Effects on growth, hemoglobin metabolism and paralogous gene expression resulting from disruption of genes encoding the digestive vacuole plasmepsins of Plasmodium falciparum J. Alfredo Bonilla a , Pedro A. Moura b , Tonya D. Bonilla a , Charles A. Yowell a , David A. Fidock b , John B. Dame a, * a Department of Infectious Diseases and Pathology, University of Florida, PO Box 110880, 2015 SW 16th Ave., Gainesville, FL 32611-0880, USA b Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461, USA Received 7 September 2006; received in revised form 7 November 2006; accepted 14 November 2006 Abstract Four of the plasmepsins of Plasmodium falciparum are localised in the digestive vacuole (DV) of the asexual blood stage parasite (PfPM1, PfPM2, PfPM4 and PfHAP), and each of these aspartic proteinases has been successfully targeted by gene disruption. This study describes further characterisation of the single-plasmepsin knockout mutants, and the creation and characterisation of double- plasmepsin knockout mutants lacking complete copies of pfpm2 and pfpm1 or pfhap and pfpm2. Double-plasmepsin knockout mutants were created by transfecting pre-existing knockout mutants with a second plasmid knockout construct. PCR and Southern blot analysis demonstrate the integration of a large concatamer of each plasmid construct into the targeted gene. All mutants have been characterised to assess the involvement of the DV plasmepsins in sustaining growth during the asexual blood stage. Analyses reaffirmed that knockout mutants Dpfpm1 and Dpfpm4 had lower replication rates in the asexual erythrocytic stage than the parental line (Dd2), but double-plasm- epsin knockout mutants lacking intact copies of either pfpm2 and pfpm1, or pfpm2 and pfhap, had normal growth rates compared with Dd2. The amount of crystalline hemozoin produced per parasite during the asexual cycle was measured in each single-plasmepsin knock- out to estimate the effect of each DV plasmepsin on hemoglobin digestion. Only Dpfpm4 had a statistically significant reduction in hemo- zoin accumulation, indicating that hemoglobin digestion was impaired in this mutant. In the single-plasmepsin knockouts, no statistically significant differences were found in the steady state levels of mRNA from the remaining intact DV plasmepsin genes. Disruption of a DV plasmepsin gene does not affect the accumulation of mRNA encoding the remaining paralogous plasmepsins, and Western blot anal- ysis confirmed that the accumulation of the paralogous plasmepsins in each knockout mutant was similar among all clones examined. Ó 2006 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. Keywords: Plasmepsin; Aspartic proteinase; Gene knockout; Transfection; Malaria; Plasmodium; Hemoglobin 1. Introduction Asexual development of Plasmodium falciparum occurs within the human erythrocyte where the parasite con- sumes most of the host cell hemoglobin for protein bio- synthesis (Krugliak et al., 2002), osmotic stability (Lew et al., 2003, 2004) and room for growth (Ginsburg, 1996). Because the digestion of hemoglobin is presum- ably an essential catabolic function performed by the blood stage parasites, the proteinases participating in this pathway have been proposed as targets for the develop- ment of novel antimalarial drugs (Olliaro and Goldberg, 1995; Rosenthal, 1998; Rosenthal et al., 2002). Protein- ases of several mechanistic classes are found within the digestive vacuole (DV) including aspartic proteinases, plasmepsins PfPM1, PfPM2, PfHAP and PfPM4 (Berry et al., 1999; Banerjee et al., 2002; Egan, 2002; Omara- Opyene et al., 2004), three cysteine proteinases, falcipains 0020-7519/$30.00 Ó 2006 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2006.11.008 * Corresponding author. Tel.: +1 352 392 4700x5818; fax: +1 352 392 4700. E-mail address: damej@mail.vetmed.ufl.edu (J.B. Dame). www.elsevier.com/locate/ijpara International Journal for Parasitology 37 (2007) 317–327