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Trypanosoma cruzi-Induced Host Immune System Dysfunction: A Rationale for Parasite Immunosuppressive Factor(s) Encoding Gene Targeting.

Ouaissi A, Da Silva AC, Guevara AG, Borges M, Guilvard E - J. Biomed. Biotechnol. (2001)

Bottom Line: In this paper, we review the data concerning the immunoregulatory effects of T. cruzi Tc24 (a B cell activator antigen) and Tc52 (an immunosuppressive protein) released molecules on the host immune system.Moreover, targeted Tc52 replacement allowed the obtention of parasite mutants exhibiting low virulence in vitro and in vivo.Thus, the generation of a complete deficiency state of virulence factors by gene targeting should provide a means to assess the importance of these factors in the pathophysiological processes and disease progression.

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ABSTRACT
An intense suppression of T cell proliferation to mitogens and to antigens is observed in a large number of parasitic infections. The impairment of T cell proliferation also occurred during the acute phase of Chagas' disease, caused by the intracellular protozoan parasite Trypanosoma cruzi. A wealth of evidence has accumulated that illustrates the ability of T. cruzi released molecules to influence directly a variety of diverse immunological functions. In this paper, we review the data concerning the immunoregulatory effects of T. cruzi Tc24 (a B cell activator antigen) and Tc52 (an immunosuppressive protein) released molecules on the host immune system. The gene targeting approach developed to further explore the biological function(s) of Tc52 molecule, revealed interesting unexpected functional properties. Indeed, in addition to its immunusuppressive activity a direct or indirect involvement of Tc52 gene product alone or in combination with other cellular components in T. cruzi differentiation control mechanisms have been evidenced. Moreover, targeted Tc52 replacement allowed the obtention of parasite mutants exhibiting low virulence in vitro and in vivo. Thus, the generation of a complete deficiency state of virulence factors by gene targeting should provide a means to assess the importance of these factors in the pathophysiological processes and disease progression. It is hoped that such approaches might allow rational design of tools to control T. cruzi infections.

No MeSH data available.


