Archives of Medical Research 35 (2004) 199–208

ORIGINAL ARTICLE

A 63 kDa VSP9B10A-Like Protein Expressed in a C-8Giardia duodenalis Mexican Clone

Rosa Marıa Bermudez-Cruz,a Guadalupe Ortega-Pierres,a Vıctor Ceja,a Ramon Coral-Vazquez,a,b

Rocıo Fonseca,a Lourdes Cervantes,a Alejandra Sanchez,a Francisco Depardon,a

George Newportc and Cecilia Montaneza

aDepartamento de Genetica y Biologıa Molecular, Centro de Investigacion y de Estudios Avanzados del InstitutoPolitecnico National (Cinvestav del IPN), Mexico City, Mexico

bUnidad de Investigacion Medica en Genetica Humana, Hospital de Pediatrıa, Centro Medico Nacional Siglo XXI (CMN-SXXI),Instituto Mexicano del Seguro Social (IMSS), Mexico City, Mexico

cDepartment of Stomatology, University of California at San Francisco (UCSF), San Francisco, CA, USA

Received for publication October 14, 2003; accepted December 10, 2003 (03/170).

Background. It is well documented that Giardia duodenalis undergoes surface antigenicvariation both in vivo and in vitro. Proteins involved have been characterized and referredto as VSP (variable surface protein).

Methods. Two cloned cDNA inserts of 0.45 and 1.95 kb were obtained from G.duodenalis expression library and sequenced. Comparison sequence analyses were madeagainst Genbank. PCR analysis was performed on G. duodenalis isolates to identifyisolates bearing genes encoding such a peptide. Specific antiserum was prepared against450-bp encoded peptide and tested by Western blot, immunofluorescence, and inhibitionof adhesion of G. duodenalis to target cells.

Results. We cloned and characterized a G. duodenalis 450-bp DNA fragment; its DNAsequence analysis revealed that this fragment displayed 99% identity with vsp9B10Agene. Predicted amino acid sequence for this fragment also had significant (99%) identityto VSP9B10A. A second 1.95-kb insert, which encompassed the 450-bp cDNA fragment,was also isolated; its DNA and amino acid sequence displayed 99.5% identity withvsp9B10A gene and 99.2% with the corresponding inferred protein, respectively. Thisinferred protein contained 24 Cys-X-X-Cys motifs and long ORF of 642 aminoacids. PCRanalysis showed that DNA sequence encoding a fragment of this gene was present in P1,CIEA:0487:2-C-8 clone and in INP:180800-B2 G. duodenalis human isolates, while itwas absent in sheep isolate of G. duodenalis INP:150593-J10.

Conclusions. Immunofluorescence analysis using antibodies raised against the peptideencoded by 450-bp fragment showed that expression of this epitope varies ontrophozoite surface of the C-8 Mexican clone and is involved in parasite adhesion totarget epithelial cells. � 2004 IMSS. Published by Elsevier Inc.

Key Words: Adhesion, Antigenic variation, Cysteine-rich variable surface protein, Giardiaduodenalis.

Introduction

The protozoan parasite Giardia duodenalis, which sharesprokaryotic and eukaryotic features (1,2), is a major cause

Address reprint requests to: Cecilia Montanez, Ph.D., Departamento deGenetica y Biologıa Molecular, Cinvestav del IPN, Apdo. Postal 14-170,07360 Mexico, D.F., Mexico. Phone: (�52) (55) 5061-3334; FAX: (�52)(55) 5747-7100; E-mail: cecim@mail.cinvestav.mx

0188-4409/04 $–see front matter. Copyright � 2004 IMSS. Published by Elsevdoi : 10.1016/ j . arcmed.2003.12.005

of human diarrhea in underdeveloped countries; in addition,epidemic outbreaks of giardiasis have been reported in devel-oped countries (3–5). This parasite inhabits small intestineof humans as well as of many other mammals. Clinicalmanifestations of infections are highly variable, the majorityof individuals remaining asymptomatic. In symptomatic pa-tients, duration of infection can last from weeks to yearsand several reports indicated that immunity to the parasitedeveloped after primary infection (6).

ier Inc.

