INFECTION AND IMMUNITY,0019-9567/99/$04.0010

June 1999, p. 3073–3081 Vol. 67, No. 6

Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Chitinase Secretion by Encysting Entamoeba invadens and TransfectedEntamoeba histolytica Trophozoites: Localization of Secretory

Vesicles, Endoplasmic Reticulum, and Golgi ApparatusSUDIP K. GHOSH,1 JESSICA FIELD,1 MARTA FRISARDI,1 BENJAMIN ROSENTHAL,1

ZHIMING MAI,1 RICK ROGERS,2 AND JOHN SAMUELSON1*

Department of Immunology and Infectious Diseases1 and BioMedical Imaging Institute,2

Harvard School of Public Health, Boston, Massachusetts 02115

Received 8 October 1998/Returned for modification 10 November 1998/Accepted 23 February 1999

Entamoeba histolytica, the protozoan parasite that phagocytoses bacteria and host cells, has a vesicle/vacuole-filled cytosol like that of macrophages. In contrast, the infectious cyst form has four nuclei and a chitin wall.Here, anti-chitinase antibodies identified hundreds of small secretory vesicles in encysting E. invadens para-sites and in E. histolytica trophozoites overexpressing chitinase under an actin gene promoter. Abundant smallsecretory vesicles were also identified with antibodies to the surface antigen Ariel and with a fluorescent sub-strate of cysteine proteinases. Removal of an N-terminal signal sequence directed chitinase to the cytosol.Addition of a C-terminal KDEL peptide, identified on amebic BiP, retained chitinase in a putative endoplasmicreticulum, which was composed of a few vesicles of mixed sizes. A putative Golgi apparatus, which was Bre-feldin A sensitive and composed of a few large, perinuclear vesicles, was identified with antibodies to ADP-ribosylating factor and to «-COP. We conclude that the amebic secretory pathway is similar to those of othereukaryotic cells, even if its appearance is somewhat different.

Entamoeba histolytica is a protozoan parasite that causesdysentery and liver abscess in developing countries which can-not prevent its fecal-oral spread (56). Amebae have three vir-ulence-associated properties, which are of interest to cell biol-ogists (43). First, amebae survive anaerobic conditions in thelumen of the colon and tissue abscesses by means of fermen-tation enzymes that resemble those of anaerobic bacteria (31,48, 57, 60). Indeed, amebae lack enzymes of oxidative phos-phorylation and have an atrophic mitochondrion-derivedorganelle, which resembles the petite mitochondria of yeastcells grown under anaerobic conditions (11, 20, 41). Second,amebae have a vesicle/vacuole-filled cytosol like that of mac-rophages (63). Amebae phagocytose bacteria in the coloniclumen and epithelial cells and erythrocytes (RBC) when par-asites invade tissues and cause dysentery (46, 56). Third, ame-bae form a chitin-walled cyst, which is the infectious form ofthe parasite, because cysts are resistant to stomach acids (5,15).

Phagocytosis is the best studied of the amebic virulencemechanisms. Parasites attach to bacteria, epithelial cells, andRBC via amebic lectins, which recognize Gal or GalNAc sug-ars on the surface of target cells (42, 45). Amebae kill bacteriaor lyse host cells within their phagolysosomes via oxygen-inde-pendent mechanisms including lysozyme, cysteine proteases,and amebapores (also known as pore-forming peptides) (8, 34,64). Lysosomal proteins, which have been identified in super-natants of cultured trophozoites, include cysteine proteases,acid phosphatase, collagenases, glycosidases, and esterases butnot pore-forming peptides (1, 35, 68). One cysteine proteinase(CP-5) is present on the surface E. histolytica trophozoites butis absent from the surface of avirulent E. dispar (26). Amebicphagocytosis is disrupted by wortmannin and by overexpres-

sion of hyperactive amebic p21rac, a ras-family protein involvedin the site selection of actin polymerization (18, 23, 36). Inanimal models, phagocytosis mutants of amebae, selected byconsumption of bromodeoxyuridine-loaded bacteria, are lessvirulent than wild-type parasites (58).

Secretion by amebae is much less well understood thanphagocytosis. Amebae have on their surface vaccine candi-dates, including the Ser-rich E. histolytica protein (SREHP)and Gal or GalNAc lectin, which presumably get there whensecretory vesicles fuse with the plasma membrane (42, 45, 51,52, 61, 67, 73). Small and large subunits of the Gal or GalNAclectin have signal sequences that are cleaved at sites predictedby the “23,21” rule of von Heijne (Table 1) (50). Signalsequences as well as propeptide sequences are cleaved frompore-forming peptides and cysteine proteases (Table 1) (8, 34).An E. histolytica gene has been cloned that encodes a 54-kDapeptide of the signal recognition particle, a ribonuclear proteinthat binds N-terminal signal sequences on secreted proteins(55). An amebic gene has also been cloned that encodes anendoplasmic reticulum (ER) retention receptor (ERD2), acis-Golgi-associated transmembrane protein (62). ERD2 bindsC-terminal KDEL peptides on proteins such as the 70-kDaheat shock protein BiP, which is a chaperonin in the lumen ofthe ER (10, 22). We recently cloned the E. histolytica bip geneand confirmed that the predicted BiP has a C-terminal KDELpeptide (16a). Although amebae lack a Golgi with tight lamel-lae, a putative Golgi was identified by confocal microscopy withNBD-ceramide and by transmission electron microscopy withthiamine-pyrophosphatase (44).

