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Infection and Immunity, August 2005, p. 5208-5211, Vol. 73, No. 8
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.8.5208-5211.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Identification of Variant-Specific Surface Proteins in Giardia muris Trophozoites

Andrea S. Ropolo, Alicia Saura, Pedro G. Carranza, and Hugo D. Lujan^*

Cátedra de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad Nacional de Córdoba, CP 5000 Córdoba, Argentina

Received 15 October 2004/ Returned for modification 22 November 2004/ Accepted 3 March 2005

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Giardia lamblia undergoes antigenic variation, a process that might allow the parasite to evade the host's immune response and adapt to different environments. Here we show that Giardia muris, a related species that naturally infects rodents, possesses multiple variant-specific surface proteins (VSPs) and expresses VSPs on its surface, suggesting that it undergoes antigenic variation similar to that of G. lamblia.

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Giardiasis is one of the most common human intestinal diseases worldwide. Although species of Giardia inhabit the intestinal tracts of almost all classes of vertebrates, Giardia lamblia is the only recognized species found in humans and most other mammals (2). G. lamblia is known to undergo antigenic variation (AV), a process that might allow the parasite to evade the host's immune response. In Giardia, AV may contribute to the variable and/or persistent course of some infections, the propensity for multiple reinfections, and the large variety of hosts (21, 22). AV in this parasite involves variant-specific surface proteins (VSPs), which cover the entire surface of the trophozoites and are the major antigens recognized by the host immune system (6, 18, 20). VSPs vary in size from 20 to 200 kDa and are rich in cysteine, an amino acid found primarily in Cys-X-X-Cys motifs distributed nonrandomly throughout the extracellular region of the polypeptide (1, 17). In addition, VSPs contain a carboxy-terminal region that includes a putative transmembrane domain as well as a five-amino-acid-long cytoplasmic tail (CRGKA), which are highly conserved among all known G. lamblia VSPs (12). Spontaneously or in response to the host's immune system, one VSP is replaced by other antigenically distinct VSPs on the surface of the trophozoites (18).

Studies of the immunology and biology of Giardia infections have been hampered because of limitations of the available models systems. Neonatal mice usually accept G. lamblia but then become resistant as they mature (7). Gerbils are easily infected with G. lamblia, but they are neither inbred nor pathogen free, and few immunological reagents are available (3). One type of G. lamblia, the GS isolate, a clone that expresses a major surface antigen, VSPH7, infects adult mice and has been used to study AV (4, 5). However, infection does not resemble most infections in humans. The numbers of trophozoites diminish rapidly after the second week, and a barely detectable infection continues for an extended period of time. Giardia muris, which infects rodents but not humans, has been used as a model of giardiasis and the development of immunity, but its use is limited because of the inability to maintain the organism in vitro and the poor knowledge of its biology. Whether G. muris undergoes surface AV is unknown.

To investigate the presence of VSPs in G. muris, trophozoites were isolated from small bowels of naturally infected Wistar rats. Isolated intestines from infected rats were rinsed with ice-cold phosphate-buffered saline, and the intestinal washings were collected, filtered through a fine screen mesh, and centrifuged for 10 min at 700 x g. The supernatant fraction was discarded, and the pellet was placed in a sterile borosilicate test tube filled with medium (1.0 g glucose, 1.0 g yeast extract, 2.0 g casein, 200 mg cysteine-HCl monohydrate, 20 mg ascorbic acid, 2.28 mg ferric ammonium citrate, 50 mg dehydrated bovine bile, 2 mg bathocuproine, 10 ml inactivated fetal calf serum in 90 ml RPMI 1640). After 1 h, the supernatant was discarded, replaced with fresh medium, and chilled for 15 min to detach trophozoites. Trophozoites were used to isolate genomic DNA, RNA, and proteins as described previously (8, 13), as well as to infect pathogen-free rats for subsequent studies.