Related in: MedlinePlus

(A) Elution profiles of T. cruzi Tc52-bound toS-hexyl glutathione columns with either S-hexyl glutathione (1)or the following amino acids: L-cysteine (2), L-methionine (3),L-glycine (4), glutamic acid (5). (B) After elution as describedin A, the columns from which the eluates analysed in lanes 2, 3,4, and 5 were recovered, washed with the column buffer, andloaded with the same buffer containing S-hexyl glutathione, thecorresponding eluates were analysed in lanes 6, 7, 8, and 9,respectively. As shown in lane 6, residual Tc52 could berecovered from the column which was first loaded with L-cysteine(lane 2). In contrast, Tc52 which still bound to the glutathionematrix after the elution with L-methionine (3), L-glycine (4) orGlutamic acid (5), could be efficiently eluted by S-hexylglutathione (lanes 7, 8, 9, respectively). (C) Aliquots of theeluted samples in A (Ia) and B (IIa) (the lanes 1, 2, 3, 4, 5,6, 7, and 8, corresponded to the products eluted and analyzed inlanes 2, 3, 4, 5, 6, 7, and 9, respectively), were separated bySDS-PAGE, transferred to nitrocellulose filters and probed using arabbit immune serum to Tc52 fusion protein (cDNA encoding the Tc52protein, initially named TcAc2, was subcloned in pGEX-2T vector,and the protein was produced in fusion with Schistosoma japonicum  26 kDa glutathione S-transferase (Tc52-Sj26GST)). Thefusion protein and the corresponding antibodies were obtained asdescribed in a previous report [10]. Ib and IIb representcontrol tests using samples analysed in Ia and IIa,respectively, and treated with anti-Sj26GST rabbit serum.Methods: T. cruzi (Y strain) epimastigotes weremaintained in culture medium as described in [10]. Parasitesoluble antigens were prepared and adjusted to 5 mgproteins/ml in buffer (20 mM HEPES, pH 7.25, 1 mM EDTA,O.15 M KCl supplemented with 0.5 mM final concentrationof PMSF), and passed through S-hexyl glutathione affinity matrix.The column was then washed with 30 ml of the same buffer,bound material was eluted using 20 ml of buffer containing2.5 mM S-hexyl glutathione (1), or 1 mg/ml of either ofthe following amino acids: L-cysteine (2), L-methionine (3),L-glycine (4), or L-glutamic acid (5). The columns corresponding tothe lanes 2, 3, 4, and 5 were extensively washed with buffer andloaded with 20 ml of buffer containing 2.5 mM S-hexylglutathione. All the eluates were dialysed against distilledwater and analysed by SDS-PAGE. The proteins were visualizedusing the silver staining method.
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Figure 1: (A) Elution profiles of T. cruzi Tc52-bound toS-hexyl glutathione columns with either S-hexyl glutathione (1)or the following amino acids: L-cysteine (2), L-methionine (3),L-glycine (4), glutamic acid (5). (B) After elution as describedin A, the columns from which the eluates analysed in lanes 2, 3,4, and 5 were recovered, washed with the column buffer, andloaded with the same buffer containing S-hexyl glutathione, thecorresponding eluates were analysed in lanes 6, 7, 8, and 9,respectively. As shown in lane 6, residual Tc52 could berecovered from the column which was first loaded with L-cysteine(lane 2). In contrast, Tc52 which still bound to the glutathionematrix after the elution with L-methionine (3), L-glycine (4) orGlutamic acid (5), could be efficiently eluted by S-hexylglutathione (lanes 7, 8, 9, respectively). (C) Aliquots of theeluted samples in A (Ia) and B (IIa) (the lanes 1, 2, 3, 4, 5,6, 7, and 8, corresponded to the products eluted and analyzed inlanes 2, 3, 4, 5, 6, 7, and 9, respectively), were separated bySDS-PAGE, transferred to nitrocellulose filters and probed using arabbit immune serum to Tc52 fusion protein (cDNA encoding the Tc52protein, initially named TcAc2, was subcloned in pGEX-2T vector,and the protein was produced in fusion with Schistosoma japonicum 26 kDa glutathione S-transferase (Tc52-Sj26GST)). Thefusion protein and the corresponding antibodies were obtained asdescribed in a previous report [10]. Ib and IIb representcontrol tests using samples analysed in Ia and IIa,respectively, and treated with anti-Sj26GST rabbit serum.Methods: T. cruzi (Y strain) epimastigotes weremaintained in culture medium as described in [10]. Parasitesoluble antigens were prepared and adjusted to 5 mgproteins/ml in buffer (20 mM HEPES, pH 7.25, 1 mM EDTA,O.15 M KCl supplemented with 0.5 mM final concentrationof PMSF), and passed through S-hexyl glutathione affinity matrix.The column was then washed with 30 ml of the same buffer,bound material was eluted using 20 ml of buffer containing2.5 mM S-hexyl glutathione (1), or 1 mg/ml of either ofthe following amino acids: L-cysteine (2), L-methionine (3),L-glycine (4), or L-glutamic acid (5). The columns corresponding tothe lanes 2, 3, 4, and 5 were extensively washed with buffer andloaded with 20 ml of buffer containing 2.5 mM S-hexylglutathione. All the eluates were dialysed against distilledwater and analysed by SDS-PAGE. The proteins were visualizedusing the silver staining method.

Mentions: Tc52 native protein was eluted from S-hexyl glutathione affinitycolumn by using glutathione (GSH) or cysteine and not other aminoacids including those in GSH (e.g., glutamic acid andglycine) (Figure 1). We have hypothesized that T cellresponse could be affected by Tc52 through its interaction withGSH and cysteine. Indeed, it is interesting to remind that thediverse responses of T cells appear to involve a complex seriesof secondary signals in addition to the triggering of T cellantigen specific receptor. Moreover, it has been shown that theextracellular concentration of cysteine could be a limitingfactor for T cell activation [11]. Indeed, the plasmacysteine concentration is 10 times lower than that of itsoxidized form cystine. In addition, the T cell membrane transportfor cystine is 10 times less efficient than for cysteine. Incontrast, macrophages have the capacity to take up cystineefficiently and release cysteine, therefore increasing thedelivery of this amino acid to T cells which in turn couldincrease their own GSH intracellular level [20].