We recently used molecular cloning methods to identifyamebic chitinases, which are secretory proteins expressed byencysting parasites (14). Each amebic chitinase contains a se-ries of acidic and hydrophilic repeats between an N-terminalsignal sequence and a C-half catalytic domain (50). As E.histolytica trophozoites are difficult to encyst in axenic culture,cyst formation has for the most part been studied by using thereptilian pathogen E. invadens, which converts to chitin-walled

* Corresponding author. Mailing address: Department of Immunol-ogy and Infectious Diseases, Harvard School of Public Health, 665Huntington Ave., Boston, MA 02115. Phone: (617) 432-4670. Fax:(617) 738-4914. E-mail: jsamuels@hsph.harvard.edu.

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cysts within 2 days when deprived of glucose (15). During E.invadens cyst formation, chitin synthase and chitinase are bothexpressed, and cyst formation is inhibited by the chitin syn-thase inhibitors polyoxin D and Nikkomycin and by the chiti-nase inhibitor allosamidin (6, 13, 72).

The goal of the present studies was to visualize structuresinvolved in secretion (secretory vesicles, ER, and Golgi) inamebic trophozoites and encysting organisms. These studieswere patterned after similar studies of Giardia lamblia, a sec-ond intestinal protozoan parasite, which forms cysts with anacid-resistant wall (19, 39). Secretory vesicles and Golgi appa-ratus of G. lamblia trophozoites (motile forms) are very diffi-cult to visualize. In contrast, secretory vesicles, which containcyst wall proteins, are so prominent in encysting giardia thatthey were given a special name, encystation-specific vesicles orESV (47). Similarly, encysting G. lamblia show increased ex-pression of Golgi proteins and the ER-associated protein BiP(22, 37, 38). Here amebic secretory vesicles, putative ER, andputative Golgi, which were visualized by the various probespresented in Table 2, were prominent in trophozoites, as wellas in encysting parasites (secretory vesicles and Golgi). Fur-ther, targeting of chitinase in transfected parasites was modi-fied by removal of an N-terminal signal sequence or addition ofa C-terminal, ER retention peptide (KDEL).

MATERIALS AND METHODS

Parasites used and conditions for pinocytosis, phagocytosis, and encystation.The HM-1 strain of E. histolytica, from which chitinase, ariel, arf, and bip geneswere identified, was used to study secretion in amebic trophozoites (14, 40). TheIP-1 strain of E. invadens, a generous gift of Dan Eichinger of New YorkUniversity, was used to study secretion during encystation (15). E. histolytica andE. invadens were grown at 37 and 25°C, respectively, in the same axenic TYI-S33medium.

Pinocytosis by E. histolytica and E. invadens was observed by incubating tro-phozoites for 30 min with fluorescein isothiocyanate (FITC)-dextran (1 mg/ml) inculture medium (18). Amebae were washed in phosphate-buffered saline (PBS),fixed in 2% paraformaldehyde, and passed through a fluorescence-activated cellsorter (Becton Dickinson). Negative controls included paraformaldehyde-fixedparasites not exposed to FITC-dextran.

Phagocytosis by E. histolytica and E. invadens was studied by using Escherichia

coli expressing recombinant green fluorescent protein (GFP) under an isopropyl-b-D-thiogalactopyranoside-inducible promoter (70). Trophozoites (105/ml) wereincubated in culture medium with GFP-labeled bacteria (107/ml) for 30 min andthen fixed with 2% paraformaldehyde in PBS (18).

E. invadens encystation was induced by simultaneously reducing the osmolar-ity, glucose, and serum in the cultures (15). Encystating organisms after 1 to 3days induction were identified in three ways: (i) observation of rounded organ-isms with a refractile wall, which were resistant to lysis in 1% Triton X-100; (ii)staining parasites with Calcofluor; or (iii) staining organisms with anti-chitinaseantibodies (see below). To determine the effects of various inhibitors on cystformation, we incubated E. invadens in encysting medium containing 100 mg ofBrefeldin A per ml, 0.3 mM okadoic acid, or 100 nM wortmannin (18, 25, 29).

