Petra Frosch

Cloning and characterisation of an immunodominant major surface antigen of Echinococcus multilocularis.

A lambda gt11 cDNA expression library from mRNA of Echinococcus multilocularis protoscolices has been constructed in Escherichia coli Y1090. Immunoscreening with pooled sera obtained from patients suffering from E. multilocularis disease revealed 5 reactive clones. By partial DNA sequence comparison all clones proved to encode the same gene. The complete cDNA sequence of the clone pEM10 with the largest insert of 2.2 kb was determined and an open reading frame of 1.7 kb could be described. The derived amino acid sequence shares 42.6% identity with human microvillar cytovillin found in the membranes of placenta and carcinoma tissues. The coding region of the cDNA of pEM10 was amplified by polymerase chain reaction (PCR) and cloned in frame into expression vector pGEX-3X. Immunoblot analysis revealed the expression of a recombinant antigen of 65 kDa and a protein with the same molecular weight was also found in the lysate of E. multilocularis protoscolices. In contrast, the protein was absent from hydatid fluid or larvae of Echinococcus granulosus. By means of immunofluorescence studies this immunodominant antigen could be located in the germinal layer of brood capsules and in the tegument of E. multilocularis protoscolices. The fusion protein was purified and used for diagnostic purposes in immunoblot. The diagnostic value of this antigen is discussed.

Sequence heterogeneity of the echinococcal antigen B

Antigen B is a thermostable lipoprotein of 160 kDa [1] and a major antigen of the hydatid fluid of Echinococcus granulosus metacestodes [2], which is strongly immunogenic in echinococcal disease. About 90% of patients suffering from cystic echinococcosis, caused by the metacestodes of E. granulosus, and 40% of patients suffering from alveolar echinococcosis, caused by Echinococcus multilocularis larvae, exhibited antibodies against antigen B [3]. This suggests that antigen B is expressed in both echinococcal species, although this antigen was previously described as an E. granulosus-specific antigen [4]. In SDS-PAGE, antigen B dissociates and 3 bands of 8, 16 and 24 kDa can be found. Lightowlers et al. [5] observed additional higher-molecular weight molecules, which differed in their masses by 8 kDa, suggesting that antigen

Paramyosin of Echinococcus granulosus: cDNA sequence and characterization of a tegumental antigen.

A λZAPII cDNA library ofEchinococcus granulosus larvae was expressed inEscherichia coli SURE cells. Screening of the library with a rabbit antiserum raised against total larval antigen yielded several immunoreactive clones. For analysis of the nucleotide sequence, in vivo excision into pBlueskript was carried out and the 3′ end of the cloned insert was sequenced. Three of these clones exhibited identical nucleotide sequences, suggesting expression of identical genes. The complete nucleotide sequence of the largest clone, EG36, with a 3.4-kb insert was determined, presenting an open reading frame of 2.59 kb. The predicted amino acid sequence showed 71.4% identity to theSchistosoma mansoni paramyosin and a significant homology to a 17 amino-acid peptide sequence from antigen B ofTaenia solium. From these data we conclude that EG36 is the paramyosin ofE. granulosus. For protein purification, the coding sequence of the cDNA was amplified by polymerase chain reaction and ligated in frame into the expression vector pGEX-3X. Affinity-chromatography-purified GST fusion protein was used to induce a polyclonal rabbit antiserum. Immunoblot analysis revealed the expression of a 97-kDa protein by theE. coli clone and that of a protein with a similar molecular weight in protoscolices fromE. granulosus andE. multilocularis as well as inE. granulosus cyst fluid. Immunofluorescence studies showed that EG36 was localized throughout the tegument ofE. granulosus andE. multilocularis larvae. Sera from patients suffering from echinococcosis, schistosomiasis, and neurocysticercosis reacted with the purified fusion protein when tested in an enzyme-linked immunosorbent assay.

Diagnosis of Echinococcus multilocularis infection by reverse-transcription polymerase chain reaction

Alveolar echinococcosis is a life-threatening parasitosis occurring in countries in the Northern hemisphere. The diagnosis of an Echinococcus multilocularis infection is routinely performed by radiological techniques and by the detection of specific antibodies in the sera of infected patients. However, because serodiagnosis fails in 5%-10% and radiological techniques are difficult to interpret in some cases, we developed a polymerase chain reaction for the detection of echinococcal-specific messenger RNA from fine-needle biopsy specimens. This technique was applied to the diagnosis of alveolar echinococcosis in a 20-year-old seronegative woman. Detection of messenger RNA not only allowed the diagnosis of echinococcal disease but also proved to be a reliable measure for determining the efficacy of an antiparasitic therapy.

Characterization of the genomic locus expressing the ERM-like protein of Echinococcus multilocularis ☆

is the causative agent of alveolarechinococcosis in man, a life-threatening parasito-sis usually affecting the liver of the intermediatehost by an infiltrative, tumor-like growth [1]. Inrecent years, molecular biological approaches ledto the identification of several cDNA cloneswhich provided useful tools for serodiagnosis ofalveolar echinococcosis, but insight into thepathogenesis of echinococcal disease was notachieved. Several of these cDNA clones expressedstructural antigens common to all eukaryotic cells[2–5]. For other antigens, no function could beassigned due to a lack of sequence homologies toknown genes and gene products [6,7]. Further-more, there is no knowledge available about thegenomic organization of echinococcal genes andabout the regulation of their expression. In conse-quence, no appropriate model system exists allow-ing the performance of reverse genetics forfunctional analysis of echinococcal gene products.To get insight into the genomic organization ofechinococcal genes and to study the regulation ofgene expression in this parasite, we analysed as afirst target sequence, the chromosomal locus en-coding the ezrin-radixin-moesin (ERM)-likeprotein, in the following referred to as ELP, of

