Replacement of the essential Dictyostelium Arp2 gene by its Entamoeba homologue using parasexual genetics

Background: Cell motility is an essential feature of the pathogenesis and morbidity of amoebiasis caused by Entamoeba histolytica. As motility depends on cytoskeletal organisation and regulation, a study of the molecular components involved is key to a better understanding of amoebic pathogenesis. However, little is known about the physiological roles, interactions and regulation of the proteins of the Entamoeba cytoskeleton. Results: We have established a genetic strategy that uses parasexual genetics to allow essential Dictyostelium discoideum genes to be manipulated and replaced with modified or tagged homologues. Our results show that actin related protein 2 ( Arp2) is essential for survival, but that the Dictyostelium protein can be complemented by E. histolytica Arp2, despite the presence of an insertion of 16 amino acids in an otherwise highly conserved protein. Replacement of endogenous Arp2 with myc- tagged Entamoeba or Dictyostelium Arp2 has no obvious effects on growth and the protein incorporates effectively into the Arp2/ 3 complex. Conclusion: We have established an effective two- step method for replacing genes that are required for survival. Our protocol will allow such genes to be studied far more easily, and also allows an unambiguous demonstration that particular genes are truly essential. In addition, cells in which the Dictyostelium Arp2 has been replaced by the Entamoeba protein are potential targets for drug screens.