Trypanosoma cruzi-Induced Host Immune System Dysfunction: A Rationale for Parasite Immunosuppressive Factor(s) Encoding Gene Targeting.

Ouaissi A, Da Silva AC, Guevara AG, Borges M, Guilvard E - J. Biomed. Biotechnol. (2001)

(A) Elution profiles of T. cruzi Tc52-bound toS-hexyl glutathione columns with either S-hexyl glutathione (1)or the following amino acids: L-cysteine (2), L-methionine (3),L-glycine (4), glutamic acid (5). (B) After elution as describedin A, the columns from which the eluates analysed in lanes 2, 3,4, and 5 were recovered, washed with the column buffer, andloaded with the same buffer containing S-hexyl glutathione, thecorresponding eluates were analysed in lanes 6, 7, 8, and 9,respectively. As shown in lane 6, residual Tc52 could berecovered from the column which was first loaded with L-cysteine(lane 2). In contrast, Tc52 which still bound to the glutathionematrix after the elution with L-methionine (3), L-glycine (4) orGlutamic acid (5), could be efficiently eluted by S-hexylglutathione (lanes 7, 8, 9, respectively). (C) Aliquots of theeluted samples in A (Ia) and B (IIa) (the lanes 1, 2, 3, 4, 5,6, 7, and 8, corresponded to the products eluted and analyzed inlanes 2, 3, 4, 5, 6, 7, and 9, respectively), were separated bySDS-PAGE, transferred to nitrocellulose filters and probed using arabbit immune serum to Tc52 fusion protein (cDNA encoding the Tc52protein, initially named TcAc2, was subcloned in pGEX-2T vector,and the protein was produced in fusion with Schistosoma japonicum  26 kDa glutathione S-transferase (Tc52-Sj26GST)). Thefusion protein and the corresponding antibodies were obtained asdescribed in a previous report [10]. Ib and IIb representcontrol tests using samples analysed in Ia and IIa,respectively, and treated with anti-Sj26GST rabbit serum.Methods: T. cruzi (Y strain) epimastigotes weremaintained in culture medium as described in [10]. Parasitesoluble antigens were prepared and adjusted to 5 mgproteins/ml in buffer (20 mM HEPES, pH 7.25, 1 mM EDTA,O.15 M KCl supplemented with 0.5 mM final concentrationof PMSF), and passed through S-hexyl glutathione affinity matrix.The column was then washed with 30 ml of the same buffer,bound material was eluted using 20 ml of buffer containing2.5 mM S-hexyl glutathione (1), or 1 mg/ml of either ofthe following amino acids: L-cysteine (2), L-methionine (3),L-glycine (4), or L-glutamic acid (5). The columns corresponding tothe lanes 2, 3, 4, and 5 were extensively washed with buffer andloaded with 20 ml of buffer containing 2.5 mM S-hexylglutathione. All the eluates were dialysed against distilledwater and analysed by SDS-PAGE. The proteins were visualizedusing the silver staining method.
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Related In: Results  -  Collection

