Date of Award
Master of Science
Jeffrey K. Beetham
Leishmania spp. promastigotes that have been cultured in vitro exhibit variable resistance to complement mediated lysis (CML) that is dependent on their stage in development and on the culture growth stage. Leishmania chagasi promastigotes in logarithmic (log) phase of growth are sensitive to CML while promastigotes in the stationary phase of growth are resistant to CML. Two different types of PAGE were utilized to asses serum-exposed log and stationary phase promastigotes for evidence of interaction (i.e. change in protein gel mobility) between abundant L. chagasi surface proteins and serum complement proteins. Analysis by reducing SDS-PAGE and Blue Native PAGE failed to detect any differences in the gel mobility of two abundant parasite surface proteins, major surface protease (MSP) and promastigote surface antigen (PSA), after cell exposure to serum. Similarly, identical isoforms of C5 were observed in log and stationary phase cells that were assessed by both PAGE methods. C9 had identical isoforms in log and stationary phase parasite fractions when resolved with both PAGE systems; however, a low mobility C9 band observed in Blue Native PAGE was stronger in stationary phase cell fractions than in log phase cells. Resolution of C3 with Blue Native PAGE detected multiple bands including one that shifted in mobility between stationary and log phase cells, while SDS-PAGE analysis detected a number of banding differences in log versus stationary cell extracts. In additional experiments using both PAGE techniques, C9 isoforms were determined in serum that was pre-exposed to log or stationary phase cells. These serum-only fractions contained a number of different C9 isoforms that exhibited variability under Blue Native PAGE analysis but not under SDS-PAGE analysis. By Blue Native PAGE, serum pre-exposed to stationary cells contained almost exclusively a largely immobile isoform, serum pre-exposed to log cells contained a lesser amount of that low mobility isoform as well as a number of higher mobility isoforms, and control serum (not pre-exposed to cells) contained almost exclusively higher mobility isoforms that migrated equivalently to those in log-exposed serum but were at higher abundance.
Eric Joseph Scolaro
Scolaro, Eric Joseph, "Analyzing the relationship of Leishmania chagasi and complement resistance through protein-protein interactions, genetic complementation, and innovative techniques" (2009). Graduate Theses and Dissertations. Paper 10944.