Major cysteine peptidases of Entamoeba histolytica are required for aggregation and digestion of erythrocytes but are dispensable for phagocytosis and cytopathogenicity

Cysteine peptidases of Entamoeba histolytica (EhCPs) are considered to be important pathogenicity factors. It has been described that under standard axenic culture conditions, only three ( ehcp-a1 , ehcp-a2 and ehcp-a5 ) out of approximately 50 cysteine peptidase genes present in the E. histolytica genome are substantially expressed, thus representing the set of major EhCPs. In this study, transcriptional silencing of the major peptidase genes was used to characterize their physiological role in more detail. Analysing the transfectants a fourth major cysteine peptidase activity belonging to EhCP-A7 could be characterized. Neither cytopathic activity nor phagocytosis of erythrocytes was altered in CP-inactivated amoebae. However, a significant difference in haemolytic activity was observed. EhCP-A1 and EhCP-A7 apparently had no influence on haemolytic activity, whereas transfectants silenced for ehcp-a5 as well as those silenced for all major peptidases showed a significant reduction in their haemolytic activity. Furthermore, cells silenced for ehcp-a1 and ehcp-a7 and more effectively cells silenced in all major ehcp s were impaired in digesting of phagocytosed erythrocytes. Moreover, amoebae silenced for all major peptidase genes lost the ability to form aggregates of erythrocytes prior to phagocytosis.