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Figure 1: (A) Elution profiles of T. cruzi Tc52-bound toS-hexyl glutathione columns with either S-hexyl glutathione (1)or the following amino acids: L-cysteine (2), L-methionine (3),L-glycine (4), glutamic acid (5). (B) After elution as describedin A, the columns from which the eluates analysed in lanes 2, 3,4, and 5 were recovered, washed with the column buffer, andloaded with the same buffer containing S-hexyl glutathione, thecorresponding eluates were analysed in lanes 6, 7, 8, and 9,respectively. As shown in lane 6, residual Tc52 could berecovered from the column which was first loaded with L-cysteine(lane 2). In contrast, Tc52 which still bound to the glutathionematrix after the elution with L-methionine (3), L-glycine (4) orGlutamic acid (5), could be efficiently eluted by S-hexylglutathione (lanes 7, 8, 9, respectively). (C) Aliquots of theeluted samples in A (Ia) and B (IIa) (the lanes 1, 2, 3, 4, 5,6, 7, and 8, corresponded to the products eluted and analyzed inlanes 2, 3, 4, 5, 6, 7, and 9, respectively), were separated bySDS-PAGE, transferred to nitrocellulose filters and probed using arabbit immune serum to Tc52 fusion protein (cDNA encoding the Tc52protein, initially named TcAc2, was subcloned in pGEX-2T vector,and the protein was produced in fusion with Schistosoma japonicum 26 kDa glutathione S-transferase (Tc52-Sj26GST)). Thefusion protein and the corresponding antibodies were obtained asdescribed in a previous report [10]. Ib and IIb representcontrol tests using samples analysed in Ia and IIa,respectively, and treated with anti-Sj26GST rabbit serum.Methods: T. cruzi (Y strain) epimastigotes weremaintained in culture medium as described in [10]. Parasitesoluble antigens were prepared and adjusted to 5 mgproteins/ml in buffer (20 mM HEPES, pH 7.25, 1 mM EDTA,O.15 M KCl supplemented with 0.5 mM final concentrationof PMSF), and passed through S-hexyl glutathione affinity matrix.The column was then washed with 30 ml of the same buffer,bound material was eluted using 20 ml of buffer containing2.5 mM S-hexyl glutathione (1), or 1 mg/ml of either ofthe following amino acids: L-cysteine (2), L-methionine (3),L-glycine (4), or L-glutamic acid (5). The columns corresponding tothe lanes 2, 3, 4, and 5 were extensively washed with buffer andloaded with 20 ml of buffer containing 2.5 mM S-hexylglutathione. All the eluates were dialysed against distilledwater and analysed by SDS-PAGE. The proteins were visualizedusing the silver staining method.
Mentions: Tc52 native protein was eluted from S-hexyl glutathione affinitycolumn by using glutathione (GSH) or cysteine and not other aminoacids including those in GSH (e.g., glutamic acid andglycine) (Figure 1). We have hypothesized that T cellresponse could be affected by Tc52 through its interaction withGSH and cysteine. Indeed, it is interesting to remind that thediverse responses of T cells appear to involve a complex seriesof secondary signals in addition to the triggering of T cellantigen specific receptor. Moreover, it has been shown that theextracellular concentration of cysteine could be a limitingfactor for T cell activation [11]. Indeed, the plasmacysteine concentration is 10 times lower than that of itsoxidized form cystine. In addition, the T cell membrane transportfor cystine is 10 times less efficient than for cysteine. Incontrast, macrophages have the capacity to take up cystineefficiently and release cysteine, therefore increasing thedelivery of this amino acid to T cells which in turn couldincrease their own GSH intracellular level [20].

Bottom Line: In this paper, we review the data concerning the immunoregulatory effects of T. cruzi Tc24 (a B cell activator antigen) and Tc52 (an immunosuppressive protein) released molecules on the host immune system.Moreover, targeted Tc52 replacement allowed the obtention of parasite mutants exhibiting low virulence in vitro and in vivo.Thus, the generation of a complete deficiency state of virulence factors by gene targeting should provide a means to assess the importance of these factors in the pathophysiological processes and disease progression.

View Article: PubMed Central - HTML - PubMed

ABSTRACT
An intense suppression of T cell proliferation to mitogens and to antigens is observed in a large number of parasitic infections. The impairment of T cell proliferation also occurred during the acute phase of Chagas' disease, caused by the intracellular protozoan parasite Trypanosoma cruzi. A wealth of evidence has accumulated that illustrates the ability of T. cruzi released molecules to influence directly a variety of diverse immunological functions. In this paper, we review the data concerning the immunoregulatory effects of T. cruzi Tc24 (a B cell activator antigen) and Tc52 (an immunosuppressive protein) released molecules on the host immune system. The gene targeting approach developed to further explore the biological function(s) of Tc52 molecule, revealed interesting unexpected functional properties. Indeed, in addition to its immunusuppressive activity a direct or indirect involvement of Tc52 gene product alone or in combination with other cellular components in T. cruzi differentiation control mechanisms have been evidenced. Moreover, targeted Tc52 replacement allowed the obtention of parasite mutants exhibiting low virulence in vitro and in vivo. Thus, the generation of a complete deficiency state of virulence factors by gene targeting should provide a means to assess the importance of these factors in the pathophysiological processes and disease progression. It is hoped that such approaches might allow rational design of tools to control T. cruzi infections.

No MeSH data available.


Related in: MedlinePlus