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Several studies showed that G. duodenalis isolates wereheterogeneous and polymorphisms among organisms of thisgroup were identified (1,7,8–11). Significant genetic differ-ences were detected among isolates and changes in surfaceantigens of this parasite were extensively studied. G. duo-denalis isolates and clones from some isolates showedmarked surface antigen variability both in vivo and in vitroby switching major surface antigens (12–17). Antigenic vari-ation was shown both by spontaneous appearance of variantsin cloned cultures (16,18,19) and by depletion experiments ofclones using cytotoxic mAbs and analysis of surviving tro-phozoites (20,21). In experimental infections of humans(18), gerbils (22), and neonatal mice (23) with clones of G.duodenalis, initial variable surface protein (VSP) was lostand replaced by a heterologous mixture of variable specificsurface proteins (12,13). Recently, this switching was ob-served on the surface of G. duodenalis where VSP1267antigen was initially expressed and then replaced byVSP9B10A. Although dual expression was observed only asa transient process in this report (17), simultaneous ex-pression of VSP9B10B and VSP1267 was recently reported(24). Experimental infections in the mother/offspring mousesystem demonstrated that surface antigen alterations withinparasite populations were initiated upon antibody produc-tion in response to parasite infections (23). Additionally,nonimmunologic factors such as intestinal proteases may in-terfere in the process of antigen variation in that they favoredproliferation of these variant antigen-type populations thatresisted hostile physiologic conditions within intestine (25).

Proteins involved in antigenic variation referred to asVSPs [also known as cysteine-rich proteins (CRP)] are anti-genically distinct and cover the surface of the parasite(14,18,26). Interestingly, approximately 2.4% of a samplingof the Giardia genome were vsp genes (24,27). VSPs aresparsely glycosylated, cysteine-rich, unique proteins withmolecular weights documented from 22.3 kDa to �200 kDaand are immunologically different. Number of VSPs perGiardia genome was estimated as at least ca. 150 by twoindependent methods (24). Sequences of VSPs reported re-vealed a family of cysteine-rich (11–12%) proteins (CRP)that characteristically contained Cys-X-X-Cys motifs. AllVSPs had a well-conserved hydrophobic tail of approxi-mately 38 amino acids terminated by invariant hydrophilicamino acids CRGKA (24).

Function of VSPs is unknown; however, it was speculatedthat cysteines were not reactive without reduction andprobably existed as disulfide bonds; this accounted for highresistance of the majority of VSPs to effects of intestinalproteases (24), which would allow the trophozoite to thrivein small intestine. Also, by binding Zn2� or other metalsin intestine, VSPs may contribute to Zn2� malnutritionor inhibition of metal-dependent intestinal enzymes, whichwould lead to malabsorption, a well-known consequence ofgiardiasis (28).

In this work we cloned a G. duodenalis 450-bp DNAfragment, which is part of a 1.95-kb DNA clone. BothDNA fragments and predicted peptides shared characteris-tics with VSP genesand displayed significant identity with therecently described VSP9B10A G. duodenalis surface antigen(17). Expression of the peptide encoded by the 450-bp frag-ment varied on the surface of the CIEA:0487:02-C-8 Mexi-can clone. This epitope was involved in adherence of parasiteto target epithelial cells.

Materials and Methods

Growth of parasites. G. duodenalis Portland 1 (P1) andWB trophozoites were obtained from E. Weinbach (NationalInstitutes of Health, Bethesda, MD, USA). G. duodenalisclone C-8 was obtained by limiting dilution from isolateCIEA:0487:2, which originated from a 4-year-old Mexicansymptomatic patient (29). Axenic isolates of G. intestinalis—INP:150593-J10 [human isolate] (30) and INP:180800-B2[sheep isolate]—were established at the Instituto Nacionalde Pediatrıa (INP) in Mexico City.

