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The Trypanosoma cruzi trans-sialidase, through its COOH-terminal tandem repeat, upregulates interleukin 6 secretion in normal human intestinal microvascular endothelial cells and peripheral blood mononuclear cells.

Saavedra E, Herrera M, Gao W, Uemura H, Pereira MA - J. Exp. Med. (1999)

Bottom Line: Most interesting, HIMECs infected with a trypomastigote population expressing trans-sialidase effectively released IL-6, but did not upon infection with the counterpart trypomastigote population expressing low trans-sialidase levels.The unique findings presented here suggest that trans-sialidase is a major inducer of IL-6 secretion in T. cruzi infection, independently of immune cell activation.Such IL-6 secretion might underlie some features of Chagas's disease, such as pyrexia, neuroprotection, and fibrosis, and might result in the undermining of normal acquired immunity against T. cruzi.

View Article: PubMed Central - PubMed

Affiliation: Parasitology Research Center, Department of Pathology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.

ABSTRACT
The Trypanosoma cruzi trans-sialidase can sensitize mice to become highly susceptible to T. cruzi invasion, through mechanisms that remain unknown. In pursuing this observation, we found that purified trans-sialidase induces the selective release of biologically active interleukin (IL)-6 in naive human intestinal microvascular endothelial cells (HIMECs), peripheral blood mononuclear cells (PBMCs), and bladder carcinoma cells. The trans-sialidase action was independent of its catalytic activity, as demonstrated with a genetically engineered trans-sialidase mutant, an enzymatically active polypeptide, and cocultures of PBMCs with epimastigotes and trypomastigotes. Instead, the trans-sialidase action was reproduced with a recombinant COOH-terminal tandem repeat and with synthetic peptides modeled on the tandem repeat. Most interesting, HIMECs infected with a trypomastigote population expressing trans-sialidase effectively released IL-6, but did not upon infection with the counterpart trypomastigote population expressing low trans-sialidase levels. IL-6 is a key factor in the regulation and symptom formation of infection caused by several types of viruses, such as HIV and influenza A virus. However, the function of IL-6 in protozoan and other parasitic diseases remains unclear. The unique findings presented here suggest that trans-sialidase is a major inducer of IL-6 secretion in T. cruzi infection, independently of immune cell activation. Such IL-6 secretion might underlie some features of Chagas's disease, such as pyrexia, neuroprotection, and fibrosis, and might result in the undermining of normal acquired immunity against T. cruzi.

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The catalytic activity of TS does not mediate IL-6 release in PBMCs and T-24 cells. Diagram of TS (Silvio strain, clone 7F; see reference 22) and of TS-154 and TS-H32 constructs (derived from Y strain, clones 154 and 121, respectively; see reference 29). NU-17, -18, -19, and -20 refer to primers used to make the constructs (see Materials and Methods). H6 refers to the 6xHis tag. Insert shows the sequence comparison between TS-154 and TS-H32 in the BglII-PstI fragment of TS. The sequence of TS-H32 is identical to the sequence of TS-154 except where indicated with the amino acid letter code. (B) Catalytic activity for each TS construct. (C) IL-6 secretory activity of TS and its respective derivatives for PBMCs. Similar results were obtained with T-24 cells.
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Figure 4: The catalytic activity of TS does not mediate IL-6 release in PBMCs and T-24 cells. Diagram of TS (Silvio strain, clone 7F; see reference 22) and of TS-154 and TS-H32 constructs (derived from Y strain, clones 154 and 121, respectively; see reference 29). NU-17, -18, -19, and -20 refer to primers used to make the constructs (see Materials and Methods). H6 refers to the 6xHis tag. Insert shows the sequence comparison between TS-154 and TS-H32 in the BglII-PstI fragment of TS. The sequence of TS-H32 is identical to the sequence of TS-154 except where indicated with the amino acid letter code. (B) Catalytic activity for each TS construct. (C) IL-6 secretory activity of TS and its respective derivatives for PBMCs. Similar results were obtained with T-24 cells.