Identification of a cDNA clone from the larval stage ofEchinococcus granulosus with homologies to theE. multilocularis antigen EM10-expressing cDNA clone

Molecular characteriztion of the tapeworm Echinococcus multilocularis, the causative agent of alveolar echinococcosis in man, resulted, as determined independently by several research groups, in the identification of fulllength or partial cDNA clones with almost identical nucleotide sequences termed EM10 (Frosch et al. 1991), antigen II/3 (Felleisen and Gottstein 1993; Vogel et al. 1988), and EM4 (Hemmings and McManus 1991), respectively. The full-length cDNA of clone EM10 expressed a protein, which was found in the germinal layer of brood capsules and in the tegument of protoscolices (Frosch et al. 1991). This antigen was also most recently described to be expressed by the adult E. muItilocularis worm (Felleisen and Gottstein 1993). Antigen EM10 exhibits a strong homology to proteins of the ERM (ezrin, radixin, moesin) family, the members of which are involved in the linkage of the plasma membrane with the cytoskeleton in eukaryotic cells (Bretscher 1993). EM10 migrates as a 65-kDa band, which is in accordance with the molecular mass predicted from the cDNA sequence. However, a 55-kDa polypeptide is also found when lysates of protoscolices are analyzed by immunoblotting (Frosch et al. 1991). The origin of this smaller protein is not known, but it might be a degradation product. Furthermore, when used for serodiagnosis of echinococcal disease, this echinococcal antigen provides great potential for a specific diagnosis of E. multilocularis infections (Helbig et al. 1993). About 95% of patients suffering from alveolar echinococcosis exhibit antibodies against antigen EM10, but only about 1% of sera from patients suffering from E. granulosus disease elicit an EM10-specific antibody response (Helbig et al. 1993). These observations correlate well with the previ-

Molecular cloning of an echinococcal microtrichal antigen immunoreactive in Echinococcus multilocularis disease

A cDNA expression library of the larval stage of the cestode worm Echinococcus multilocularis has been established in the phage lambda ZAPII system. By immunoscreening with pooled sera from patients with alveolar echinococcosis an immunoreactive clone, termed pEM13, was isolated. EM13 was expressed using the expression vector pGEX-3X, resulting in the synthesis of a glutathione S-transferase fusion protein. 82% of the sera from 28 patients suffering from E. multilocularis disease had antibodies against EM13, whereas none of the 55 sera obtained from Echinococcus granulosus-infected patients and none of the 15 sera from patients with other helminthic infections reacted with recombinant EM13. By use of a polyclonal rabbit anti-EM13 hyperimmune serum native EM13 protein could be detected only in the protoscolices of E. multilocularis, but not in E. granulosus larvae or hydatid fluid. Immunoelectron microscopy suggested that EM13 is located in the microtriches on the surface of the larvae. In contrast, EM13 mRNA could be detected by Northern blot analysis in both E. multilocularis and E. granulosus larval RNA preparations. Nucleotide and amino acid sequence analysis of a cDNA clone coding for the corresponding antigen of E. granulosus larvae, termed EG13, revealed a 21-bp insertion, a 51-bp deletion and additional 22 nucleotide exchanges resulting in a 96.3% identity at the nucleotide sequence level and a 96.6% identity at the amino acid sequence level to the coding region of the cDNA pEM13. Cross-reactivity of the polyclonal anti-EM13 serum with the recombinant EG13 indicates a posttranscriptional regulation mechanism, resulting in an EG13 negative phenotype in E. granulosus.

Molecular cloning and characterization of an Echinococcus multilocularis and Echinococcus granulosus stress protein homologous to the mammalian 78 kDa glucose regulated protein

Abbreviations: hsp, heat shock protein(s); ER, endoplasmatic reticulum; GST, glutathione Stransferase; PCR, polymerase chain reaction; HPLC, high-performance liquid chromatography; bp, base pairs; IPTG, isopropyl-P-o-thiogalactopyranoside. Note: The nucleotide sequences reported here were deposited in the GenBanWEMBL data bank under accession numbers M63604 (EM-Grp78) and M63605 (EG-Grp78), respectively. ’ Presenr address: Department of Microbiology and Immunology, Georgetown University Medical Center, Washington, DC, USA. * Corresponding author, Tel.: +49 51 I 5324352; Fax: +49 511 5324366. However, stimulators of grp78 expression include tunicamycin via inhibition of glycosylation, glucose starvation and calcium ionophores [3,4]. Important features of Grp78 that are absent in other hsp70 proteins include a hydrophobic signal sequence facilitating the translocation of Grp78 into the ER and a carboxy terminal ER retention signal (KDEL) maintaining the protein in the lumen of the ER [5]. Grp78 is believed to function as a molecular chaperone’ in the ER, promoting the correct folding and/or assembly of polypeptides in an ATP-dependent manner [6]. Members of the hsp70 family have been shown to be highly immunogenic in several parasitic diseases [7]. This includes the Plasmodium falciparum Grp78 [8] and the recently described Grp78 of Trypanosoma cruzi [9]. Furthermore hsp70-like polypeptides induce high levels of antibodies in infections caused by P. falciparum, Schistosoma mansoni, S. japonicum [7,10] and Brugia malayi [ll]. By screening of a cDNA library of the larval