Trophozoites were axenically cultured under anaerobicconditions at 37�C in Diamond’s modified TYI-S-33 medium(31) supplemented with 10% heat-inactivated calf serum andantibiotics (penicillin and streptomycin at 250 µg/mL). Cellswere harvested in logarithmic phase by centrifugation at250 × g for 10 min and washed three times with phosphatebuffered saline pH 7.2 (PBS). The pellet from the final washwas suspended in a small volume of 10 mM TRIS pH 8.1,25 mM NEM (N-ethylmaleimide), and 1 mM PMSF (phenyl-methyl-sulfonylfluoride). Soluble extracts were obtained bysonication using 0.4% Triton X-100 as solubilizing agentand protein determination was performed by modified Lowrymethod (32).

Isolation of clones from an expression library. A cDNAexpression library in λgt11 was obtained after RNA isolationfrom G. duodenalis P1 according to the method previouslydescribed (33). Giardia trophozoites were disrupted with aTenbroek tissue grinder containing 4 M guanidine isothiocy-anate/20 mM sodium acetate (pH 5.2)/1 mM mercaptoetha-nol/0.5% sarcosyl and centrifuged over a cushion of 5.7 MCsCl for 12 h (34). The pellet was resuspended in water,and polyA� RNA was selected by passage over an oligo-dT column. Blunt-ended cDNAs were synthesized accordingto the method of Gubler and Hoffman (35), incubated withEcoRI methylase, then ligated to excess EcoRI linkers anddigested with EcoRI. cDNAs were cloned into EcoRI siteof bacteriophage lambda gt11. After in vitro packaging ofcDNAs into phage, recombinants were plated on Escherichiacoli Y1088 (36), yielding approximately 150,000 plaquesthat were amplified and maintained as liquid stocks. Recom-binant clone λgt11VSP63-1 was identified by using a rabbitpolyclonal antiserum against surface antigens of G. duode-nalis P1 strain as previously described (37).

Giardia duodenalis 63 kDa VSP9B10A-Like Protein 201

DNA cloning and sequencing. Cloned 0.45-kb cDNA insertof G. duodenalis λgt11VSP63-1 was excised using EcoR1and subcloned into Bluescript SK vector. DNA sequence ofVSP63-1 fragment was performed on double-stranded tem-plate using protocol for DNA sequencing by chain termina-tion method (38) and Sequenase 2.0 (U.S. Biochemical,Cleveland, OH, USA). DNA and inferred amino acid se-quences were analyzed and compared using the programsFASTA (39), BLAST 2.0.11 (40), and LALIGN (41). Asecond clone, VSP63-2 (1.95-kb) was obtained after screen-ing the expression library mentioned previously by hybrid-ization with 32P-labeled cDNA insert of VSP63-1 clone.This 1.95-kb cDNA fragment was subcloned and sequencedas previously described for VSP63-1 clone using Ampli-TaqTM DNA polymerase (PE Applied Biosystems, FosterCity, CA, USA).

Isolation of fusion proteins. Lac Z fusion protein (rVSP63-1) was obtained after infection of 20 mL of E. coli Y1090with λgt11VSP63-1. Cultures were grown overnight in Luriabroth (50 µg/mL ampicillin) using 5 × 108 phages/mL toinfect bacteria. Induction was performed by adding IPTG toa final concentration of 2 mM and incubating for an addi-tional 5–6 h at 40�C. Extracts were obtained and bacterialproteins from cultures were separated in 7.5% PAGE-SDSgels according to Laemmli (42). The polypeptide repre-sented by 137 kDa band was excised after matching anunstained gel with a Coomassie blue (Bio Rad, Hercules,CA, USA)-stained section. This polypeptide was electroe-luted, homogenized, and injected into mice. The polypep-tide was specifically recognized by rVSP63-1-specificantibodies by Western blot.

Production of antisera against fusion peptide. An antiserumagainst rVSP63-1 was raised in BALB/c mice. Mice wereinjected intraperitoneally (i.p.) with the electroeluted 137kDa (rVSP63-1) protein previously excised from the gel(approximately 3–4 µg per mouse). Mice were boosted i.p.four times 1 week apart with a similar dose and were bled7 days after final immunization. Reactivity of immune mousesera was determined by ELISA (43).

Western blot. Extracts of G. duodenalis C-8 trophozoitesprepared as described (44) were subjected to electrophore-sis in SDS on 7% polyacrylamide gels and resolved polypep-tides were transferred to nitrocellulose. Immunoblotting wasperformed as previously described (45). A serum against β-galactosidase was used as control at 1:60 dilution.

