Antigenic variation of a cysteine-rich protein in Giardia lamblia.

The WB isolate of Giardia lamblia expresses a cysteine-rich 170-kD surface antigen (CRP170) that undergoes antigenic variation. An (6E7), cytotoxic for isolates expressing CRP170, was used in another study to select antigenic variants from clones of the WB isolate of Giardia. CRP170 was replaced by surface-labeled bands ranging in size from approximately 50 to 170 kD. In this study, mAb 6E7 was used to isolate a 1-kb portion of the CRP170 gene (M2-1) from a lambda gt 11 expression library. The M2-1 clone hybridized to a 5.4-kb transcript from isolates expressing CRP170 but did not hybridize to RNA from antigenic variants. Evidence was found for frequent rearrangements at the CRP170 gene locus. DNA sequencing of the M2-1 clone revealed the presence of long tandem repeats. The putative amino acid sequence of M2-1 reveals a 12% cysteine content, and CRP170 is readily labeled in vivo with cysteine.

Mechanisms of Giardia lamblia differentiation into cysts.

Microbiologists have long been intrigued by the ability of parasitic organisms to adapt to changes in the environment. Since most parasites occupy several niches during their journey between vectors and hosts, they have developed adaptive responses which allow them to survive under adverse conditions. Therefore, the life cycles of protozoan and helminthic parasites are excellent models with which to study numerous mechanisms involved in cell differentiation, such as the regulation of gene expression, signal transduction pathways, and organelle biogenesis. Unfortunately, many of these studies are very difficult because the conditions needed to elicit developmental changes in parasites remain undetermined in most cases. Recently, several interesting findings were reported on the process of differentiation of Giardia lamblia trophozoites into cysts. G. lamblia is a flagellated protozoan that inhabits the upper small intestine of its vertebrate host and is a major cause of enteric disease worldwide. It belongs to the earliest identified lineage among eukaryotes and therefore offers a unique insight into the progression from primitive to more complex eukaryotic cells. The discovery of a specific stimulus that induces trophozoites to differentiate into cysts, the identification and characterization of encystation-specific molecules, the elucidation of novel biochemical pathways, and the development of useful reagents and techniques have made this parasite an excellent model with which to study differentiation in eukaryotic cells. In this review, we summarize the most recent fundings on several aspects of Giardia differentiation and discuss the significance of these findings within the context of current knowledge in the field.

The cysteine-rich protein gene family of Giardia lamblia: loss of the CRP170 gene in an antigenic variant.

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.

Telomeric location of Giardia rDNA genes.

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.

Revised diagnostic criteria for neurocysticercosis.

BACKGROUND: A unified set of criteria for neurocysticercosis (NCC) has helped to standardize its diagnosis in different settings. METHODS: Cysticercosis experts were convened to update current diagnostic criteria for NCC according to two principles: neuroimaging studies are essential for diagnosis, and all other information provides indirect evidence favoring the diagnosis. Recent diagnostic advances were incorporated to this revised set. RESULTS: This revised set is structured in absolute, neuroimaging and clinical/exposure criteria. Absolute criteria include: histological confirmation of parasites, evidence of subretinal cysts, and demonstration of the scolex within a cyst. Neuroimaging criteria are categorized as major (cystic lesions without scolex, enhancing lesions, multilobulated cysts, and calcifications), confirmative (resolution of cysts after cysticidal drug therapy, spontaneous resolution of single enhancing lesions, and migrating ventricular cysts on sequential neuroimaging studies) and minor (hydrocephalus and leptomeningeal enhancement). Clinical/exposure criteria include: detection of anticysticercal antibodies or cysticercal antigens by well-standardized tests, systemic cysticercosis, evidence of a household Taenia carrier, suggestive clinical manifestations, and residency in endemic areas. Besides patients having absolute criteria, definitive diagnosis can be made in those having two major neuroimaging criteria (or one major plus one confirmative criteria) plus exposure. For patients presenting with one major and one minor neuroimaging criteria plus exposure, definitive diagnosis of NCC requires the exclusion of confounding pathologies. Probable diagnosis is reserved for individuals presenting with one neuroimaging criteria plus strong evidence of exposure. CONCLUSIONS: This revised set of diagnostic criteria provides simpler definitions and may facilitate its more uniform and widespread applicability in different scenarios.