Mentions: Construct TS-154 is derived from enzymatically active trans-sialidase clone 154 of the Y strain of T. cruzi 29. The NH2 terminus of clone 154 was amplified by PCR using primers NU-17 (27 mer, 5′-GCCCATGGCACCCGATCGAGCCGAGTT-3′) and NU-18 (20 mer, 5′-CGGAATTTTCATCACCAATG-3′) with restriction sites shown in Fig. 4 A. NU-17 was designed to introduce starting ATG codon just before the NH2 terminus of mature TS protein, and a NcoI site for subcloning. The PCR product of NU-17 and NU-18 was treated with NcoI and BglII, and subcloned into the NcoI and BamHI sites of pET-21d (Novagen). Most of the 12 amino acid repeats and hydrophobic region at the COOH terminus were removed by PCR using primers NU-19 (24 mer, 5′-GTTCCGAACGGTTTGAAGTTTGCG-3′) and NU-20 (25 mer, 5′-CTGTCGACGGGAGTTGAGGGCGTAC-3′). NU-20 corresponds to the partial sequence of the TR and, in addition, contains a SalI site. The PCR product with the minimum numbers of repeats (i.e., five repeats) was selected and used to replace the original COOH terminus of clone 154 at unique MluI site (see Fig. 4 A). The DNA fragment with unique BamHI-SalI sites was ligated to the pET-21d plasmid containing the NH2-terminal region of TS using the BamHI and XhoI sites of the vector, to yield construct TS-154 (see Fig. 4 A). To generate construct TS-H32, the BglII-PstI DNA fragment of pTS-154 was replaced with the corresponding fragment from the gene 121 29. The trans-sialidase gene 121 is catalytically inactive due to a single amino acid difference in the BglII-PstI fragment, with histidine (H374) replacing tyrosine in the catalytically active TS gene 154 29. Thus, construct TS-H32 should be enzymatically inactive, as determined experimentally (see Fig. 4 B). All constructs were verified by automated sequencing (ABI Perkin Elmer) using BigDye terminator. Production and purification of TS-154 and TS-H32 proteins were identical to the method described above for the CD construct.


The Trypanosoma cruzi trans-sialidase, through its COOH-terminal tandem repeat, upregulates interleukin 6 secretion in normal human intestinal microvascular endothelial cells and peripheral blood mononuclear cells.

Saavedra E, Herrera M, Gao W, Uemura H, Pereira MA - J. Exp. Med. (1999)

The catalytic activity of TS does not mediate IL-6 release in PBMCs and T-24 cells. Diagram of TS (Silvio strain, clone 7F; see reference 22) and of TS-154 and TS-H32 constructs (derived from Y strain, clones 154 and 121, respectively; see reference 29). NU-17, -18, -19, and -20 refer to primers used to make the constructs (see Materials and Methods). H6 refers to the 6xHis tag. Insert shows the sequence comparison between TS-154 and TS-H32 in the BglII-PstI fragment of TS. The sequence of TS-H32 is identical to the sequence of TS-154 except where indicated with the amino acid letter code. (B) Catalytic activity for each TS construct. (C) IL-6 secretory activity of TS and its respective derivatives for PBMCs. Similar results were obtained with T-24 cells.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2195715&req=5