DNA purification. Trophozoites from axenic cultures werewashed in HBSS (pH 7.5), concentrated, and incubated over-night at 42�C in a lysis solution (10 mM Tris-HCl, pH 7.4;10 mM EDTA; 150 mM NaCl; 0.4% sodium dodecylsulfate,and 200 µg/mL proteinase K). The lysate was treated withphenol/chloroform (1:1) and nucleic acids were precipitatedwith 0.3 M sodium acetate pH 7 and ethanol at �20�Cand then dissolved in 10 mM Tris-HCl, 1 mM EDTA (pH

8.0). RNA was removed by incubating with 20 µg/mL RNaseA at 37�C for 30 min and DNA was re-extracted with phenol.DNA was again precipitated in ethanol and finally redis-solved in 10 mM Tris-HCl pH 8.0, 1 mM EDTA.

PCR amplification. Specific oligonucleotides 5′ CTGCAC-TGCACTCGGTGTAAC 3′ and 5′ GGAGCCTCTACTTG-GCAGGTA 3′ were designed based on vsp9B10A DNAgene sequence and purchased from GIBCO. These oligoswere used to amplify a 1283-bp segment from vsp9B10Agene. PCR reaction was performed in a Perkin Elmer model2400 thermocycler using 50 µL reaction mixtures containing50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2,0.2 mM dATP, dGTP, dCTP, and dTTP, 5′ and 3′ primers(each 0.25 µM), 100 ng of Giardia DNA, and 1.5 U of TaqDNA polymerase (TaqGold, Perkin Elmer Co., Boston, MA,USA). Reactions involved an initial 3-min denaturation at94�C followed by 30 cycles comprising 1 min at 94�C, 1min at 55�C, and 1 min at 72�C, and a final 7-min extensionstep at 72�C. A control lacking template DNA was includedin the experiment. Amplification products were identifiedafter electrophoresis in 1% agarose gels (Tris-Borate-EDTAbuffer) and staining with ethidium bromide (0.5 µg/mL).Phage ΦX174 Hae III restriction fragments (Boehringer-Mannheim, Mannheim, Germany) were used as size markers.

Immunofluorescence assays. Immunofluorescence assayswere performed using trophozoites of clone C-8 of G. duode-nalis according to Riggs et al. (46). [rVSP63-1]-specificantibody was used at 1:50 dilution and incubated with form-aldehyde fixed trophozoites at room temperature (approxi-mately 22�C) for 30 min in presence of 10% bovine serum.Antiserum against λgt11 was also used as negative control.Afterward, trophozoites were washed by centrifugation andincubated with 1:1,000 dilution of goat anti-mouse Ig-coupled to FITC. Fluorescent cells were visualized using aZeiss (Mexico City, Mexico) optical microscope equippedwith an epifluorescence system. C8 and other clones werederived from the same parental and because the latter didnot express VSP63 they were also used as negative control(data not shown).

Adhesion inhibition assay. Effect of anti-[rVSP63-1] mouseserum on adhesion of C-8 trophozoites to Madin–Darbycanine kidney (MDCK) cells was examined using in vitroadhesion assay. In this, 7.5 × 105 trophozoites from cloneC-8 previously shown to bear vspB910A-like gene and todisplay surface epitopes similar to the fusion peptide (byindirect immunofluorescence) were labeled for 48 h with3H thymidine and incubated with 1:5 dilution of anti-β-galactosidase or [rVSP63-1]-specific antibodies at roomtemperature for 30 min. Then, trophozoites were added toconfluent MDCK cell monolayers grown in 2-cm2 wellsand cultures were incubated for 2 h at 37�C in a 5% CO2

atmosphere. Subsequently, radioactivity was determined as

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TCA-precipitable material of nonadherent trophozoites har-vested on a 3 MM Whatman filter paper as described (47).Adhesion inhibition was determined as percentage of TCA-precipitable cpm in nonadherent trophozoites with regard tototal number of TCA-precipitable cpm. To verify total cpm,adherent trophozoites were washed with 200 µL of coldPBS and then incubated 1 h at 4�C to obtain TCA-precipita-ble material and cpm were determined. Net inhibition wasdetermined by subtracting nonspecific inhibition in presenceof anti-β-galactosidase serum from this inhibition obtainedin presence of [rVSP63-1]-specific antibodies.

