Degradation of human immunoglobulins and cytotoxicity on HeLa cells by live <i>Trichomonas vaginalis</i>

, Min, D Y; , Ryu, J S; , Park, S Y; , Shin, M H; , Cho, W Y

doi:1997.35.1.39

Korean J Parasito > Volume 35(1):1997 > Article

Original Article


Korean J Parasitol. 1997 Mar;35(1):39-46. English. Published online Mar 20, 1997. http://dx.doi.org/10.3347/kjp.1997.35.1.39
Copyright © 1997 by The Korean Society for Parasitology


Degradation of human immunoglobulins and cytotoxicity on HeLa cells by live Trichomonas vaginalis
D Y Min,¹J S Ryu,^*¹S Y Park,¹M H Shin,² and W Y Cho¹
¹Department of Parasitology, College of Medicine, Hanyang University, Seoul 133-791, Korea.

Received February 28, 1997; Accepted March 03, 1997.
Abstract
The present study was undertaken to determine whether live T. vaginalis degrades human secretory IgA, serum IgA and IgG molecules. Human immunoglobulins were exposed to live trophozoites, parasite lysate, and excretory-secretory product (ESP) of T. vaginalis. To determine the fragmentation of immunoglobulins, the reaction sample was subjected to SDS-PAGE and EITB, and peroxidase conjugated antihuman IgA and IgG were used as probes. Live trophozoites degraded secretory IgA. Serum IgA and IgG, and degradation were pressed forward by the prolongation of the incubation time and by increasing the number of trichomonads respectively. Also the lysates and ESP of trichomonads degraded IgA and IgG. The cysteine and serine proteinase inhibitors such as E-64, antipain, iodoacetic acid, iodoacetamide, TLCK reduced the ability of cleaving immunoglobulins. The proteinase activity and cytotoxicity of T. vaginalis to HeLa cells were decreased when live T. vaginalis was treated with metallo-proteinase inhibitor as well as cysteine and serine proteinase inhibitors. These results suggest that proteinase secreted from live T. vaginalis may play a part role in host pathogenesis by T. vaginalis, and the cleaving ability of host immunoglobulins by the proteinase may contribute as a one of immune evasion mechanism for parasite survival in the host.

Figures


	Fig. 1 Degradation of secretory IgA by live T.vaginalis. Live T. vaginalis digest secretory IgA in a dose-dependent manner. Lane 1, secretory IgA alone; Lane 2-5, secretory IgA incubated with 2 × 10⁵, 5 × 10⁵, 2 × 10⁵ and 5 × 10⁶ of T. vaginalis. (▸; heavy chain)


	Fig. 2 Degradation of serum IgA by live T. vaginalis. Live T. vaginalis digest serum IgA in a time-dependent and dose-dependent manner. Lane 1, serum IgA alone; Lane 2-4, serum IgA incubated with T. vaginalis (2 × 10⁶) for 30 min, 1 hr and 2 hrs. Lane 5-8 serum IgA incubated for 2 hrs with 5 × 10⁴, 2 × 10⁵, 5 × 10⁵ and 1.5 × 10⁶ of T. vaginalis. (▸; heavy chain)


	Fig. 3 Degradation of serum IgG by live T. vaginalis. Live T. vaginalis digest serum IgG in a time-dependent and dose-dependent manner. Lane 1, serum IgG alone; Lane 2-4, serum IgG incubated with T. vaginalis (2 × 10⁶) for 30 min, 1 hr, 2 hrs and 22 hrs. Lane 6-9 serum IgG incubated for 2 hrs with 5 × 10⁴, 2 × 10⁵, 5 × 10⁵ and 1.5 × 10⁶ of T. vaginalis. (▸; heavy chain)


	Fig. 4 Degradation of human immunoglobulins by T. vaginalis. lysate (50 µg) and excretory-secretory product (ESP) (50 µg). A: Degradation of secretory IgA by T. vaginalis lysate (lane 2) and ESP (lane 3). B: Degradation of serum IgA by T. vaginalis lysate (lane 2) and ESP (lane 3). C: Degradation of IgG by T. vaginalis lysate (lane 2) and ESP (lane 3).


	Fig. 5 Degradation of secretory IgA by live T. vaginalis treated with following proteinase inhibitors. Lane 1, secretory IgA alone; Lane 2, DTT; Lane 3, bestatin; Lane 4, EDTA; Lane 5, TLCK; Lane 6, E-64, Lane 7, leupeptin; Lane 8, antipain; Lane 9, IAA; Lane 10, iodoacetamide. (▸; heavy chain)

Tables


	Table 1 Effect of proteinase inhibitor on the cytotoxicity of Trichomonas vaginalis to HeLa cells


	Table 2 Effect of proteinase inhibitor on the of proteinase activity of Trichomonas vaginalis

References


1.	Alderete JF, Pearlman E. Pathogenic Trichomonas vaginalis cytotoxicity to cell culture monolayers. Br J Vener Dis 1984;60(2):99–105.

2.	Alderete JF, Garza GE. Specific nature of Trichomonas vaginalis parasitism of host cell surfaces. Infect Immun 1985;50(3):701–708.