Cloning of E. histolytica and E. invadens arf genes. Segments of the E. invadensand E. histolytica arf genes were isolated from DNA of E. invadens IP-1 strain andE. histolytica HM-1:IMSS strain genomic DNAs by using the PCR and degen-erate primers to conserved peptides in ADP-ribosylating factor (ARF) of othereukaryotes (3, 21, 28, 32). A degenerate sense primer [AGAAT(CT)(CT)T(AT)ATGGT(AT)GG] was to RILMVG, while an antisense primer [GG(AT)A(AG)(AG)TCTTGTTT(AG)TT(AT)(AG)CG] was to F(AV)NKQDL. PCR productswere cloned in TA-vector and sequenced by dideoxy methods. As expected, theE. histolytica arf PCR product was more AT-rich in the third position (89%) thanthe E. invadens arf PCR product (60%) (8, 14, 31, 36, 40, 53, 55, 60, 62). Thepredicted E. histolytica ARF was compared with proteins in the GenBank data-base by using BLAST2 (2). An alignment of ARF and ARF-like sequences wasmade by using PIMA-2, and a neighbor-joining (NJ) tree was constructed byusing Treecon (66, 71). The reproducibility of the resulting topology was deter-mined by replicating the analysis on 100 bootstrap resamplings of each align-ment.

RT-PCR of arf, erd2, and chitinase mRNAs of E. histolytica and E. invadensparasites. Total RNA was extracted from E. histolytica trophozoites and fromencysting E. invadens by lysing organisms in saturated guanidinium and centri-fuging the lysate through a cesium chloride cushion. First-strand cDNA synthesiswas performed with E. histolytica RNA by using reverse transcriptase (RT) andantisense primers specific for arf (ATTTCTCATCTCATCTTC), erd2 (TCAATATGGCAAAACGAATTT), or chitinase (TTAACATTTCTCAATTAG) genes(14, 62). PCRs were then performed with these antisense primers and senseprimers specific for E. histolytica arf (GGACTTGATGCTGCCGG), erd2 (ATGGTGTTTAATCTTTTTAGA), and chitinase (ATGTCACTACTGGCATTAAT) genes, respectively. Controls included PCR without prior RT. RT-PCR wasperformed in a similar manner with RNAs from encysting E. invadens by usingsense (GTGACATCGTCCCAAGAA) and antisense (GGGCACAGTACAGACAAA) primers to E. invadens chitinase genes, and the same sense and antisenseprimers were used to amplify E. histolytica arf cDNAs (14).

Sources of antibodies against chitinases, Ariel antigens, ADH1, ARF, and«-COP. E. histolytica and E. invadens chitinases each have a series of degener-ative repeats, which are hydrophilic and acidic, between signal peptides andcatalytic domains (14). Because the chitinase repeats differ from each other, twosets of rabbit polyclonal, monospecific anti-chitinase antibodies were prepared(Table 2) (24). The first antibodies were against a multiantigenic peptide (MAP)containing E. histolytica chitinase repeats (HESSEIKPDSSESKHESSEK). Thesecond antibodies were against a MAP containing E. invadens chitinase repeats(PKPEESSEQPKPEESSEEKK). Rabbit antibodies were purified on affinity col-umns containing each chitinase MAP and checked against Western blots ofamebic proteins.

Anti-Ariel antibodies were made by immunizing a rabbit with a glutathioneS-transferase (GST) fusion protein containing octapeptide repeats from theAriel 1 protein, an E. histolytica surface protein (40, 65). Anti-alcohol dehydro-genase 1 (ADH1) antibodies were made by immunizing a rabbit with a GST-ADH1 fusion protein (31, 41). Anti-Ariel and anti-ADH1 antibodies were af-finity purified on columns made of each fusion protein. Mouse ID9 monoclonalantibodies to ARF, a Golgi-associated coatomer protein, and polyclonal rabbit

TABLE 1. N-terminal signal sequences of E. histolytica plasmamembrane, secretory, ER, or lysosomal proteins

Protein Sequencea

Plasma membrane proteinsLectin small subunit ...........miilililfsysfg KTQ...Lectin large subunit............mkllllnillccla DKL...SREHPb ...............................mfafllfiaftsaTNI...Arielb ....................................mfafllfiafttaTTNV...

Secretory or ER proteinsChitinase ..............................msllaliyranaHNC...E. invadens chitinase...........msamafvcfvlalansQTCEG...BiP ........................................mltflftllfcitvfsEDT...ALDH1 ................................meayvlslsdvlfnilfigvcilsvllishaLKY...

Lysosomal proteinsPore-forming peptide Ac....mkaivfvlifavafavtathq gEIL...Cysteine protease 1c ...........mftfilmfyigygIDFNTWVANNNKHFTAVESLR

RRAIFNMNARIVAENNRKETFKLSVDGPFAAMTNEEYNSLLKLKRSGEEKGEVRYLNIQ APKA...

a Predicted signal sequences (lowercase letters) at the amino terminus of eachprotein were identified by the 23,21 rule of von Heijne and confirmed byN-terminal sequencing (spaces) for the large and small subunits of the lectin(50). The remainder of each protein is indicated in capital letters. Pro sequence(underlined) of cysteine protease 1 was identified by N-terminal sequencing.

b The SREHP and Ariel proteins are genetically related.c The pore-forming peptide A is representative of two other pore-forming pep-

tides, while cysteine protease 1 is representative of five other cysteine proteases.