Results

cDNA clone-encoding part of a trophozoite surfaceprotein. Clone λgt11VSP63-1 was identified within the Gi-ardia cDNA λgt11 expression library by immunoblottingusing rabbit antiserum raised against P1 isolate surface anti-gens. This antiserum recognized antigens from P1 G. duode-nalis with molecular weights of approximately 65, 55, and 40kDa by Western blot analysis (data not shown). Nucleotidesequence of cloned 0.45-kb cDNA insert was determinedafter subcloning it in a Bluescript plasmid (Figure 1A).VSP63-1 sequence was 60% G/C and encoded a hydrophilic

Figure 1. A) Homology between VSP63-2 clone and vsp9B10A gene DNA. VSP63-2 sequence is placed on bottom strand, while vsp9B10A gene sequenceis above it. Symbol “:” indicates identity. Region whereVSP63-1 clone is located is shown by highlighting(EMBL nucleotidesequence database accessionnumberAJ320503). Differences in nucleotide sequence from VSP9B10A can be found by lack of the symbol “:”; *** indicates location of translation initiationcodon; ••• indicates location of translation termination codon. Arrows mark position of specific primers for vsp9B10A gene used for PCR assay; B)Nucleotide and predicted amino acid sequence of Giardia duodenalis VSP63-2 cDNA fragment (EMBL nucleotide sequence database accession numberAJ457171). Sequence shown is right after EcoRI site. Predicted amino acid sequence is shown below nucleotide sequence. C-X-X-C motifs are indicatedin bold.

Giardia duodenalis 63 kDa VSP9B10A-Like Protein 203

Figure 1. Continued.

peptide 149 amino acids long. Recombinant phage expresseda fusion protein of approximately 137 kDa (data not shown).Inferred peptide sequence contained cysteine residues anddisplayed characteristic C-X-X-C motifs of VSP (Figure 1B,motifs in bold). It was also rich in alanine and glutamicacid, while tryptophan was not found in the sequence.Comparison of VSP63-1 sequence to these G. duodenalisDNA sequences contained in the GenBank database showedclosest similarity to Giardia cysteine-rich VSP genes.Among these, the most closely related nucleotide sequencewas found with VSP9B10A (17) (99.5% identity) (Figure1A), VSP9B10B (24) (89.98%), tsa417-2 (48,49) (67.6%identity), and tsa417-1 (50) (58.98% identity) G. duodenalismajor surface antigens.

To identify a cDNA clone representing a more completeportion of the coding sequence of the parent gene, VSP63-1insert was used as a probe to re-screen the G. duodenaliscDNA library. Thus, a second clone was identified and desig-nated as VSP63-2. This clone contained a 1.95-kb cDNAinsert. After DNA sequence was determined, it was com-pared with vsp9B10A gene sequence and high identity(99.5%) was also found (Figure 1A). VSP63-1 is part ofVSP63-2 (Figure 1A), which suggests that VSP63-1 andVSP63-2 inserts may represent overlapping parts of the samegene. A potential translation initiation codon was found atposition 44 (position 44 corresponded to position 804 in

VSP9B10A); however, because N-terminal segment re-quired for protein translocation was absent, clone VSP63-2might be an internal part of a vsp9B10A-like gene. An openreading frame expanding 1930 bp and beginning at position 1was obtained. A termination codon was located at position1930, allowing expression of a 642-amino acid peptide with99.2% identity with VSP9B10A-inferred protein sequence,this sequence containing 24 C-X-X-C motifs (Figure 1B).VSP63-2 sequence contained nine differences fromvsp9B10A gene sequence; however, these were changes andnot deletions or insertions (Figure 1A, see Legend) and onlyfive differences were found in inferred protein sequence(Figure 1B). Due to high homology with VSP9B10A, VSP63could be a vsp9B10A-like gene or at least part of vsp9B10Agene.