Figure 4: The catalytic activity of TS does not mediate IL-6 release in PBMCs and T-24 cells. Diagram of TS (Silvio strain, clone 7F; see reference 22) and of TS-154 and TS-H32 constructs (derived from Y strain, clones 154 and 121, respectively; see reference 29). NU-17, -18, -19, and -20 refer to primers used to make the constructs (see Materials and Methods). H6 refers to the 6xHis tag. Insert shows the sequence comparison between TS-154 and TS-H32 in the BglII-PstI fragment of TS. The sequence of TS-H32 is identical to the sequence of TS-154 except where indicated with the amino acid letter code. (B) Catalytic activity for each TS construct. (C) IL-6 secretory activity of TS and its respective derivatives for PBMCs. Similar results were obtained with T-24 cells.
Mentions: Construct TS-154 is derived from enzymatically active trans-sialidase clone 154 of the Y strain of T. cruzi 29. The NH2 terminus of clone 154 was amplified by PCR using primers NU-17 (27 mer, 5′-GCCCATGGCACCCGATCGAGCCGAGTT-3′) and NU-18 (20 mer, 5′-CGGAATTTTCATCACCAATG-3′) with restriction sites shown in Fig. 4 A. NU-17 was designed to introduce starting ATG codon just before the NH2 terminus of mature TS protein, and a NcoI site for subcloning. The PCR product of NU-17 and NU-18 was treated with NcoI and BglII, and subcloned into the NcoI and BamHI sites of pET-21d (Novagen). Most of the 12 amino acid repeats and hydrophobic region at the COOH terminus were removed by PCR using primers NU-19 (24 mer, 5′-GTTCCGAACGGTTTGAAGTTTGCG-3′) and NU-20 (25 mer, 5′-CTGTCGACGGGAGTTGAGGGCGTAC-3′). NU-20 corresponds to the partial sequence of the TR and, in addition, contains a SalI site. The PCR product with the minimum numbers of repeats (i.e., five repeats) was selected and used to replace the original COOH terminus of clone 154 at unique MluI site (see Fig. 4 A). The DNA fragment with unique BamHI-SalI sites was ligated to the pET-21d plasmid containing the NH2-terminal region of TS using the BamHI and XhoI sites of the vector, to yield construct TS-154 (see Fig. 4 A). To generate construct TS-H32, the BglII-PstI DNA fragment of pTS-154 was replaced with the corresponding fragment from the gene 121 29. The trans-sialidase gene 121 is catalytically inactive due to a single amino acid difference in the BglII-PstI fragment, with histidine (H374) replacing tyrosine in the catalytically active TS gene 154 29. Thus, construct TS-H32 should be enzymatically inactive, as determined experimentally (see Fig. 4 B). All constructs were verified by automated sequencing (ABI Perkin Elmer) using BigDye terminator. Production and purification of TS-154 and TS-H32 proteins were identical to the method described above for the CD construct.

Bottom Line: Most interesting, HIMECs infected with a trypomastigote population expressing trans-sialidase effectively released IL-6, but did not upon infection with the counterpart trypomastigote population expressing low trans-sialidase levels.The unique findings presented here suggest that trans-sialidase is a major inducer of IL-6 secretion in T. cruzi infection, independently of immune cell activation.Such IL-6 secretion might underlie some features of Chagas's disease, such as pyrexia, neuroprotection, and fibrosis, and might result in the undermining of normal acquired immunity against T. cruzi.

View Article: PubMed Central - PubMed

Affiliation: Parasitology Research Center, Department of Pathology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.

ABSTRACT
The Trypanosoma cruzi trans-sialidase can sensitize mice to become highly susceptible to T. cruzi invasion, through mechanisms that remain unknown. In pursuing this observation, we found that purified trans-sialidase induces the selective release of biologically active interleukin (IL)-6 in naive human intestinal microvascular endothelial cells (HIMECs), peripheral blood mononuclear cells (PBMCs), and bladder carcinoma cells. The trans-sialidase action was independent of its catalytic activity, as demonstrated with a genetically engineered trans-sialidase mutant, an enzymatically active polypeptide, and cocultures of PBMCs with epimastigotes and trypomastigotes. Instead, the trans-sialidase action was reproduced with a recombinant COOH-terminal tandem repeat and with synthetic peptides modeled on the tandem repeat. Most interesting, HIMECs infected with a trypomastigote population expressing trans-sialidase effectively released IL-6, but did not upon infection with the counterpart trypomastigote population expressing low trans-sialidase levels. IL-6 is a key factor in the regulation and symptom formation of infection caused by several types of viruses, such as HIV and influenza A virus. However, the function of IL-6 in protozoan and other parasitic diseases remains unclear. The unique findings presented here suggest that trans-sialidase is a major inducer of IL-6 secretion in T. cruzi infection, independently of immune cell activation. Such IL-6 secretion might underlie some features of Chagas's disease, such as pyrexia, neuroprotection, and fibrosis, and might result in the undermining of normal acquired immunity against T. cruzi.

Show MeSH
Related in: MedlinePlus