Evasion of the alternative complement pathway by metacyclic trypomastigotes of Trypanosoma cruzi: dependence on the developmentally regulated synthesis of surface protein and N-linked carbohydrate.
Epimastigotes (EPI) of Trypanosoma cruzi are highly sensitive to lysis in fresh normal human serum by the alternative complement pathway (ACP). In contrast, metacyclic trypomastigotes (CMT) derived from EPI in stationary culture fail to activate the ACP and are thus resistant to serum-mediated lysis. To investigate the nature of the parasitic surface molecules which enable infective metacyclic trypomastigotes to evade the ACP, CMT were treated with a variety of different proteolytic and glycosidic enzymes, and their sensitivity to ACP-dependent lysis was tested. Pretreatment with pronase was found to cause a near complete reversal in the resistance of CMT to serum lysis, whereas trypsin or chymotrypsin induced smaller increases in complement sensitivity. Similarly, pretreatment with N-glycanase or neuraminidase also partially abrogated the resistance of CMT to ACP-dependent lysis. The effect of these enzymes on susceptibility to complement-mediated lysis was paralleled in increased C3 and C9 deposition on the organism. In addition, electrophoretic analysis of parasite-bound C3 indicated that the hemolytically inactive fragment, iC3b, was the major form of the molecule on CMT, while the hemolytically active fragment, C3b, predominated on pronase-treated CMT. Furthermore, when C3 was deposited on the parasite surface by means of purified ACP components, 80% of C3b on pronase-pretreated CMT but only 14% of the C3b on CMT bound the amplification protein factor B with high affinity, a prerequisite for efficient ACP activation. When cultured at 37 degrees C after pronase treatment, CMT gradually regained their resistance to ACP-mediated lysis. This process was blocked if puromycin, cycloheximide, or tunicamycin were included in the culture medium. The above findings suggest that evasion of the ACP by CMT is dependent on the developmentally regulated synthesis of protein as well as N-linked carbohydrate chains. A stage-specific 90,000 to 115,000 m.w. glycoprotein doublet present on the surface of CMT was shown to be uniquely sensitive to pronase digestion. Thus, this complex, which is also recognized by a CMT-specific monoclonal antibody, may be the glycoprotein component responsible for control of ACP activation
D000818:Animals; D000953:Antigens, Protozoan / Q000276:immunology*; D000954:Antigens, Surface / Q000276:immunology; D003167:Complement Activation; D003176:Complement C3 / Q000378:metabolism; D003186:Complement C9 / Q000378:metabolism; D003170:Complement Pathway, Alternative; D003513:Cycloheximide / Q000494:pharmacology; D006023:Glycoproteins / Q000096:biosynthesis / Q000276:immunology*; D006026:Glycoside Hydrolases; D008565:Membrane Proteins / Q000276:immunology; D008970:Molecular Weight; D009439:Neuraminidase; D010447:Peptide Hydrolases; D014349:Trypanosoma cruzi / Q000276:immunology*; D014415:Tunicamycin / Q000494:pharmacology
D000953:Antigens, Protozoan; D000954:Antigens, Surface; D003176:Complement C3; D003186:Complement C9; D006023:Glycoproteins; D008565:Membrane Proteins; D014415:Tunicamycin; D003513:Cycloheximide; D006026:Glycoside Hydrolases; C020668:glycanase; D009439:Neuraminidase; D010447:Peptide Hydrolases