Proline incorporation into protein by Rickettsia prowazekii during growth in Chinese hamster ovary (CHO-K1) cells

Abstract

Both the requirement of Rickettsia prowazekii for the amino acid proline for growth and rickettsial proline incorporation were determined in Chinese hamster ovary (CHO-K1) cells auxotrophic for proline. Incubation of cells in Dulbecco modified Eagle medium supplemented with various concentrations of proline resulted in a range of host intracellular proline pools, as determined by both dansylation and equilibration of specific radioactivities. Maximal rickettsial growth was observed only in host cells with an intracellular proline pool of 1.0 mM or greater. Protein synthesis by rickettsiae in infected cells was determined to be the difference between emetine-resistant proline incorporation in the presence and absence of chloramphenicol. After density gradient centrifugation in Percoll, a rickettsial band with associated radioactivity was observed in lysates of infected cells treated with emetine but not in lysates of infected cells treated with both emetine and chloramphenicol. The average amount of proline incorporated into protein in situ was determined to be 6.3 +/- 0.8 amol per rickettsia. These results, obtained with a system which allows the study of rickettsiae in their natural habitat, are discussed in light of existing information about protein synthesis in isolated rickettsiae.