vsp9B10A DNA is not present in all G. duodenalisisolates. To determine whether a VSP9B10A-like sequenceis present in several G. duodenalis isolates, PCR amplifica-tion reaction using specific primers for vsp9B10A gene (lo-cation of these primers is shown in Figure 1A) was performedusing DNA from G. duodenalis human isolates and clonesWB, P1, CIEA:0487:2-C-8, INP:150593-J10, and G. duode-nalis sheep isolate INP:180800-B2. Figure 2 shows resultsof PCR amplification of the vsp9B10A-like gene fragmentfrom plasmid pVSP63-2 (control), and isolates P1, C-8, WB,

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Figure 2. PCR amplification of vsp9B10A-like gene segments from G.duodenalis isolates genomic DNA. PCR amplification products fromvsp9B10A-like gene were resolved in a 1% agarose gel stained withethidium bromide. Negative image of the gel is shown. PCR products fromplasmid pVSP63-2 (lane 1) and DNA from P1 (lane 2), C-8 (lane 3), WB(lane 5), B2 (lane 6), and J10 (lane 7) G. duodenalis isolates and clonesare shown. Arrowhead indicates 1283-bp specific fragment for vsp9B10Agene. Numbers to the right are shown to indicate sizes in bp of PhageΦX174/Hae III molecular weight markers (lane 4).

INP:180800-B2 (Figure 2, lanes 1–3, 5, 6, respectively),which indicated that these isolates bore a vsp9B10A-likegene. Interestingly, either this sequence was not present inisolate INP:150593-J10 (Figure 2, lane 7) as observed in otherisolates where vsp9B10A was not expressed (24), or se-quence contained in the primer was not present due togene polymorphism and therefore no amplification was ob-tained when PCR was performed.

Analysis of epitopes expressed by VSP63. C-8 was clonedfrom isolate CIEA:0487 obtained from a 4-year-old symp-tomatic patient, which does bear the vsp9B10A-like vsp63gene. Therefore, C-8 was used to characterize VSP63-encoded protein. Antiserum against (pVSP63-1)-encodedfusion peptide (rVSP63-1) purified by polyacrylamide gelelectrophoresis was prepared in mice. This antiserum wastested by Western blot analysis and immunofluorescenceanalysis using trophozoites of G. duodenalis C-8 clone asdescribed in Materials and Methods. Results of Western blotanalysis (Figure 3) showed specific recognition of a 63-kDaG. duodenalis component by [rVSP63-1]-specific antibod-ies. Due to its VSP features (see later), this protein isreferred to as VSP63.

Analysis of trophozoites from C-8 clone by indirect im-munofluorescence assay using [rVSP63-1]-specific antibod-ies revealed presence of cells expressing exposed epitopes.This expression was not associated with any condition or

Figure 3. Western blot analysis of G. duodenalis C-8 proteins. Extracts ofC-8 clone were obtained as described by Ortega-Pierres et al. (43). [rVSP63-1]-specific antibody was diluted 1:5 (lane 2) and mouse polyclonal antise-rum raised against β-galactosidase diluted at 1:60 (lane 1) was used ascontrol. Molecular weight in kDa of proteins used as standards is shownto the left of the figure.

presence of any specific factor. Antibody reactivity was lo-calized as patches on trophozoite surface (data not shown).Variation in expression of epitopes recognized by [rVSP63-1]-specific antibodies was observed by indirect immunoflu-orescence assay during continuous parasite culture. Figure4 shows percentage of fluorescent cells in parasites testedevery 3–4 days for a period of 35 days of continuous culture.Percentage of cells expressing VSP63-1 epitope among tro-phozoites of G. duodenalis clone C-8 varied from 2 to 90%.Immunofluorescence signal correlated with Western blot re-sults using [rVSP63-1]-specific antibodies where C-8 cul-tures with 2% of cells expressing VSP63-1 showed noVSP63 corresponding band while those with 67–90% per-centage of cells expressed showed a specific VSP63 band.These analyses were performed at 18, 21, and 32 days aftercultures were initiated. Control trophozoites showing noreactivity with antiserum raised against VSP63-1 were alsoincluded (data not shown).

