Biomedical subjects
Publications and source records attributed to T E Nash.
An adult mouse-Giardia lamblia model was developed and used to study host-parasite interactions, including antigenic variation. The H7/1 clone of isolate GS infected mice consistently and produced infections in 14 mouse strains tested. Infection patterns were mouse strain and Giardia isolate dependent. Antigenic variation occurred in immunocompetent mice but not in mice with severe combined immunodeficiency.
Giardia lamblia undergoes surface antigenic variation. The variant-specific surface proteins (VSPs) are a distinct family of cysteine-rich proteins. Characteristically, cysteine residues occur mostly as CXXC tetrapeptides. Four of the reported five VSPs contain a putative metal-binding domain that resembles other metal-binding motifs; the fifth is closely related but lacks an essential histidine. Three different native VSPs bound Zn2+. Co2+, Cu2+, and Cd2+ inhibited Zn2+ binding. Analysis of recombinant VSP fusion proteins showed that the putative binding motif bound Zn2+. Surprisingly, peptide fragments from other regions of the VSP contain numerous CXXCXnCXXC motifs that also bound Zn2+. Analysis of deduced amino acid sequences showed well-conserved CXXC spacing in three out of five VSPs, suggesting conservation of structure despite amino acid sequence divergence. The function of VSPs is unknown, but by binding Zn2+ or other metals in the intestine, VSPs may contribute to Zn2+ malnutrition or inhibition of metal-dependent intestinal enzymes, which would lead to malabsorption, a well-known consequence of giardiasis.
Giardia lamblia is a protozoan intestinal parasite that has characteristics of both eukaryotes and prokaryotes. To determine whether genes for guanine nucleotide-binding proteins are present in Giardia, genomic DNA and cDNA libraries were screened by polymerase chain reaction and by hybridization with mixed oligonucleotide probes complementary to sequences encoding conserved GTP-binding domains. A gene with a high degree of sequence identity with mammalian ADP-ribosylation factors (ARFs), believed to be important in vesicular transport, was identified. The Giardia ARF gene had a 573-base open reading frame encoding 191 amino acids which are 63-70% identical with known mammalian and yeast ARFs. Sequence conservation among ARFs was greatest in putative GTP-binding domains. A single ARF mRNA species of approximately 750 bases was found in two different Giardia isolates. Primer extension and RNA sequencing of the Giardia ARF transcript revealed a short (6-base) 5'-untranslated region similar in size to those found in other Giardia transcripts. Giardia extracts contained ARF activity, as shown by stimulation of cholera toxin-catalyzed ADP-ribosylation and a Giardia ARF expressed in Escherichia coli as a fusion protein likewise exhibited biochemical activity. Its presence in Giardia is consistent with the view that ARF emerged before the divergence of this protozoan from other eukaryotes (approximately 1.5 billion years ago), and that an ARF-like protein may have been the ancestor of several other classes of signal-transducing guanine nucleotide-binding proteins, including the alpha subunits of the heterotrimeric G proteins.
Giardia lamblia consist of heterogeneous isolates that can be divided into at least three groups. Differential screening of a cDNA library with isolate-specific antisera identified a gene which is expressed and found only in Group 3 isolates. This gene, GLORF-C4, is 597 bp in length and predicts a deduced protein of 198 amino acids that is characterized by a polyserine motif. Giardia can also be grouped by their ability to express certain variant-specific surface proteins (VSPs), expression of which is restricted among groups. In Southern blots, probes specific to two VSPs were used to characterize isolates. Failure to detect VSP genes correlated with inability to express the same VSP. Analysis of isolates with these new probes complements and confirms the groupings previously suggested using other criteria. These genetic differences should allow differentiation of isolates and permit the application of basic epidemiological techniques to determine the manner of spread and the presence of animal reservoirs.