TABLE 2. Antibodies and transformation constructsused to localize amebic organelles

Structure Antibodies to: Constructs

Secretory Chitinasea ChitinaseVesicles ArielER BiP-myc,

chitinase (1) KDELCytosol ADH1 Chitinase (2) signalGolgi Mammalian ARF, mammalian

ε-COP

a Encystation-specific chitinase in E. invadens. All constructs were in the pJST4vector and transfected into E. histolytica trophozoites. In addition, pinosomes werelocalized with FITC-dextran, phagosomes with GFP-labeled bacteria, and lyso-somes with the fluorescent substrate (Arg–Arg–4-methoxy-2-naphthylamide).

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antibodies to ε-COP were a generous gift of Victor Hsu of the Brigham andWomen’s Hospital (4).

Expression of chitinase genes under an actin promoter in transfected E. his-tolytica trophozoites. E. histolytica trophozoites, which do not encyst in axenicculture, do not express their chitinase gene (14). To identify secretory vesicles inE. histolytica trophozoites, we expressed the E. histolytica chitinase gene in trans-fected amebae under an E. histolytica actin 1 gene promoter (Table 2) (17, 18).Briefly, the coding region of the E. histolytica chitinase gene was isolated fromgenomic DNA by PCR. The sense primer (S1 5 GCGGTACCATGTCACTACTGGCATTAAT) contained a KpnI site (italics) and encoded the first six aminoacids at the amino terminus of the parasite’s chitinase (MSLLAL [underlined]).The antisense primer (A1 5 GCGGATCCTTAACATTTCTCAATTAG) con-tained a BamHI site (italics) and encoded five amino acids at the C terminus ofthe organism’s chitinase (LIEKC [underlined]). The chitinase gene was clonedinto the pJST4 amebic transformation vector and electroporated into E. histo-lytica HM-1 strain amebae (17). Parasites were step selected in 10 to 100 mg ofG418 per ml (4 to 6 weeks), when the expression of chitinase mRNA was checkedby RT-PCR. Overexpression of intact chitinase or modified chitinases (describedbelow) had no effect on amebic viability, growth rate, pinocytosis, or phagocy-tosis.

N-terminal signal sequences on amebic secretory, ER, or plasma membraneproteins were identified by using the SignalP algorithm at the website of theCenter for Biological Sequence Analysis at the Technical University of Denmark(Table 1) (50). A truncated E. histolytica chitinase gene encoding a chitinaselacking its 12-amino-acid signal sequence was made with the PCR (14). Theantisense primer was A1 (described above), while the sense primer (GCGGTACCATGGCTCACAACTGTGAAG) contained a BamHI site (italics) and en-coded Met and Ala13 to Glu19. To determine whether a C-terminal KDELpeptide would cause chitinase to be retained in the lumen of the ER of trans-fected parasites, we made a modified chitinase gene by using PCR. The senseprimer was S1 (described above), while the antisense primer (GGGGATCCTTAAAGTTCATCTTTTAGACTCTTAATGTATTTTG) contained a BamHI site

(italics) and encoded KYIKSLKDEL (underlined) at the C terminus of thechitinase instead of the wild-type C-terminal sequence (KYIKSLIEKC) (14).The truncated chitinase and the chitinase-KDEL PCR products were cloned intopJST4 and expressed in amebae as described above.

As a control for localization of the ER, amebic BiP was overexpressed with amyc tag in E. histolytica trophozoites (10, 16, 22, 38). The coding region of theE. histolytica bip gene was isolated from genomic DNA by using PCR. A senseprimer (GCGGTACCATGTTAACTTTCTTATTC) contained a KpnI site (ital-ics) and encoded the first six amino acids at the amino terminus of the parasite’sBiP (MLTFLF [underlined]). An antisense primer (GCGGATCCTTAAAGTTCATCTTTTAAATCTTCTTCTGAAATTAATTTTTGTTCTTCATATTCTTCATAATT) contained a BamHI site (italics) and encoded the C-terminal KDEL(underlined), the myc epitope EQKLISEEDL (boldface), and the BiP hexapep-tide NYEEYE adjacent to the C terminus (not underlined). The BiP constructswere cloned into the pJST4 amebic transformation vector and electroporatedinto E. histolytica HM-1 strain amebae as described above.

