Chlamydia infection of epithelial cells expressing dynamin and Eps15 mutants: clathrin-independent entry into cells and dynamin-dependent productive growth

Chlamydiae enter epithelial cells via a mechanism that still remains to be fully elucidated. In this study we investigated the pathway of entry of C, psittaci GPIC and C, trachomatis LGV/L2 into HeLa cells and demonstrated th at it does not depend on clathrin coated vesicle formation. We used mutant cell lines defective in clathrin-mediated endocytosis due to overexpression of dominant negative mutants of either dynamin I or Eps15 proteins. When c lathrin-dependent endocytosis was inhibited by overexpression of the dyn(K4 4A) mutant of dynamin I (defective in GTPase activity), Chlamydia entry was not affected. However, in these cells there was a dramatic inhibition in t he proliferation of Chlamydia and the growth of the chlamydia vacuole (incl usion), When clathrin-dependent endocytosis was inhibited by overexpression of an Eps15 dominant negative mutant, the entry and growth of Chlamydia wa s unaltered. These results indicate that the effect on the growth of Chlamy dia in the dyn(K44A) cells was not simply due to a deprivation of nutrients taken up by endocytosis, Instead, the dominant-negative mutant of dynamin most likely affects the vesicular traffic between the Chlamydia inclusion a nd intracellular membrane compartments. In addition, cytochalasin D inhibit ed Chlamydia entry by more than 90%, indicating that chlamydiae enter epith elial cells by an actin-dependent mechanism resembling phagocytosis. Finall y, dynamin is apparently not involved in the formation of phagocytic vesicl es containing Chlamydia.