Giardia lamblia undergoes surface antigenic variation. The variant-specific surface proteins (VSPs) of isolate WB are cysteine-rich, can vary dramatically in size, contain Cys-X-X-Cys motifs, and are differentially expressed. GS/M(H7) is a Giardia clone from a different isolate which expresses a VSP epitope not found in WB. The VSP gene encoding this epitope was selected by differential hybridization using radiolabeled cDNA from H7 and variant sibling trophozoite lines from the same isolate that express other VSPs. The VSPH7 gene probe detects an 1800 nucleotide transcript abundant in H7 but undetectable in variant siblings. Primer extension directly from RNA was used to complete the gene sequence which predicted a protein with a molecular weight of 56,832. The protein showed many of the characteristics of 2 previously sequenced WB VSPs including many Cys-X-X-Cys tetrapeptides and a conserved carboxy-terminal region. Genomic Southern analysis indicated the presence of 2 distinct VSPH7 genes in H7. An oligonucleotide from the conserved region was used in combination with one specific for the VSPH7 gene to estimate the VSP repertoire size at between 133 and 151. VSPs, even from isolates expressing unique epitopes, constitute a family of related proteins.
A sensitive and specific polymerase chain reaction-based assay has been developed to detect and analyze polymorphism in the Giardia lamblia 18S ribosomal RNA gene. Efficient amplification required the inclusion of cosolvents (glycerol and dimethyl sulfoxide) in the reaction. Following the optimization of conditions for amplification and subsequent hybridization of amplified product with radiolabeled oligonucleotide probe, a detection limit of less than one organism's worth of DNA was achieved. Thirty-five different G. lamblia strains obtained from various human and animal host types and geographic locations were analyzed by this method. The strains could be divided into 3 groups on the basis of defined nucleotide substitutions within the 183-bp amplified DNA fragment of the 18S ribosomal RNA gene. The groupings based upon the 18S ribosomal RNA gene sequence correlated with groupings previously assigned based upon patterns of surface antigens and restriction enzyme analysis. Analysis of the G. lamblia 18S ribosomal RNA gene sequences present in fecal specimens obtained from giardiasis patients revealed the presence of the different sequence types in these specimens. Some specimens contained more than one sequence type. The identification of subgroups of G. lamblia may facilitate studies of virulence, infectivity, and the epidemiology of giardia infection.
Giardia lamblia trophozoites demonstrate variable expression of a repertoire of cysteine-rich surface antigens in vitro and in vivo. The size of the repertoire has been estimated at 20 to 184, and specific variants can be detected after approximately 12 generations of in vitro growth for the WB isolate. In earlier studies, we cloned a portion of the gene for a 170-kDa surface antigen (CRP170) and demonstrated by DNA sequencing that it was cysteine rich (12%) and contained 2.6 copies of a tandemly repeated 195-bp pair sequence. The clone hybridized to multiple bands on a Southern blot of G. lamblia DNA in a pattern that was variable among the cloned lines but did not correlate with expression of CRP170. We have now cloned a nearly full length cDNA as well as genomic clones for CRP170 from the WBA6 cloned isolate. In addition, we have isolated a cDNA clone from the WB1269 line (expressing CRP72), an antigenic variant which was derived from WBA6. Sequence analysis of the CRP170 and CRP72 genes revealed marked C-terminal amino acid homology, suggesting a conserved functional role such as membrane anchoring. The CRP170 repeat oligonucleotide hybridized to a stairstep of bands approximately 6 kb in size on HindIII-digested WBA6 DNA representing the expressed copy(ies) of CRP170. In contrast, there was no hybridization to a fragment of similar size in WB1269, suggesting that WB1269 trophozoites have lost the expressed copy of the CRP170 gene.
Antigenic variation in the parasitic protozoan Giardia lamblia was studied by characterizing the expression and genomic organization of a variant-specific surface protein (VSP) gene. Transcripts from this gene, vsp1267, were abundant in the cloned variant WB/1267, but undetectable in the parental clone from which WB/1267 was derived or in variant progeny of WB/1267. Two identical copies of vsp1267 exist in the WB/1267 genome, separated by 3 kb and arranged as convergent transcription units. Primer extension sequencing and S1 nuclease protection analysis suggested that the 5' untranslated region (UTR) of VSP1267 mRNA consists of a single nucleotide (nt). Primer extension sequencing mapped the site of VSP1267 transcript polyadenylation 25 nt beyond the termination codon. vsp1267 contained no introns and predicted a cysteine-rich polypeptide with features common to other VSPs. Comparison of vsp1267 with another VSP gene sequence revealed striking conservation, both at the nucleotide and amino acid levels, and the 3' ends of the genes. An oligonucleotide derived from this region detected size-variant VSP transcripts in 4 of 5 G. lamblia clones analyzed, suggesting the general utility of this probe in studying VSP genes and their expression.