Trophozoites showed typical G. muris morphology, and species identification was confirmed by rRNA gene amplification and sequencing (11, 23) (data not shown). For PCR, generic primers for G. lamblia VSPs were generated. Sequences from four previously known VSPs belonging to G. lamblia isolates WB (VSP1267, VSP9B10, and VSPA6) and GS (VSPH7) were selected and aligned, and conserved regions useful for primer design were identified (data not shown). Sense primers were S1 (5'-CVT GTG CHR RST GCA A-3'), S2 (5'-TGC ACS RSC TGC YAB CC-3'), S3 (5'-TAG TGY DSY VMV TGY AA-3'), and S4 (5'-CGA TCA TGA CGG GCT TCT-3'), and antisense primers were R1 (5'-CCB ACG AGG CCY CCS ACG AC-3') and R2 (5'-CGC CTT CCC KCK RCA KAY GA-3'). Combinations of those primers were then employed in PCR analysis of genomic DNA derived from G. muris. PCR conditions were as follows: denaturation at 94°C for 40 s, annealing at 50°C for 40 s, and elongation at 72°C for 90 s for a total of 35 cycles. Extreme care was taken in order to abolish contamination with G. lamblia DNA. Several DNA fragments were amplified regardless of the primer combination used (data not shown). Sequence analysis of these PCR products showed characteristic VSP signature motifs (10). Alignment of the deduced amino acid sequences of the PCR products from G. muris with G. lamblia VSPs by using Clustal W 1.8 showed a high degree of similarity among them (Fig. 1) and typical G. lamblia VSP structure and motifs. These included 10 to 13% cysteine (mostly as multiple CXXC motifs), GGCY motifs, a variable hydrophilic amino-terminal region, and a highly conserved 34-amino-acid sequence at the C terminus with the conserved amino acids CRGKA.

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FIG. 1. Homology of VSPs identified in G. muris with VSPH7 from G. lamblia. Alignment of selected VSP fragments was carried out using Clustal W 1.8 and shading with GeneDoc software, allowing residue similarity. The percentage of similarity among sequences is represented as boxes: black, 100%; dark grey, 99 to 80%; light gray, 79 to 50%. VSPH7 is at the bottom. The five-amino-acid cytoplasmic tail present in all known VSPs is boxed.

To evaluate the expression of VSP genes in G. muris trophozoites, Northern blot analysis using total RNA from G. muris and the conserved 3' region of vsps (oligonucleotide R2) as a probe were performed (9). The results indicated that VSPs are expressed in the G. muris populations isolated from infected rats (data not shown).

To address whether VSPs were expressed on the trophozoite cell surface, monoclonal antibodies (MAbs) specific for G. lamblia and G. muris VSPs were generated. Six-week-old female BALB/c mice were immunized intraperitoneally on days 0, 7, 14, and 21 with 25 µg of either a trophozoite extract of G. muris or a mixed extract of several clones of G. lamblia (isolates WB-1267, WB-C6, PC-9B10, and GS-H7), emulsified in Ribi adjuvant (Sigma). Mice were boosted on day 28 intravenously with 25 µg of the antigen preparation. Three days later, mice were euthanatized and the spleen cells used for fusion to NSO myeloma cells (ECACC 85110503). MAbs were screened by indirect immunofluorescence with G. muris or G. lamblia trophozoites (13). The results indicated that MAbs generated against G. muris (Fig. 2A) or G. lamblia trophozoites (Fig. 2B) labeled the surface of a fraction of G. muris trophozoites in a pattern similar to that seen with MAbs directed to VSPs of G. lamblia, further supporting the presence of VSPs in G. muris. Moreover, the frequency of particular VSPs recognized by the MAbs was highly variable in G. muris trophozoites isolated from infected rats (Fig. 3) or at different time points during infection (not shown), suggesting that G. muris undergoes antigenic variation similar to that of G. lamblia. To demonstrate that the pattern observed in immunofluorescence assays using these MAbs was due to the fact that these reagents recognize G. muris VSPs, we performed Western blot analysis on recombinant VSPs expressed in Escherichia coli. Recombinant proteins were produced by cloning each VSP fragment on the pGEX vector (8). Our results indicated that of all the MAbs generated against G. muris trophozoites, only MAb 2D10 was able to recognize one of the VSPs previously identified in this parasite (Fig. 4A). For this reason, we decided to generate new MAbs against recombinant VSPs expressed as glutathione S-transferase fusion proteins. Recombinant VSP-expressing bacteria were plated, transfer to nitrocellulose disks, and assayed for reactivity with the MAbs. Positive controls using an anti-glutathione S-transferase MAb and negative controls using secondary antibody only were always performed. Several of these novel MAbs were specific to the VSP used as the immunogen (Fig. 4B, left panels) and showed a pattern of expression in immunofluorescence assays similar to that observed on G. muris trophozoites with the original MAbs (Fig. 4B, right panels). Taken together, these results confirmed that the MAbs were directed to VSP molecules present on the trophozoite surface. The occurrence of homologous VSPs in G. muris implies that VSPs and AV are attributes of all Giardia species. Thus, G. muris infection in mice is expected to be a reliable laboratory model to study AV.