Confocal microscopy. To immunolocalize secretory proteins on the surface ofE. histolytica and E. invadens, we fixed parasites with 2% paraformaldehyde for10 min at 4°C. To visualize secretory vesicles or Golgi-associated proteins, wepermeabilized amebae by incubation with 0.1% Triton X-100 for 5 min at roomtemperature. Similar results were obtained when parasites were permeabilizedwith 0.1% saponin. Amebae were immunostained for 60 min with polyclonalrabbit antibodies to chitinases, Ariel, or ε-COP, diluted 1:100 or 1:200 in PBSwith 1 mg of bovine serum albumin per ml (24). Organisms were washed fourtimes and immunodecorated for 60 min with a Texas red-conjugated goat anti-rabbit antiserum. Controls included parasites stained with preimmune rabbitserum. Similar methods were performed with mouse monoclonal anti-ARF andanti-myc antibodies, each diluted 1:200. Lysosomes were visualized by incubatingliving parasites in Arg-Arg-4-methoxy-2-naphthylamide, a substrate of cysteineproteinases that fluoresces when it is cleaved (64). Fluorescently labeled para-sites were observed with a Leica NT-TCS confocal microscope fitted with argonand krypton lasers. Images of amebae were recorded in 512 image size formatwith a 340 or 3100 Planapo objective. The number of vesicles per ameba labeledwith anti-ARF, anti-ε-COP, or anti-chitinase antibodies in parasites transfectedwith the chitinase-KDEL construct was determined by making serial opticalsections through the parasites with the confocal microscope. To illustrate someof these structures, composite figures were made that combined multiple opticalsections. The number of secretory vesicles, which were identified with antibodiesto intact chitinase or Ariel, was too many to count accurately.

Nucleotide sequence accession numbers. Nucleotide and derived amino acidsequences of E. histolytica and E. invadens arf gene segments have been submit-ted to GenBank under accession numbers AF082517 and AF082518.

RESULTS

Chitinase is an appropriate reporter protein for studyingsecretion in transfected E. histolytica because it is not ex-pressed by trophozoites. GFP, which is a popular epitope tagused to localize proteins in living eukaryotic cells, does notwork in transfected amebae (unpublished observations) (54).This is because GFP fluorescence is dependent upon the pres-ence of free oxygen, which is not present in amebic cultures.Amebic chitinase was chosen here to study secretion in trans-fected trophozoites because chitinase contains a series of an-tigenic repeats and is normally secreted by encysting parasites

FIG. 1. RT-PCR of arf, erd2, and chitinase (chit) mRNAs in nontransfectedE. histolytica trophozoites and E. invadens encysting for 0 to 48 h. Ethidiumstaining of PCR products, separated on agarose gels, was electronically reversedfor ease of reproduction.

FIG. 2. Confocal micrographs of chitinase-associated secretory vesicles of encysting E. invadens identified with anti-chitinase antibodies. (A) Chitinase-associatedsecretory vesicles were absent from trophozoites (data not shown) but were widely distributed in amebae encysting for 24 h. (B) Anti-chitinase antibodies innonpermeabilized amebae mark secreted products on the parasite surface. Bars, 5 mm.

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(14). Chitinase mRNAs, detected by RT-PCR, were present inextracts of encysting E. invadens parasites but were absent inextracts of either E. histolytica or E. invadens trophozoites (Fig.1). In contrast, mRNAs of the Golgi-associated protein ARFwere present in extracts of trophozoites of E. histolytica andE. invadens and in extracts of encysting E. invadens (Fig. 1 andsee further discussion below) (12). ERD2 mRNAs were alsopresent in extracts of E. histolytica trophozoites (Fig. 1) (62).

Chitinase-associated secretory vesicles in encysting E. in-vadens parasites are numerous and small. Antibodies toantigenic repeats of the E. invadens chitinase were used todemonstrate hundreds of mostly small secretory vesicles inencysting E. invadens parasites (Fig. 2 and Table 2) (14, 61). Inmost cells, these chitinase-associated secretory vesicles were soabundant that their exact number could not be determined.Chitinase was also present in patches on the surface of encyst-ing parasites. Chitinase was absent from trophozoites, whichlack detectable messages for this gene (as above). The chiti-nase-associated vesicles of encysting amebae are similar intheir appearance to ESV previously described in encysting G.lamblia (19, 39, 47).

When encysting parasites were incubated with FITC-dextran

or GFP-labeled bacteria prior to fixation and staining withanti-chitinase antibodies, two results were apparent. First, par-asites with many chitinase-associated secretory vesicles, whichwere well into encystation, pinocytosed much less FITC-dex-tran and phagocytosed many fewer bacteria than trophozoites.For example, after 24 h, most encysting parasites pinocytosedthe same amount of FITC-dextran as the control parasitesfixed before incubation with FITC-dextran (data not shown).After 24 h, most encysting parasites no longer phagocytosedbacteria. Second, chitinase-associated secretory vesicles werenot overlapping with pinosomes and phagosomes, so double-staining (orange vesicles) was rare or absent (data not shown).