Athymic nude mice of the outbred Zur:ICR-nu and inbred BALB/c strain and scid mice were infected with a cloned human isolate of Giardia lamblia (GS/M-83-H7). Changes in the expression of the major surface epitope of the intestinal trophozoites (characterized by the binding capacity of monoclonal antibody MoAbG10/4) as well as cellular and humoral immune parameters of the hosts were followed during the course of infection. Self-cure was observed in heterozygous (nu/+) BALB/c mice by day 22 post-infection (p.i.) and in heterozygous (nu/+) Zur:ICR-nu strain by day 65 p.i. Homozygous (nu/nu) mice of both strains remained chronically infected until end of the experiments (day 45 p.i. for BALB/c mice and day 122 p.i. for Zur:ICR-nu mice, respectively). Only heterozygous (nu/+) mice were able to mount a gut-associated (Peyer's patch) lymphoproliferative response to G. lamblia antigen. Therefore, T-cell dependent mechanisms were necessary for a self-cure. Antigenic variation occurred in all nu/+ and nu/nu animals of both strains. Trophozoites expressing the major surface epitope (assessed by direct immunofluorescence with FITC-labelled MoAb G10/4) decreased to zero by day 22 p.i. In contrast, the proportion of trophozoites expressing the major surface epitope in infected scid mice remained at the initial level (greater than 99%) until termination of the experiment (day 25 p.i.); therefore, antigenic variation did not occur. All nu/nu and nu/+ mice but not scid mice demonstrated a humoral immune response to G. lamblia antigen. These experiments suggest functional B-cell dependent mechanisms are most likely responsible for the surface antigen switch. Transfer of infection occurred naturally from experimentally infected scid-mice to their mother, proving the initial antigenic surface variant remains unchanged after encystment and subsequent excystment followed by infection in a new host.
The surface antigens of Giardia lamblia differ. To determine whether the unique surface antigens found in variants and isolates could differentially protect the parasite from digestion by intestinal protease, G. lamblia clones WB-2X (WB), GS/M-H7 (GS/M), and B6, each of which expresses a unique surface variant antigen, were exposed to alpha-chymotrypsin and trypsin at concentrations up to 20 mg/ml in culture medium. The number of surviving trophozoites and morphologic changes were assessed over time. After 24 h, there was a significant decrease in the number of surviving trophozoites of WB (80.5 and 94.2% for trypsin and alpha-chymotrypsin treatments, respectively, compared with controls) and B6 (78.9 and 95.5% for trypsin and alpha-chymotrypsin treatments, respectively, compared with controls) at 10 mg of enzyme per ml compared with culture medium alone. Cytotoxicity was prevented by the presence of soybean trypsin inhibitor, indicating the effects were due to protease activity. In contrast, there was no significant cytotoxicity after exposure of GS/M to either enzyme at the same enzyme concentration. After exposure to alpha-chymotrypsin, susceptible G. lamblia became rounded and then lysed, but after exposure to trypsin, G. lamblia appeared plastered onto the surface of the well and was intertwined and surrounded by finely granular material. Effects were concentration and time dependent; at least 6 h of treatment was required to observe changes 12 to 18 h later. Trophozoites surviving alpha-chymotrypsin or trypsin exposure became stably resistant to protease treatment. In vitro, the variant surface antigen of GS/M, but not those of WB or B6, resisted digestion by trypsin or alpha-chymotrypsin, suggesting that the variant surface antigens impart susceptibility or resistance to digestion. The initial surface variant antigens of WB and B6 were replaced in resistant cultures. Trophozoites differ in their ability to survive after exposure to intestinal proteases, which may enable certain G. lamblia isolates or isolates possessing certain surface variant antigens to survive in the small intestine.