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FIG. 2. Immunostaining of Giardia trophozoites with monoclonal antibodies against Giardia VSPs. G. muris trophozoites were subjected to immunofluorescence assays using MAbs obtained from mice immunized with membrane fractions from either (A) G. muris or (B) G. lamblia. Regardless of the MAb, a typical pattern indicative of antigenic variants can be observed (some cells show surface labeling, while others are not labeled).

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FIG. 3. Immunostaining of Giardia muris trophozoites with MAb 2D10. G. muris trophozoites isolated from two naturally infected rats were subjected to immunofluorescence assays using MAb 2D10. The frequency of positive cells is 6/9 in A and 2/15 in B.

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FIG. 4. Specific MAbs against G. muris VSPs. (A) Western blotting analysis of recombinant G. muris VSPs using MAb generated against G. muris and G. lamblia trophozoites. Of all the MAbs, only MAb 2D10 was able to detect G. muris VSP02. Other VSPs were not detected by the MAbs (not shown). (B) New MAbs were generated against recombinant G. muris VSP03, -04, and -06 (left panels) (only four MAbs are shown; one of them recognizes two VSP). By immunofluorescence assays, MAbs showed surface labeling in a percentage of the cells (in green), clearly demonstrating that these MAbs are specific to G. muris VSPs. Nuclei were counterstained with DAPI (4',6'-diamidino-2-phenylindole) (in blue).

Although it is generally assumed that AV in Giardia is a mechanism developed by the parasite to evade the host's immune system, this assumption might need to be reconsidered, since VSP switching is evident even in the absence of an adaptive immune response (18). Here we provide evidence that VSPs may have additional biological functions. One VSP identified in G. muris, VSP03, possesses homology (95% at the protein level) with one G. lamblia VSP, the VSPH7 from the clone GS/M-83 (Fig. 1). This implies that VSPs from these two species are not very different and might explain infection with this clone in the adult mouse model (5). G. lamblia isolates are heterogeneous, and there are biochemical as well as biological differences among them. In particular, the GS/M isolate belongs to the most distant and heterogeneous group 3 of human isolates (15, 16). Our results suggest that G. lamblia GS is more closely related to G. muris than to other strains of Giardia. Additional questions arise: why could only the G. lamblia GS isolate colonize the intestines of mice, and how important are the VSPs expressed by the trophozoite for colonization of the small intestine? When the murine model of G. lamblia was developed, several clones from isolates WB and GS were tested for infectivity, but only the GS isolate clone H7 could infect adult mice (5). It is then reasonable to hypothesize that VSPs play a role in the colonization of the intestine by the trophozoites (14, 19). The high similarity between VSPH7 and VSP03 of G. muris might indicate the significance of the VSP expressed on the surface of either species rather than the species itself for parasite infectivity. In this regard, we are testing whether trophozoites from the WB isolate expressing VSPH7 are able to infect adult mice.

In summary, we show here that G. muris undergoes AV similarly to G. lamblia and that there are equivalences between the VSPs of the two species, indicating that AV is likely a feature common to all Giardia species. Increased knowledge of the biology of G. muris will allow a better understanding and rational use of the parasite in model infections in mice.

Nucleotide sequence accession numbers. The sequences of six novel G. muris VSPs were deposited in GenBank under accession numbers AY754877 to AY754882.

 
This work was supported by Agencia Nacional para la Promoción de la Ciencia y la Tecnología (FONCYT), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Universidad Nacional de Cordoba, Ministerio de Salud de Argentina, and the Howard Hughes Medical Institute (HHMI).

We thank Theodore E. Nash for support and critical reading of the manuscript.

 
* Corresponding author. Mailing address: Cátedra de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad Nacional de Córdoba, Haya de la Torre S/N, Pabellón Argentina 2do piso, Ciudad Universitaria, CP 5000 Córdoba, Argentina. Phone: 54-351-4333024, ext. 108. Fax: 54-351-4333072. E-mail: hlujan{at}biomed.uncor.edu.

Editor: W. A. Petri, Jr.

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Infection and Immunity, August 2005, p. 5208-5211, Vol. 73, No. 8
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.8.5208-5211.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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