Analysis of the role of VSP63-1 peptide in adhesion oftrophozoites to MDCK cells. With the aim to analyze partici-pation of VSP63 epitope in adherence of parasite to epithelialcells, C-8 clone trophozoites with high expression of clonedepitope (69% fluorescent cells) were incubated prior to theirinteraction with MDCK monolayer cells with a subagglutina-tion dose of antibodies (anti-β-galactosidase or [rVSP63-1]-specific antibodies, stippled and stripped bars, respectively)performed as previously reported (47). Figure 5 shows in-hibition of adhesion of trophozoites to MDCK cells. Therewas nonspecific inhibition (24%) by anti-β-galactosidaseantibody of adherence of trophozoites to epithelial cells.However, when trophozoites were incubated with [rVSP63-1]-specific antibodies, higher inhibition (45%) of their adhe-sion to MDCK cells was observed; therefore, net adhesion

Giardia duodenalis 63 kDa VSP9B10A-Like Protein 205

Figure 4. Kinetics of reactivity of [rVSP63-1]-specific antibodies with trophozoites from clone C-8. Reactivity of C-8 clone cells with [rVSP63-1]-specificantibodies was analyzed at 3- to 4-day intervals during 35 days of culture by indirect immunofluorescence assays. Percentage of fluorescent trophozoiteswas determined by epifluorescence microscopy. Average percentage obtained in three independent assays at each time point is shown. � indicates cells labeledwith antibody against λgt11 and ■ indicates cells labeled with [rVSP63-1]-specific antibodies.

inhibition was 21% in these assays. The possibility of Giar-dia-specific antibodies resulting from Giardia infections canbe ruled out because control mouse serum did not recognizeVSP63 antigen as determined by Western blot (data notshown). Also, control mouse serum was used as negativecontrol and 2% inhibition of adhesion was obtained.

Discussion

Several microorganisms developed a variety of mechanismsthat allowed them to evade host immune response. These

Figure 5. Inhibition of adhesion of G. duodenalis C-8 clone to MDCKcells. Inhibition of adhesion of G. duodenalis C-8 to MDCK cells wasperformed as indicated in Materials and Methods. Percentage of nonadher-ent trophozoites under each condition was determined as described inMaterials and Methods. Stippled box corresponds to trophozoite culturesincubated with mouse antiserum against β-galactosidase; striped box cor-responds to trophozoites incubated with [rVSP63-1]-specific antibodies,as explained in the text. Value reported represents mean value of threeindependent adhesion assays carried out by duplicate. Error bars indicate4.5% deviation.

mechanisms included antigenic variation of major surfaceantigens and it was shown that this antigenic variation wasused by various organisms such as African trypanosomes,Plasmodium spp., Neisseria gonorrhea, Borrelia hermsii,and Giardia duodenalis (51).

CRVSP genes of Giardia consist of a family of genes en-coding cysteine-rich variable surface proteins. Putative rolesfor these proteins include resistance to proteases, bile salt,and heavy metals and binding to Zn2� (28,52). Polymor-phisms in the physical map of cysteine-rich surface proteingenes among cloned cell lines were previously described;however, these genes were not present in rearranging Giardiatelomeres or polymorphic chromosomes (53,54). G. duode-nalis possessed a repertoire of variant specific surface protein(VSP) genes ranging from 133 to 151 (24,55); nonetheless,little is known with regard to organization and mechanisms ofexpression of CRVSP genes. It was suggested that VSP geneswere not telomeric (54,56) and head-to-head orientation oftwo expression-linked genes was described (56). Recentstudies, however, reported a new family of genes encodingcysteine-rich proteins that is located at telomeres of Giardiachromosomes (57,58). Despite this finding, VSP genes werenot generally telomeric and apparently no evidence ofrecombination was documented for VSP relocation to otherparts of the genome (2).