Giardia lamblia telomeres have been isolated from a library enriched for repaired chromosome ends by (i) screening with a Plasmodium falciparum telomere and (ii) differential hybridization with Bal 31-digested and total G. lamblia DNA. Analysis of three clones isolated by this strategy has identified multiple tandem repeats of the 5-mer TAGGG. An oligonucleotide containing these repeats recognizes Bal 31-sensitive bands in Southern hybridizations and detects all G. lamblia chromosomes in pulsed-field gel electrophoresis separations. An abrupt transition from the G. lamblia rDNA sequence to telomeric repeats has been found in all three clones. In two of the clones the transition occurs at the same site, near the beginning of the large subunit rDNA sequence. In the third clone the transition occurs at a site in the intergenic spacer sequence between the rDNA genes. Hybridization of an rDNA probe to a pulsed-field separation of G. lamblia chromosomes indicates that rDNA genes are present on several chromosomes but vary in location from isolate to isolate. These results suggest that rRNA genes are clustered at telomeric locations in G. lamblia and that these clusters are mobile.
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To determine if Giardia surface Ag vary in human infections volunteers were inoculated enterally with trophozoites of uncloned GS/M-85 and in later experiments with two clones derived from GS/M. The surface Ag of trophozoites reisolated from 6/6 volunteers differed from the inoculum. To determine if the surface Ag of trophozoites derived from clones would also change, volunteers were inoculated with two clones, B6 or H7. B6 possesses a 200-kDa surface Ag recognized by mAb 3F6 and H7 has a 72-kDa surface Ag recognized by mAb G10/4. One of thirteen B6 and four of four H7-inoculated volunteers became infected. Analysis of Giardia obtained on day 22 from the intestines of the four H7-infected volunteers and cultures derived from these trophozoites revealed loss of the initial major surface Ag as determined by surface IFA using mAb, surface radiolabeling and loss of cytotoxicity to mAb, and Western blots. Loss of the 72-kDa Ag began after day 14 and was practically complete by day 22. The 200-kDa surface Ag was almost totally absent from the surface of Giardia isolated from the single B6-infected volunteer. Serum surface-reactive antibodies, as measured by IFA and cytotoxicity to H7 and the day 22 isolates, showed high levels of antibodies to H7, primarily to the 72-kDa surface Ag, but negligible or low levels of late-appearing antibodies to the day 22 isolates. These studies document antigenic variation of Giardia in human infections and show that humoral responses are in part isolate-specific.
Giardia lamblia undergoes antigenic variation. The rate of antigenic variation and the size of the variant antigen repertoire were estimated in clones of Giardia lamblia which reexpresses surface variant antigens that are characteristics of its parent. Calculations were based on determinations of the number of trophozoites expressing defined or nondefined epitopes as well as the total number of trophozoites in newly established clones. The rate of appearance of variant antigens containing defined epitopes was expressed as the number of generations until the first trophozoite expressing a defined epitope appeared. In clones of isolate WB, tested because their major surface variant antigens were largely nondefined, variants expressing epitopes recognized by Mabs 6E7 or 3F6 appeared after approximately 12 generations. Variants expressing epitopes recognized by Mab 5C1 appeared at about 13 generations, significantly greater than for the other epitopes. The rate of antigenic variation was studied in another isolate, GS/M, whose surface epitope repertoire differs from that of isolate WB. A single epitope recognized by Mab G10/4 was tested. Trophozoites reexpressing this epitope first appeared after about 6.5 generations, significantly less than in WB. Therefore, the single epitope studied in isolate GS/M is reexpressed much more frequently than those of WB. In isolate WB, the epitopes recognized by Mab 6E7 and 3F6 tended to appear at the same time. The median number of variant antigens in WB was estimated to lie between 20.5 and 184.
