function extracellularly to cleave nucleic acids (reviewed in Ref. 32). These properties are in good agreement with those we determined experimentally for the native, wild-type E. histolytica-secreted nucleases.

Having identified and cloned the genes encoding EhNucI and EhNucII, it was important to determine whether or not both genes were actually transcribed by parasites. To that end, we developed primers specific to each of these genes and used them in RT-PCR with total E. histolytica RNA as template, and the products of such reactions were cloned and sequenced. Results of these experiments demonstrated that both EhNucI and EhNucII were indeed transcribed by E. histolytica trophozoites during their growth in vitro.

Although the foregoing data suggested that the cloned EhNucI and EhNucII genes were both transcribed by E. histolytica, and both possessed sequences characteristic of nucleases, it was necessary to demonstrate that these genes actually encoded functionally active nucleases. To accomplish this, we used a homologous E. histolytica episomal expression system to produce HA-tagged EhNucI and EhNucII chimeric proteins. Results of our Western blot analyses using an anti-HA antibody demonstrated that both EhNucI-HA- and EhNucII-HA-transfected parasites synthesized and released/secreted the 28-kDa EhNucI-HA and EhNucII-HA chimeric proteins into their culture medium during their growth in vitro. Furthermore, results of our coupled immunoprecipitation/SDS-PAGE, poly(A)-zymogram activity gel assays showed that both the EhNucI-HA- and EhNucII-HA-expressed proteins did in fact possess functionally active, DTT-sensitive nuclease activities. Moreover, results of these coupled immunoprecipitation/zymogram gel analyses also demonstrated that the EhNucI-HA and EhNucII-HA chimeric enzymes could hydrolyze, in addition to poly(A), a variety of other synthetic polynucleotide substrates (i.e. poly(U) and poly(I) but not poly(G)). The apparent inability of the parasite EhNucI-HA and EhNucII-HA enzymes to hydrolyze poly(G) may be due to the secondary structure of this polynucleotide substrate. Interestingly, similar results have been reported for several other T2 ribonuclease family members (58-61). Although the use of synthetic polynucleotide substrates to demonstrate nuclease activity is well established, it was essential to demonstrate that the parasite EhNucI-HA- and EhNucII-HA-expressed enzymes could in fact hydrolyze naturally occurring nucleic acids. In that regard, the results of our coupled anti-HA immunoprecipitation/substrate hydrolysis assays clearly demonstrated that the EhNucI-HA- and EhNucII-HA-expressed nucleases were capable of hydrolyzing RNA. Furthermore, the RNA substrate was more efficiently hydrolyzed by EhNucI-HA and EhNucII-HA at acidic pH, a property characteristic of many ribonuclease T2 family member enzymes (reviewed in Ref. 32). In contrast, however, neither EhNucI-HA nor EhNucII-HA showed any detectable activity with ssDNA or dsDNA as substrates. The absence of nuclease activity with DNA substrates is also typical of T2 ribonucleases (reviewed in Ref. 32).

In summary, in this study, we showed for the first time that the human intestinal pathogen E. histolytica synthesizes and releases extracellular nucleases. By using a molecular approach, we identified and characterized genes coding for two closely related nucleases, EhNucI and EhNucII, that are constitutively transcribed by parasites during their in vitro culture. Furthermore, we showed when episomally expressed in parasites, the functionally active protein products of these genes are constitutively released/secreted by E. histolytica. As indicated above, E. histolytica parasites are both purine and pyrimidine auxotrophs that reside and multiply primarily within the large intestine of their infected human host and extra-intestinally in more advanced disease states. We hypothesize that within such environments, these secretory nucleases could function at a distance away from the parasite to hydrolyze host-derived nucleic acids. Presumably, these nucleases could act in concert with other purine/pyrimidine salvage enzymes and nucleoside/nucleo-base transporters to facilitate the acquisition of such essential nutrients by these parasites during the course of human disease. In that regard, it has been shown recently using micro-array technology that the nucleases annotated in Entamoeba Gene Data Base as 177.m00126 and 100.m00129, and characterized here as EhNucI and EhNucII, were expressed by parasites isolated directly from experimental animal infections (62). In that context, results of our preliminary experiments have shown that sera derived from acute human amebiasis patients recognized and immunoprecipitated both the EhNucI-HA and EhNucII-HA nucleases expressed and released/secreted by transfected E. histolytica trophozoites. Cumulatively, these data suggest that EhNucI and EhNucII are expressed by parasites in situ during the course of both experimental and naturally occurring (human) host infections. Taken together, the results of this study should facilitate future investigations into the role(s) that the EhNucI and EhNucII secretory nucleases might play in the survival, growth, and development of this important pathogen.


   FOOTNOTES
 
* This work was supported in part by the intramural research program of the Division of Intramural Research, NIAID, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1. Back

1 Supported by an intramural postdoctoral research fellowship from the NIAID, National Institutes of Health. Back

2 Supported by an appointment to the Oak Ridge Institute for Science and Education, Research Specialist Program at the National Institutes of Health. Back

3 To whom correspondence should be addressed. Tel.: 301-496-5969; Fax: 301-402-0079; E-mail: ddwyer{at}niaid.nih.gov.

4 The abbreviations used are: PBS, phosphate-buffered saline; aa, amino acid; DTT, dithiothreitol; ORF, open reading frame; RT, reverse transcription; ssDNA, single-stranded DNA; dsDNA, double-stranded DNA; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; HA, hemagglutinin; FITC, fluorescein isothiocyanate; nt, nucleotide; MOPS, 4-morpholinepropanesulfonic acid; MES, 4-morpholineethanesulfonic acid; gDNA, genomic DNA; Fwd, forward; Rev, reverse. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Egbert Tannich (Bernhard Nocht Insitute for Tropical Medicine, Hamburg, Germany) for the generous gift of the E. histolytica ExEhNeo expression plasmid. We thank Dr. William Petri (Department of Medicine, Infectious Diseases, University of Virginia, Charlottesville) for generously providing sera from amebiasis patients. We also thank Duane Bartley of The Johns Hopkins University DNA Analysis Facility, Baltimore, MD, for assistance with nucleotide sequencing; Dr. Owen Schwartz and the personnel of the Biological Imaging Unit, Research Technologies Branch, DIR, NIAID, National Institutes of Health, for their assistance with our immunofluorescence imaging experiments, and especially Dr. Juraj Kabat for performing the deconvolution analyses of our confocal imaging data.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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