Eimeria necatrix: Induced proteolytic sensitivity of infected chick crypt cells

Abstract

While devising a protocol for the isolation of chick crypt cells infected with Eimeria necatrix, it was observed that infected cells were readily lysed by 0.25% trypsin. Time-course studies at 17 C with 5.5 × 10 5 cells at 96 hr postinfection revealed that 0.001% trypsin effectively lysed >90% of infected cells within 10 min. Uninfected crypt cells were not lysed under these conditions. To determine the site of action of trypsin, the plasma membrane proteins from trypsin-treated and untreated infected cells were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. While the major proteins were unaffected by the trypsin treatment, some minor changes were noted: (1) three components (∼-60, ∼-52, and ∼-20 KDa) were trypsin sensitive and (2) a new band (∼-42 KDa) appeared in the membrane of trypsin-treated infected cells. Previously, it was found that the plasma membrane of infected cells, in contrast to uninfected cells, accumulated gel-phase lipid (J. E. Thompson, M. A. Fernando, and J. Pasternak, Biochimica et Biophysica Acta 555, 472–487, 1979). Here, it was examined whether trypsin would perturb the physical state of the plasma membrane of infected cells. Both X-ray diffraction patterns and transition temperature studies revealed no difference between membranes from untreated and trypsin-treated infected cells. Thus, “trypsin sensitivity” may be a secondary phenomenon that is due primarily to the cellular leakiness that accompanies the accumulation of gel-phase lipid in the plasma membrane of infected cells. The uptake of trypsin may stimulate the release of catabolic enzymes that, consequently, lyse an infected cell.