Secretory vesicles of E. histolytica trophozoites, marked byanti-chitinase antibodies in transfected parasites, are numer-ous and small like vesicles containing Ariel antigens or cys-teine proteinases. Axenic amebae, which were transfected withan unmodified chitinase gene under an actin gene promoter,had numerous small chitinase-associated vesicles (Fig. 3 andTable 2). Again, these secretory vesicles in most cells were tooabundant to make an accurate count. Double-labeling studies,which were performed with Texas red-labeled chitinase andFITC-dextran or GFP-labeled E. coli, showed that chitinase-

FIG. 3. Confocal micrographs of E. histolytica trophozoites transfected with their own chitinase gene under an actin promoter. Anti-chitinase antibodies (red)demonstrated secretory vesicles in transfected, fixed, and permeabilized E. histolytica trophozoites. These vesicles were independent of pinocytotic vacuoles marked withFITC-dextran (green in panel A) or phagocytotic vacuoles marked with GFP-labeled bacteria (green in panel B). Anti-chitinase antibodies failed to stain the surfaceof nonpermeabilized, transfected parasites, thus demonstrating that the enzyme is secreted and shed (data not shown). Chitinase-associated vesicles were about thesame size and abundance as lysosomes, as marked by the fluorescent substrate (Arg–Arg–4-methoxy-2-naphthylamide) of cysteine proteases (yellow in panel C). Bars,5 mm.

FIG. 4. Confocal micrographs of nontransfected E. histolytica trophozoites which were fixed, permeabilized, and stained with antibodies to Ariel, a homologue ofthe vaccine candidate SREHP. While most Ariel-associated secretory vesicles (red) were independent of pinocytosed FITC-dextran (green in panel A), some localizedwith phagocytotic vacuoles marked with GFP-labeled bacteria (green in panel B). Anti-Ariel antibodies outlined the surface of nonpermeabilized trophozoites (red inpanel C). Bars, 5 mm.

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associated vesicles did not fuse with pinocytotic or phagocy-totic vacuoles. Chitinase-associated secretory vesicles wereabout the same size as lysosomes (identified with Arg–Arg–4-methoxy-2-naphthylamide, a fluorescent substrate of the cys-teine proteases) (64). Because the signal from the cysteineproteinase was present in both FITC and Texas red channels,it was not possible to colocalize the lysosomes and the chiti-nase-containing vesicles. Chitinase was absent from the surfaceof transfected parasites (data not shown), suggesting that thesurface of trophozoites lacked chitin-binding activity presenton the surface of encysting parasites.

Vesicles containing the E. histolytica surface antigen Ariel

were small and numerous and so most closely resembled chiti-nase-associated vesicles of encysting E. invadens (Fig. 4 andTable 2) (40). Ariel antigens were also present in phagocytoticvacuoles and coated the surface of nonpermeabilized E. histo-lytica trophozoites. A similar appearance has been describedfor the vaccine candidate SREHP, which is encoded by a genethat belongs to the same superfamily of antigen-encodinggenes as Ariel (67).

Targeting of chitinase in transfected E. histolytica parasitesis changed when an N-terminal signal sequence is removed ora C-terminal ER retention signal is added. Amebic chitinaseand other secretory or plasma membrane proteins have at their

FIG. 5. Confocal micrographs of amebic secretory vesicles, cytosol, and putative ER. (A) Intact chitinase was targeted in transfected E. histolytica trophozoites tosecretory vesicles, which were small and numerous. (B) Similar vesicles were visualized with anti-Ariel antibodies in nontransfected parasites. (C) A truncated chitinaselacking an N-terminal signal sequence went to the cytosol, which has dark vesicles against a bright background. (D) This is the same appearance as that ofnontransfected amebae stained with antibodies to ADH1. (E) A modified chitinase with a C-terminal KDEL peptide was retained in a putative ER, which was composedof many fewer vesicles than the secretory vesicles marked by unmodified chitinase or Ariel. (F) A similar pattern of staining was observed with myc-labeled BiP, whichis retained by its native C-terminal KDEL in the putative ER. Bars, 5 mm.

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N-termini signal sequences, which fit the 23,21 rule of vonHeijne (Table 1) (50). To test the necessity of the 12-amino-acid signal sequence of the E. histolytica chitinase, we trans-fected parasites with chitinase genes that encoded an intactchitinase and a truncated chitinase lacking the signal sequence(Fig. 5 and Table 2). Anti-chitinase antibodies demonstrated avesicle-bright, cytosol-dark distribution of the intact chitinasethat was similar to that of Ariel. In contrast, anti-chitinaseantibodies demonstrated a reverse image of the truncatedchitinase, in which vesicles were dark and the cytosol wasbright. This cytosolic distribution, in which vesicles appeardark, like the holes in Swiss cheese, was also demonstratedwith antibodies to ADH1, an abundant amebic fermentationenzyme (31, 41).

To localize the amebic ER, E. histolytica trophozoites weretransfected with construct which overexpressed BiP with a mycepitope tag (Fig. 5 and Table 2). Amebic BiP, which has aC-terminal KDEL peptide, should bind ERD2 receptors in theproximal Golgi and be retained in the ER (10, 22, 62). myc-labeled BiP was present in a putative ER, which was composedof much fewer (#20 per parasite) vesicles than the secretoryvesicles marked by intact chitinase or Ariel. To determinewhether the KDEL peptide is sufficient to retain amebic pro-teins in the ER, we overexpressed chitinase with a KDEL pep-tide at its C terminus in E. histolytica trophozoites. Anti-chiti-nase antibodies demonstrated putative ER-associated vesiclesin these parasites, which resembled those identified with myc-labeled BiP. The ER was not visualized in encysting E. inva-dens because transfection methods have not been developedfor these parasites.