The abundance of sterile transcripts in Giardia lamblia.

The protozoan parasite Giardia lamblia synthesizes a diverse and surprisingly abundant array of sterile transcripts unable to code for proteins. Random sampling of cDNAs from two evolutionarily divergent Giardia strains indicates that approximately 20% of cDNAs in the libraries represent polyadenylated sterile transcripts. RNase protection analysis and northern blot hybridization of three sterile transcript loci demonstrated that both the sterile transcript and a complementary mRNA were made in each case, further categorizing these sterile transcripts as antisense transcripts. Investigation of the genomic loci for these same three sterile antisense transcripts showed typical transcription units for the sense transcripts, but still failed to reveal a usable open reading frame for the sterile antisense transcripts. 5'-RACE mapped the transcription start site for one of the sterile antisense transcripts to an AT-rich region, as is typical for GIARDIA: It is unclear whether these sterile transcripts represent errors in transcription or whether they have regulatory functions within the cell, although preliminary investigations failed to reveal evidence for a role in developmental gene regulation. In either case, the presence of such a large pool of sterile antisense transcripts is dramatic evidence of the unusual molecular machinery of the early diverging protist G.lamblia.

Uricosuric effects of niridazole.

Niridazole, an antischistosomal drug, caused a 44% decrease in the serum uric acid (SUA) concentration in 11 patients with schistosomiasis. The mean SUA (+/- SE) was 6.3 +/- 1.4 mg/100 ml at baseline and 3.5 +/- 1.5 mg/100 ml (p less than 0.01) on day 7 of treatment. There was a significant increase in the urinary uric acid/creatinine ratio, from 0.446 +/- 0.165 at baseline to 0.550 +/- 0.145 on day 3. There was no significant difference on day 7. The fractional clearance of uric acid rose from 7.2 +/- 6.8% to 13.9 +/- 17.3% (p less than 0.01), indicating a uricosuric effect. Oxypurine excretion was unchanged. In a separate study on 7 other patients, the SUA remained low for 4 to 7 days after the last dose. Niridazole, although not an organic acid, has uricosuric effects.

Edema associated with calcified lesions in neurocysticercosis.

OBJECTIVE: To determine serial MRI and CT abnormalities around calcified cysts due to cysticercosis in previously treated patients during periods of seizure activity. BACKGROUND: Some patients with calcified lesions due to cysticercosis have seizures. How and why seizures occur in this setting are unknown. METHODS: Three patients with known, treated cysticercosis were studied prospectively by serial MRI and CT before, during, and after seizure activity. RESULTS: All three patients demonstrated edema surrounding calcified lesions. Two of three patients had repeated episodes involving the same calcified lesions, and their symptoms corresponded to the location of the lesion. Enhancement was present in the lesions demonstrating edema, but was also present surrounding other nonsymptomatic calcified lesions. CONCLUSIONS: Perilesional edema surrounding calcified lesions due to cysticercosis occurs in some patients at the time of seizure activity. Repeated seizure episodes tend to be associated with the same lesions. Although the mechanisms involved are unknown, long-term antiseizure medication is likely indicated in these patients. Current evidence does not support the use of specific antiparasitic treatment in these patients.

Proposed diagnostic criteria for neurocysticercosis.

Neurocysticercosis is the most common helminthic infection of the CNS but its diagnosis remains difficult. Clinical manifestations are nonspecific, most neuroimaging findings are not pathognomonic, and some serologic tests have low sensitivity and specificity. The authors provide diagnostic criteria for neurocysticercosis based on objective clinical, imaging, immunologic, and epidemiologic data. These include four categories of criteria stratified on the basis of their diagnostic strength, including the following: 1) absolute--histologic demonstration of the parasite from biopsy of a brain or spinal cord lesion, cystic lesions showing the scolex on CT or MRI, and direct visualization of subretinal parasites by funduscopic examination; 2) major--lesions highly suggestive of neurocysticercosis on neuroimaging studies, positive serum enzyme-linked immunoelectrotransfer blot for the detection of anticysticercal antibodies, resolution of intracranial cystic lesions after therapy with albendazole or praziquantel, and spontaneous resolution of small single enhancing lesions; 3) minor--lesions compatible with neurocysticercosis on neuroimaging studies, clinical manifestations suggestive of neurocysticercosis, positive CSF enzyme-linked immunosorbent assay for detection of anticysticercal antibodies or cysticercal antigens, and cysticercosis outside the CNS; and 4) epidemiologic--evidence of a household contact with Taenia solium infection, individuals coming from or living in an area where cysticercosis is endemic, and history of frequent travel to disease-endemic areas. Interpretation of these criteria permits two degrees of diagnostic certainty: 1) definitive diagnosis, in patients who have one absolute criterion or in those who have two major plus one minor and one epidemiologic criterion; and 2) probable diagnosis, in patients who have one major plus two minor criteria, in those who have one major plus one minor and one epidemiologic criterion, and in those who have three minor plus one epidemiologic criterion.