Giardia lamblia undergoes surface antigenic variation. The capability of different isolates to express certain epitopes on the surfaces of trophozoites from different isolates and clones was determined using 4 surface-reacting monoclonal antibodies (mAbs) to variants derived from WB or WB-like Giardia (mAbs 6E7, 5C1, and 3F6) and GS/M (mAb G10/4). Of 28 isolates, 11 possessed trophozoites reactive with mAbs 6E7, 5C1 and 3F6, 6 with mAb 3F6, 2 with Mab G10/4, 1 with mAb 6E7, and 8 showed no reactivity as determined by direct or indirect immunofluorescence. Newly established clones from different isolates generated small numbers of reactive trophozoites similar to their parents. Only one epitope was found on any single trophozoite. Southern blots hybridized to a probe encoding for the epitope recognized by mAb 6E7 revealed that the inability to express the antigen in most isolates was due to lack of the gene. Analysis of the surface antigens of mAb 6E7 reactive clones from 3 isolates revealed that mAb 6E7 reacted with surface antigens of different molecular masses.
Neonatal mice (CR:NIH:S) were infected with a cloned human isolate of Giardia lamblia (GS/M-83-H7) and the surface antigens of the intestinal trophozoites, as well as the cellular and humoral immune responses, were analysed during the course of infection. Infections in mice peaked 2-3 weeks after inoculation and were self-cured by day 42 post-infection (p.i.). The proportion of trophozoites expressing the Mr 72,000 surface antigen of the initial inoculum had decreased by day 12 and approached zero by day 22 p.i., similar to infections in humans. The predominant parasite-specific humoral response was an IgM- and IgG-isotype directed to the original Mr 72,000 surface antigen as well as other antigens. T-lymphocytes (predominantly LY4(CD4)+) isolated from Peyer's patches 12 days p.i. and later showed a significant proliferative response to Giardia lamblia antigens. Spleen and lymph node cells showed no lymphoproliferative response. T-cell blot analysis revealed the presence of dominant T-cell epitopes in the areas of Mr 200,000-75,000 and less than 50,000 polypeptides. No response was demonstrated in the Mr 72,000 region (migration site of the major surface antigen), suggesting T-cell dependent mechanisms are most likely not responsible for the surface antigen switch which occurred during the course of infection. This model infection can be used to study the role of immunological mechanisms in Giardia lamblia variant antigen switching and in the control of infections.
Surface antigenic variation was previously demonstrated in vitro and in vivo using Giardia lamblia isolate WB. To determine whether other isolates undergo similar changes, isolates GS and N were cloned and exposed to cytotoxic anti-isolate sera or monoclonal antibodies (MAbs) specific to particular surface antigen. The surviving Giardia (progeny) and clones showed different surface antigens as judged by resistance to cytotoxicity, loss of antigens determined by surface radiolabeling, and failure of MAbs to recognize epitopes by immunoprecipitation. The major varying surface antigens incorporate large amounts of [35S]cysteine compared to [35S]methionine. Surface antigenic variation occurs commonly in Giardia isolates, and the major surface antigens appear to comprise a family of cysteine-rich proteins.
Giardia lamblia undergo surface antigenic variation in vitro and in vivo. The presence of variant trophozoites can be detected in clones after exposure to cytotoxic monoclonal antibodies. Surviving Giardia (progeny) no longer possess the initial major surface antigen which is replaced by new antigens. Exposure of a clone from one progeny to another cytotoxic mAb specific to one newly appearing surface antigen resulted in the loss of this antigen and replacement by another set of antigens. The frequency of change was rapid (1:100-1:1000) and was dependent upon the isolate. The presence of variant populations in clones was confirmed by direct and indirect immunofluorescence using mAbs to major surface antigens of subsequent progeny. The putative amino acid sequence of a portion of one antigen revealed a cysteine-rich composition which was confirmed in this variant protein as well as others by preferential uptake of [35S]cysteine. The mechanism(s) responsible most likely involves genomic rearrangements since Southern blots revealed a family of related genes which changed frequently compared to other areas of the genome. However, expression-linked copies have not been detected. Loss and gain of surface antigens have also been found in gerbils and humans infected with defined clones, but there does not appear to be cyclical appearance of variant populations. The biological importance of antigenic variation is not known but it may contribute to chronic and/or repeated infections.