E. histolytica and E. invadens arf genes encode a conservedGolgi-associated coatomer protein (COP) called ARF. To bet-ter understand the role of Golgi apparatus in secretion byamebic trophozoites and encysting parasites, we used the PCRand degenerate primers to conserve peptides in other ARF toclone segments of E. invadens and E. histolytica arf genes (Fig.6) (3, 21, 28, 32). The predicted 113-amino-acid open readingframe (ORF) of the amebic arf genes, which encode abouttwo-thirds of the expected 20-kDa ARF proteins, showed 96%positional identity with each other and 76 to 88% positionalidentities with other eukaryotic ARFs. Perfectly conserved inthe amebic ARFs were guanine nucleotide-binding domains,including the pyrophosphate-binding loop (GLDAAGKT),switch region (DVGG), and guanine recognition motif (NKQD)

(3, 21). In NJ trees, amebic ARF was easily distinguished fromARF-like proteins of parasites or humans (Fig. 7) (71).

The amebic Golgi and encystation are disrupted by Brefel-din A and okadoic acid. As discussed above, RT-PCR showedthat mRNAs that encode the Golgi-associated protein ARFand the ER-retention receptor (ERD2) are expressed byE. histolytica trophozoites (Fig. 1) (12, 62). ARF was also ex-pressed by E. invadens trophozoites and encysting E. invadens(Fig. 1). Anti-ARF antibodies identified a stage-independent,putative Golgi apparatus, which was composed of a few largeperinuclear vesicles (5 to 20 per cell) (Fig. 8). Similar Golgi-associated vesicles were identified with antibodies to ε-COP(Fig. 9). This putative Golgi apparatus was disrupted into tinyvesicles Brefeldin A, which targets the GTPase of ARF (29).Brefeldin A also reduced the number of E. invadens parasitesthat encyst by 60%. Okadoic acid, which targets protein phos-phatases and disrupts trans-Golgi of eukaryotic cells, disruptedthe amebic Golgi and eliminated encystation completely (25).Wortmannin, which targets phosphoinositide 3-kinases andeliminates amebic pinocytosis and phagocytosis, also eliminat-ed encystation (18). Previously, an amebic Golgi was seen withNBD-ceramide (as seen by fluorescence microscopy) and thi-amine-pyrophosphatase (as seen by electron microscopy) (44).

FIG. 6. ARF alignment. Predicted ORF of a segment of the E. histolytica(Eh) arf gene aligned with ARF segments of E. invadens (Ei), G. lamblia (Gl;GenBank accession number S29008), Plasmodium falciparum (Pf, U57370), Dic-tyostelium discoideum (Dd; AJ000063), Saccharomyces cerevisiae (Sc; M35158),and Bos taurus (Bt; A45422). Dashes indicate identities with the E. histolyticaARF, while periods indicate gaps. Vertical boxes indicate locations of conservedpyrophosphate-binding loop (GLDAAGKT), switch region (DVGG), and gua-nine-recognition motif (NKQD) (3, 34).

FIG. 7. NJ tree of ARF shown in Fig. 6 as well as ARF of Xenopus laevis (Xl;GenBank accession number U31350), Drosophila melanogaster [Dm(A); S62079],Cryptococcus neoformans (Cn; L25115), and Schizosaccharomyces pombe (Sp;L09551), and ARF-like proteins of D. melanogaster [Dm(AL); A40438], Homosapiens (Hs; L28997), P. falciparum [Pf(AL); U57370], and Leishmania tarentolae(Lt; X97072). Branch lengths are proportional to differences between adjacentsequences, while numbers at each node indicate the extent of bootstrap support(of 100 replicates). Unmarked nodes occurred in fewer than 50 replicates.

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DISCUSSION

These are the first arf genes identified in amebae, and this isthe first use of transfection methods to localize amebic pro-teins and the first experimental demonstration of N-terminalsignal and C-terminal ER retention sequences. Roughly drawn,the amebic cytosol is composed of small vesicles, which includethose of the putative ER, secretory vesicles, lysosomes, pino-cytotic vacuoles, and large vesicles, which include those of theputative Golgi and phagocytotic vacuoles.

Conservation of secretory apparatus and protein-targetingsequences in amebae. Perhaps because amebae have suchprominent pinosomes, phagosomes, and lysosomes, secretorystructures of the parasite were not obvious in the absence ofthe specific probes used here (Table 2) (18, 43, 63). Chitinaseexpression in transfected parasites was used to show that ame-bic N-terminal signal sequences and C-terminal ER retentionsignals appear to follow the same rules as those established forhigher eukaryotes (50, 61, 62). Indeed the 23,21 rule appearsto hold for all signal sequences of amebic secretory and plasmamembrane proteins which have been identified to date (Table1). The amebic Golgi and encystation were disrupted by Bre-feldin A and okadoic acid, which disrupt the Golgi of highereukaryotes (25). Disruption of the G. lamblia Golgi, which isencystation specific, also inhibits cyst formation (37).