3.	Alderete JF, Kasmala L, Metcalfe E, Garza GE. Phenotypic variation and diversity among Trichomonas vaginalis isolates and correlation of phenotype with trichomonal virulence determinants. Infect Immun 1986;53(2):285–293.

4.	Alderete JF, Demes P, Gombosova A, Valent M, Fabusova M, Janoska A, Stefanovic J, Arroyo R. Specific parasitism of purified vaginal epithelial cells by Trichomonas vaginalis. Infect Immun 1988;56(10):2558–2562.

5.	Alderete JF, Neale KA. Relatedness of structures of a major immunogen in Trichomonas vaginalis isolates. Infect Immun 1989;57(6):1849–1853.

6.	Alderete JF, Newton E, Dennis C, Neale KA. Antibody in sera of patients infected with Trichomonas vaginalis is to trichomonad proteinases. Genitourin Med 1991;67(4):331–334.

7.	Alderete JF, Newton E, Dennis C, Neale KA. The vagina of women infected with Trichomonas vaginalis has numerous proteinases and antibody to trichomonad proteinases. Genitourin Med 1991;67(6):469–474.

8.	Arroyo R, Alderete JF. Trichomonas vaginalis surface proteinase activity is necessary for parasite adherence to epithelial cells. Infect Immun 1989;57(10):2991–2997.

9.	Auriault C, et al. Immunol Lett 1980;2:135–139.

*10.*	Auriault C, Ouaissi MA, Torpier G, Eisen H, Capron A. Proteolytic cleavage of IgG bound to the Fc receptor of Schistosoma mansoni schistosomula. Parasite Immunol 1981;3(1):33–44.

*11.*	Carmona C, Dowd AJ, Smith AM, Dalton JP. Cathepsin L proteinase secreted by Fasciola hepatica in vitro prevents antibody-mediated eosinophil attachment to newly excysted juveniles. Mol Biochem Parasitol 1993;62(1):9–17.

*12.*	Chapman CB, Mitchell GF. Proteolytic cleavage of immunoglobulin by enzymes released by Fasciola hepatica. Vet Parasitol 1982;11(2-3):165–178.

*13.*	Coombs GH, North MJ. An analysis of the proteinases of Trichomonas vaginalis by polyacrylamide gel electrophoresis. Parasitology 1983;86(Pt 1):1–6.

*14.*	Dailey DC, Chang TH, Alderete JF. Characterization of Trichomonas vaginalis haemolysis. Parasitology 1990;101(Pt 2):171–175.

*16.*	Keene WE, Hidalgo ME, Orozco E, McKerrow JH. Entamoeba histolytica: correlation of the cytopathic effect of virulent trophozoites with secretion of a cysteine proteinase. Exp Parasitol 1990;71(2):199–206.

*17.*	Kelsall BL, Ravdin JI. Degradation of human IgA by Entamoeba histolytica. J Infect Dis 1993;168(5):1319–1322.

*18.*	Kong Y, Chung YB, Cho SY, Kang SY. Cleavage of immunoglobulin G by excretory-secretory cathepsin S-like protease of Spirometra mansoni plerocercoid. Parasitology 1994;109(Pt 5):611–621.

*19.*	Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227(5259):680–685.

*20.*	Lambin P. Reliability of molecular weight determination of proteins by polyacrylamide gradient gel electrophoresis in the presence of sodium dodecyl sulfate. Anal Biochem 1978;85(1):114–125.

*21.*	Lockwood BC, North MJ, Coombs GH. Trichomonas vaginalis, Tritrichomonas foetus, and Trichomitus batrachorum: comparative proteolytic activity. Exp Parasitol 1984;58(3):245–253.

*22.*	Lockwood BC, North MJ, Scott KI, Bremner AF, Coombs GH. The use of a highly sensitive electrophoretic method to compare the proteinases of trichomonads. Mol Biochem Parasitol 1987;24(1):89–95.

*23.*	Min DY, Leem MH, Kim JM, Choi YK. [Comparative antigen analysis of Trichomonas vaginalis by enzyme-linked immunoelectrotransfer blot technique]. Korean J Parasitol 1992;30(4):323–328.

*24.*	Min DY, Ryu JS, Hyun KH. [Characterization of the partially purified proteinase from Trichomonasvaginalis]. Korean J Parasitol 1996;34(1):49–57.

*25.*	North MJ. Microbiol Rev 1982;46:308–340.

*26.*	North MJ, Coombs GH, Barry JD. A comparative study of the proteolytic enzymes of Trypanosoma brucei, T. equiperdum, T. evansi, T. vivax, Leishmania tarentolae and Crithidia fasciculata. Mol Biochem Parasitol 1983;9(2):161–180.

*28.*	Provenzano D, Alderete JF. Analysis of human immunoglobulin-degrading cysteine proteinases of Trichomonas vaginalis. Infect Immun 1995;63(9):3388–3395.

*29.*	Song CY, Chappell CL. Purification and partial characterization of cysteine proteinase from Spirometra mansoni plerocercoids. J Parasitol 1993;79(4):517–524.

*31.*	Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 1979;76(9):4350–4354.

*32.*	Tsang VC, Peralta JM, Simons AR. Enzyme-linked immunoelectrotransfer blot techniques (EITB) for studying the specificities of antigens and antibodies separated by gel electrophoresis. Methods Enzymol 1983;92:377–391.