Poster Presentation Lorne Infection and Immunity 2014

Utilising novel compounds to study cyclic AMP-dependent phosphorylation of Apical Membrane Antigen 1: a key event in red blood cell invasion by Plasmodium falciparum (#151)

Katherine L Harvey 1 2 , Brittany L Howard 3 , Melissa J Buskes 4 , Nicole Ramsey 5 , Mauro Azevedo 1 , Philip E Thompson 3 , David T Manallack 3 , David JD Wilson 4 , Belinda M Abbot 4 , Lonny R Levin 5 , Jochen Buck 5 , Paul R Gilson 1 3 , Brendan S Crabb 1 2 3
  1. Burnet Institute, Melbourne, VIC, Australia
  2. University of Melbourne, Parkville, VIC, Australia
  3. Monash University, Clayton, VIC, Australia
  4. LaTrobe University, Melbourne, VIC, Australia
  5. Cornell University, New York, NY, United States of America

Entry into host red blood cells by Plasmodium falciparum merozoites entails a complex sequence of receptor ligand interactions and signal transduction events. Apical Membrane Antigen 1 (AMA1) is an essential membrane spanning ligand with a critical pre-invasion role. Previous work has shown that the cytoplasmic domain of AMA1 is phosphorylated at residue serine 610, which is important for efficient red blood cell invasion, however currently the specific role of phosphorylation remains unclear. Here we explore the opportunity to utilise novel compounds as functional probes for S610 phosphorylation by manipulating the putative constituents within the preceeding signal cascade. Protein kinase A, a 3’5’-cyclic AMP (cAMP) -dependent kinase, has been shown indirectly to phosphorylate S610 of AMA1. cAMP levels in parasites are regulated through the action of adenylyl cyclases, which catalyse the production of cAMP from ATP, and phosphodiesterases, which degrade cAMP into 5’-AMP. Both adenylyl cyclase β (ACβ) and phosphodiesterase β (PDEβ) are co-transcribed with PKA and AMA1 and are essential for blood stage infection thus are the most likely candidates to act within this signal cascade.

Aims:

We have tested 4 novel compounds designed to bind and block the PKA catalytic subunit and 3 compunds designed against ACβ, all of which should inhibit phosphorylation of S610, and one new phosphodiesterase inhibitor which may stimulate phosphorylation of S610. Here we aimed to (a) provide direct evidence that S610 is phosphorylated by PKA and (b) assess the potential of these compounds to target S610 phosphorylation. In addition to providing new biological insight into the mechanisms by which malaria parasites establish infection, this work may pave the way for the optimisation of these compounds as novel therapeutics against malaria.