Theileria annulata-infected cells produce abundant proteases whose activity is reduced by long-term cell culture

Abstract

Lysates of Theileria annulata-infected bovine lymphoblastoid cells and their uninfected counterparts were tested for protease activity using gelatin substrate SDS-PAGE. The infected cells produced a number of extra activities at pH 8.0 in the presence of Ca2+. Calcium was found to enhance the activities but was not an absolute requirement. Studies using inhibitors, including E64, 3,4-dichloroisocoumarin, pepstatin and 1, 10-phenanthroline suggested that the activities were metalloproteases. We analysed two vaccine lines; the Ode line from India and the Ankara Pendik line from Turkey. In the Ode line the later passage had very much reduced levels of the enzyme activities. In the case of the Ankara Pendik line both stages analysed had very low protease activities, but a reduction from the early to late passage was also observed. The reduction in the level of protease activity was also observed as a gradual process during on-going culture of lines derived from the Hissar and Ode stocks. In the Ode line we demonstrated a parallel decrease in the production of microschizonts upon temperature shift in vitro.

Mechanism(s) of attenuation of Theileria annulata vaccine cell lines

“…The work summarized here demonstrates that the attenuated phenotype is associated with several phenomena, namely changes in the parasite population structure, reduction in MMP activity with concomitant reduction in metastatic potential and reduction in microschizont production ( Baylis et al . 1992 ; Adamson & Hall 1996, 1997; Darghouth et al .…”

“…1994 ; Somerville et al . 1998a ), metalloproteinase profiles were assayed on gelatin substrate gels exactly as detailed by Baylis et al . (1992) .…”

The Detection of Enzyme Activity Following Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis

“…It also should be noted that substrate and cofactor molecules are not the only ones that can be diffused into the polyacrylamide gel to facilitate analysis. Enzyme inhibitors (128) or activators such as Ca 2ϩ (68,91,129) can be used as tools for the identification or characterization of certain enzyme activities. Conversely, the plasminogen-activating enzyme urokinase was detected by electrophoresing urokinasecontaining samples into an SDS-polyacrylamide gel containing a copolymerized substrate (See below), and then diffusing in plasminogen after the electrophoretic separation was complete (130,131).…”

Culture‐Derived Parasites in Vaccination of Cattle against Tick‐Borne Diseases

“…Another mechanism underlying attenuation of continuously cultured T. annulata schizonts was shown when production of metalloproteases by culture‐grown schizonts was diminished as the number of passages of the infected cell lines increased. 26…”