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. 2005 Dec;142(3):528-38.
doi: 10.1111/j.1365-2249.2005.02939.x.

Screening of an Echinococcus granulosus cDNA library with IgG4 from patients with cystic echinococcosis identifies a new tegumental protein involved in the immune escape

E Ortona  1 , P Margutti, F Delunardo, V Nobili, E Profumo, R Riganò, B Buttari, G Carulli, A Azzarà, A Teggi, F Bruschi, A Siracusano
Affiliations

Screening of an Echinococcus granulosus cDNA library with IgG4 from patients with cystic echinococcosis identifies a new tegumental protein involved in the immune escape

E Ortona et al. Clin Exp Immunol. 2005 Dec.

Abstract

The worldwide problem of chronic Echinococcus granulosus disease calls for new parasite-derived immunomodulatory molecules. By screening an E. granulosus cDNA library with IgG4 from patients with active cystic echinococcosis, we identified a cDNA that encodes a predicted partial protein that immunofluorescence studies localized in the protoscolex tegument and on the germinal layer of cyst wall. We named this protein EgTeg because the 105 amino acid sequence scored highest against a family of Schistosoma tegumental proteins. Evaluating the role of EgTeg in the human early inflammatory response we found that EgTeg significantly inhibited polymorphonuclear cell (PMN) chemotaxis. Cytometric analysis of intracellular cytokines disclosed a significantly higher percentage of cells producing IL-4 than IFN-gamma (P = 0.001, Student's t-test) in T lymphocytes from patients with cystic echinococcosis stimulated with EgTeg. EgTeg induced weak Th1-dependent proliferation in 42% of patients' peripheral blood mononuclear cells. In immunoblotting (IB) analysis of total IgG and IgG subclass responses to EgTeg in patients with cystic echinococcosis, patients with other parasitoses, patients with cystic lesions and healthy controls, total IgG specific to EgTeg yielded high sensitivity (73%) but low specificity (44%) precluding its use in immunodiagnosis. Conversely, IgG4 specific to EgTeg gave acceptable sensitivity (65%) and high specificity (89%) suggesting its use in immunodiagnosis to confirm ultrasound documented cysts suggestive of E. granulosus. Because the new tegumental antigen EgTeg inhibits chemotaxis, induces IL-4-positive T lymphocytes and noncomplement fixing antibodies (IgG4) it is an immunomodulatory molecule associated with chronic infection.

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Figures

Fig. 1
Fig. 1
Nucleotide and amino acid sequence of cDNA clone encoding EgTeg. Western blot showing the molecular mass (12 kD) of the recombinant protein encoded by the cDNA of the EgTeg clone after subcloning into pQE-31, induction with isopropyl-β-D-1-thiogalactopiranoside and purification under denaturating conditions. The purified, recombinant protein (3 µg) was subjected to SDS-PAGE and stained with Coomassie Blue (lane A) or transferred to nitrocellulose and revealed with the monoclonal antibody specific to the 6X-histidine (lane B) and with the mouse polyclonal antiserum specific to EgTeg (lane C). Molecular weights are indicated on the left.
Fig. 2
Fig. 2
Transmembrane protein topology prediction analysis (using the hidden Markov model TMHMM). The region within the 66–84 amino acids, between the hydrophilic intracellular N-terminal region and extracellular C-terminal region, exhibited high hydrophobicity. Comparison of amino acid sequence of EgTeg and, the 21·7 tegumental antigen of Schistosoma japonicum (AAD 13338), the 22·6 tegumental antigen of S. japonicum (AAA 29940) and the 22·6 tegumental antigen of S. mansoni (A54518) using the program CLUSTAL for T-COFFEE (1·41). Identity (*). Strong similarity (:). Weak similarity (.). Peptide 1 (––) and peptide 2 (- - -) were underlined. Shaded grey amino acids represent the sequence that has 100% similarity and 91% identity with the compared sequences.
Fig. 3
Fig. 3
Immunofluorescence analysis of EgTeg localization in Echinococcus granulosus. The mouse polyclonal antiserum specific to EgTeg revealed binding sites localized (a) in the tegument of protoscolex and (c) on the internal germinal membrane. (b) No EgTeg-antibody staining was observed in protoscoleces incubated with preimmune mouse serum. Original magnifications 400×. (d) Immunoblotting after SDS-PAGE of protoscoleces (lane 2) and hydatid fluid (lane 3) revealed with the mouse polyclonal antiserum specific to EgTeg. Molecular weights are indicated on lane 1.
Fig. 4
Fig. 4
Effect of EgTeg on polymorphonuclear cell random migration and chemotaxis. Data presented here are the means of triplicate experiments in three uninfected controls. Asterisks indicate statistically significant difference versus control (P < 1 × 10−3 by Student's t-test).
Fig. 5
Fig. 5
Proliferation of PBMC from 19 patients with cystic echinococcosis in response to EgTeg. Asterisks indicate statistically significant differences (P = 0·04 by Student's t-test).
Fig. 6
Fig. 6
Cytofluorimetric analysis of IFN-γ and IL-4 in PBMC specific to EgTeg obtained from three patients with cystic echinococcosis. T lymphocytes were defined by the typical forward-angle and side-scatter gate and by a CD3+ gate (logical gate). The percentage of IL-4 and/or IFN-γ producing cells are given in the respective quadrants.
Fig. 7
Fig. 7
Competitive inhibition immunoblotting of human serum pool binding to EgTeg inhibited by peptide 1 (YEGVWQVVILTGSYSAFSAY) and peptide 2 (PERLFHFKFGRFVVLVWQS). Lane 1, unabsorbed serum; lane 2, serum adsorbed with peptide 1; lane 3 serum adsorbed with peptide 2; lane 4, serum adsorbed with EgTeg; lane 5, serum adsorbed with a non parasite-related recombinant protein (Nedd5, Accession No. Q15019).
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