Characterization of a Giardia lamblia variant-specific surface protein (VSP) gene from isolate GS/M and estimation of the VSP gene repertoire size.

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.

Carboxy-terminal sequence conservation among variant-specific surface proteins of Giardia lamblia.

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.

Cysteine-rich variant surface proteins of Giardia lamblia.

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.

Episomal and integrated maintenance of foreign DNA in Giardia lamblia.

Giardia lamblia is an early diverging eukaryote which causes gastrointestinal disease throughout the world. Different subgroups of Giardia have been defined based on several biochemical and genetic criteria. We have developed a method for stably introducing DNA into the nuclei of the parasite using puromycin acetyltransferase (pac) as a dominant selectable marker. Transfected circular DNAs were maintained as episomes in the isolate WB, a representative of one Giardia subgroup. When input DNAs were linearized, integration was observed to occur by homologous recombination producing gene replacements in this isolate. In isolate GS, which represents a different subgroup, both linear and circular transfected DNAs were integrated into the genome by homologous recombination. In GS, linear DNA again produced gene replacements, while circular DNA produced duplicative integration events. The failure of GS to replicate episomes may reflect differences in the structure or recognition of DNA replication origins between these subgroups. A plasmid shuttle vector was also developed for expression of other genes in Giardia lamblia. Utilizing the green fluorescent protein as a reporter gene in the WB isolate, we show that gene expression from this vector correlated with plasmid copy number over a range of two orders of magnitude. Together these tools should greatly enhance our ability to study both the basic biology and the pathogenesis of this ubiquitous parasite.

Classification of subgroups of Giardia lamblia based upon ribosomal RNA gene sequence using the polymerase chain reaction.

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.

Variant specific epitopes of Giardia lamblia.

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.

Initiator and upstream elements in the alpha2-tubulin promoter of Giardia lamblia.

Giardia lamblia, one of the earliest diverging eukaryotes and a major cause of diarrhea world-wide, has unusually short intergenic regions, raising questions concerning its regulation of gene expression. We have approached this issue through examination of the alpha2-tubulin promoter and in particular investigated the function of an AT-rich element surrounding the transcription start site. Its placement and the ability of this sequence to direct transcription initiation in the absence of any other promoter elements is similar to the initiator element in higher eukaryotes. However, the sequence diversity of extremely short (8-10 bp) initiator elements is surprising, as is their ability to independently direct substantial levels of transcription. We also identified a large AT-rich element located between -64 and -29 bp upstream of the transcriptional start site and show using both deletions and site-specific mutations of this region that sequences between -60 and the start of transcription are important for promoter strength; interestingly this AT-rich sequence is not highly conserved among different Giardia promoters. These data suggest that while the overall structure of the core promoter has been conserved throughout eukaryotic evolution, significant variation and flexibility is allowed in element consensus sequences and roles in transcription. In particular, the short and diverse sequences that function in transcription initiation in Giardia suggest the potential for relaxed transcriptional regulation.

Isoprenylation of proteins in the protozoan Giardia lamblia.