In this work we cloned two DNA fragments encoding apeptide from G. duodenalis strain P1, which belong toCRVSP gene family based on homology with other VSPgenes (mainly vsp9B10A). Clone VSP63-2 encodes a pep-tide that displays Cys-X-X-Cys motifs and might correspondto an internal segment of VSP9B10A because N-terminaldomain required for translocation is absent. Western blotresults indicated that antiserum raised in mice against thefusion polypeptide from clone λgt11VSP63-1 reacted with

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a protein of 63 kDa from an extract of the G. duodenalisC-8 clone. This clone was shown to bear a vsp9B10A-likegene fragment by PCR analysis. With the aim of obtainingthe complete sequence for this gene, VSP63-2 was identifiedby using VSP63-1 as a probe to screen the Giardia expres-sion library. Clone VSP63-2 sequence was compared tovsp9B10A gene and results suggested that VSP63 couldbe a VSP9B10A-like protein. Predicted molecular weightcorresponding to VSP9B10A was 76 kDa; however, a 68kDa protein was detected (24), which differed from VSP63molecular weight (63 kDa). Although this could also be theresult of an incomplete denaturation process during SDSelectrophoresis, most probably the VSP63-2 clone representsan internal segment of a vsp9B10A-like gene. Interestingly,although there are few changes (nine changes in all) withinVSP63 sequence when compared with VSP9B10A sequence,there is no shift of the open reading frame. This findingwould suggest that VSP63 might be part of the VSP9B10gene family.

It is noteworthy that the majority of G. duodenalis isolatesanalyzed in this study did bear a vsp9B10A-like gene, whileone did not. It appears that one human isolate tested eitherdoes not contain a vsp9B10A-like gene or that this genehas changes in the region where primers bind, resultingin absence of amplification product during PCR.

The importance of adherence for survival of G. duode-nalis in vivo is dependent on cell attachment abilitiesbecause without these the parasite would be swept away byperistaltic movements of intestine. Mechanisms of attach-ment of trophozoites to intestinal cells have not been estab-lished definitively. Evidence supports roles for ventral disk,considered a specific attachment organelle, trophozoite con-tractile elements, hydrodynamic mechanical forces, andlectin-mediating binding.

Ultrastructural studies showed that Giardia attaches toInt-407 (human intestinal cells) monolayer predominantly byits ventral surface probably, as previously reported, throughgiardins. Int-407 cells contact trophozoites with elongatedmicrovilli, and both trophozoite imprints and interactions ofGiardia flagella with intestinal cells were also observed(59). Transmission electron microscopy showed that Giardialateral crest and ventrolateral flange were important struc-tures in the adherence process. Although a role for VSP inadhesion was not reported previously for Giardia, variablesurface proteins from other organisms such as Mycoplasmabovis play a major part as adhesion factors. Competitiveadherence assays were performed to analyze MycoplasmaVSP role in adhesion. Specific antibodies against thesevsp were analyzed, and it was shown that they also reducedMycoplasma adhesion (60,61).

In this work we present the first evidence that VSPproteins are involved in adhesion of parasite to host cells.We would like to speculate that, meanwhile, VSP9B10Bprotein is preferentially expressed during encystation pro-cess and therefore related with such a process; VSP9B10A,

a protein expressed preferentially in vegetative trophozoite[not present in extracts from cysts (24)] is involved tosome extent in adhesion process, this being only a part ofthis complex event.

Although the role of antigenic variation is not known, itis possible that it contributes to survival of the parasite,because a large amount of the organism’s resources aredevoted to this. Antibody-mediated inhibition of parasiteadhesion suggests that these molecules participate in thisphenomenon. Indeed, B-cell mechanisms have been foundto play a role in control of the infection. This together withT-cell-mediated responses as well as other factors of hostcontrol the outcome of Giardia infections.

Additionally, [rVSP63-1]-specific antibody was obtainedfrom G. duodenalis Portland 1 fusion peptide and it recog-nized a 63 kDa protein in extracts from clone C-8 fromCIEA:0487:2 isolate. In addition, the fact that VSP9B10Bprotein has only been shown as expressed as an encysting-related protein suggested that most likely [rVSP63-1]-spe-cific antibody recognized only VSP9B10A protein. Furtherinvestigation with regard to the role of VSPs in adhesionmechanisms is currently the theme of our work.

AcknowledgmentsWe thank Alison Rattray and Vicky van Santen for critical reviewof the manuscript. We also thank Adalberto Herrera, Blanca Her-rera, and Rene Lopez for technical assistance.

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