Unique properties of the amebic secretory pathway. Amebicsecretion is different from that of higher eukaryotes in at leastfour ways. First, the putative amebic Golgi apparatus does notform tightly packed lamellae but instead forms a few largevesicles, which are adjacent to the nucleus (12, 44, 79). This issimilar to the Golgi of G. lamblia, whereas Tritrichomonasfoetus, another lumenal parasite, has a Golgi with tight lamel-lae (19, 37). Second, an amebic protein disulfide isomerase(PDI) lacks a C-terminal KDEL peptide, which usually retainsother PDI in the ER (27). Third, a sharp distinction betweenamebic lysosomes and secretory vesicles has not yet been made(63). Fourth, signals that direct amebic proteins to the lyso-somes cannot be the same as those of higher eukaryotes. Ame-bic lysosomal proteins lack signals for Asn-linked glycosylation,which is necessary for the addition of mannose-6-phosphatesthat might bind to lysosomal receptors (30, 61, 69). We arepresently performing experiments to distinguish secretory ves-icles from lysosomes and to identify signals that target amebicproteins to lysosomes. We plan to use immunoelectron micros-copy to visualize amebic secretory vesicles, putative ER, andputative Golgi.

Implications of these findings for our understanding ofamebic virulence. Four implications of these data may be im-portant in understanding amebic virulence. First, the secretory

FIG. 8. Confocal micrographs of E. invadens parasites stained with heterologous anti-ARF antibodies. (A) ARF was present in large vesicles (presumed Golgiapparatus) surrounding the nucleus of trophozoites. (B) The ARF-stained vesicles of trophozoites were disrupted by Brefeldin A. (C) The Golgi apparatus of encystingparasites was similar when stained with anti-ARF. Micrographs shown in panels A and B represent a single section each, while the micrograph in panel C is a compositeof multiple sections. Bars, 5 mm.

FIG. 9. Confocal micrographs of E. histolytica and E. invadens parasites stained with antibodies to Golgi-associated coatomer protein ε-COP. Vesicles that containε-COP were relatively few and large in trophozoites of E. histolytica (A), trophozoites of E. invadens (B), or encysting E. invadens (C). The micrograph shown in panelA represents a single section, while the micrographs in panels B and C are each composed of multiple sections. Bars, 5 mm.

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apparatus, which targets proteins to the plasma membrane ofamebic trophozoites, likely releases numerous proteins intothe medium, even though to date only lysosomal enzymes havebeen identified in amebic supernatants (1, 35, 68). Second, the23,21 algorithm might be used to identify secretory proteinsin EST databases of amebic trophozoites or encysting parasites(50). Third, ARF in the cytosol of amebae may activate choleratoxin in individuals who are coinfected with Vibrio cholerae andamebae (49). Other targets for bacterial toxins previously iden-tified in amebae include elongation factor 2 (diphtheria andpseudomonas toxins) and Ras-superfamily proteins (clostridialtoxins) (36, 53). Fourth, stage-independent Golgi and ER inamebae suggest that these parasites may be susceptible tobacterial toxins, which enter the cytosol through the Golgi orER (49).

Changing image of amebae and other lumenal parasites:from primitive to divergently evolved. Lumenal parasites (E.histolytica, G. lamblia, and Trichomonas vaginalis) branchedearly from the main eukaryotic tree, lack mitochondria andenzymes of oxidative phosphorylation, and have fermentationenzymes like those of bacteria (31, 33, 48, 57). These observa-tions led to the idea that these microaerophilic parasites are“primitive” or living fossils from a time before eukaryotic cellswere exposed to oxygen and captured the mitochondrial en-dosymbiont (7, 20, 57). The experiments described here showthe similarity of the amebic secretory pathway to those of othereukaryotic cells, even if its appearance is somewhat different.Further, amebae, giardias, and trichomonads each have a geneencoding a homologue of the mitochondrial 60-kDa heat shockprotein (Hsp60) (9, 11, 59). The presence of Hsp60 genes in“amitochondriate” parasites strongly suggests that all extanteukaryotes share a common ancestor that had a mitochondrion(20). In trichomonads, the mitochondrion-derived organellewas converted over time into a fermentation factory called thehydrogenosome (9, 48). The function of an atrophic mitochon-drion-derived organelle of amebae is not known, while a mi-tochondrion-derived organelle has not yet been identified ingiardias (41, 59).

ACKNOWLEDGMENTS

This work was supported in part by National Institutes of Healthgrants AI-33492 and GM-31318 (to J.S.) and grants HL-330099 andHL-43510 (to R.R.).

We acknowledge the expert technical support of Jean Lai for con-focal microscopy and image analysis.

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