We report the ability of Giardia lamblia to modify several of its cellular proteins by isoprenylation. Trophozoites cultured in the presence of [3H]mevalonate synthesized radiolabeled proteins of approx. 50 and 21-26 kDa. Chemical analysis indicated that farnesyl and geranylgeranyl isoprenoids comprised the majority of the radiolabel covalently associated with trophozoite proteins. In addition, antibodies to human p21ras immunoprecipitated mevalonate-labelled species of approx. 21 kDa. Inhibitors of several enzymatic steps of the mevalonate pathway dramatically affected Giardia metabolism. Protein isoprenylation and cell growth were blocked by compactin and mevinolin, competitive inhibitors of HMG-CoA reductase, the rate-limiting enzyme in isoprenoid biosynthesis. In the presence of these inhibitors, Giardia growth was restored by the addition of mevalonate to the culture medium. In contrast, cell growth was blocked irreversibly by inhibitors of subsequent steps in the protein isoprenylation pathway. Trophozoite growth inhibition by limonene, perillic acid, perillyl alcohol and N-acetyl-S-farnesyl-L-cysteine was not reversed after the addition of mevalonate, dolichol, ubiquinone or cholesterol to the medium. These observations constitute the first description of protein isoprenylation in any protozoan and indicate that this post-translational modification is an important step in the regulation of the growth of this primitive eukaryote.

Antibody response to a polysaccharide antigen present in the schistosome gut. I. Sensitivity and specificity.

By using paraffin sections of adult schistosomes fixed in Rossman's fixative, specific human IgM and IgG antibodies to a polysaccharide present in the epithelial cells of the schistosome were measured by using indirect immunofluorescent techniques. Of the 49 patients, mostly infected with S. mansoni but a few infected with S. haematobium or S. japonicum, all had antibody present at a 1:8 dilution of serum. Specific IgM antibody was more sensitive than IgG, yielding 100% and 86% positivity respectively. The false positive rate was 3% in a panel of sera obtained from patients most of whom were infected with other parasites. Antibodies were detected by the 3rd week in experimentally infected animals. Unisexual infections also induced antibody production. As a diagnostic test, the measurement of antibody to the polysaccharide is an easily performed reliable test with high sensitivity and specificity.

Immune complex size determines the clearance rate of a circulating antigen in schistosome-infected mice.

Previous studies showed that the clearance rate of gut-associated proteoglycan (GASP), a specific excretory-secretory antigen, was enhanced in mice infected with low schistosome burdens compared to heavily infected and control mice. Because the size of immune complexes alters the clearance rate of antigens, in the present experiments the size of GASP-containing immune complexes was determined after injection of radiolabeled GASP into lightly and heavily infected and control mice. The size and amount of immune complexes were determined by sucrose density centrifugation, agarose electrophoresis, and (NH4)2SO4 precipitation. All three methods showed that lightly infected mice had larger and quantitatively more GASP-containing immune complexes than did heavily infected mice. The differences in clearance between lightly and heavily infected mice, therefore, appear to be due to the presence of smaller, and quantitatively less, immune complexes in the heavily infected mice.

Comparison of two antigenically distinct Giardia lamblia isolates in gerbils.

Previous studies established that some isolates of Giardia differ antigenically. In order to determine if antigenic differences resulted in altered biological behavior of host immune responses, two antigenically distinct isolates, WB and GS-E, were used to infect gerbils, and the course of infection, resistance to reinfection, and host humoral responses were measured. Maximum numbers of trophozoites were recovered on day 14 from the intestine of gerbils infected with both isolates, but by day 28, 75% of WB-infected gerbils were free of infection, while GS-E-infected animals continued to be infected until day 42. After curative metronidazole therapy, animals were challenged with the homologous or heterologous isolates. Gerbils previously infected with WB were resistant to challenge with WB and GS-E, while previously GS-E-infected gerbils were more resistant to challenge with the homologous isolate. Antibody responses were measured by ELISA to both surface and cytosol antigens and by IFA to the surface of Giardia. By IFA there was a greater reactivity using the homologous isolate, but with ELISA this was not as apparent. Complement independent cytotoxicity of sera was additionally tested against both isolates. Sera from WB-infected gerbils were cytotoxic to both WB and GS-E whereas sera from GS-E-infected gerbils were cytotoxic to GS-E only. These studies demonstrate that Giardia possessing different surface antigens have different patterns of infection and induce qualitatively and quantitatively different immune responses. Cytotoxicity of sera, most likely antibodies, correlated best with the development of resistance.