Cyclic AMP effectors in African trypanosomes revealed by genome-scale RNA interference library screening for resistance to the phosphodiesterase inhibitor CpdA

Gould, M.K., Bachmaier, S., Ali, J.A.M., Alsford, S., Tagoe, D.N.A., Munday, J.C. , Schnaufer, A.C., Horn, D., Boshart, M. and De Koning, H. (2013) Cyclic AMP effectors in African trypanosomes revealed by genome-scale RNA interference library screening for resistance to the phosphodiesterase inhibitor CpdA. Antimicrobial Agents and Chemotherapy, 57(10), pp. 4882-4893. (doi:10.1128/AAC.00508-13)

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Abstract

One of the most promising new targets for trypanocidal drugs to emerge in recent years is the cyclic AMP (cAMP) phosphodiesterase (PDE) activity encoded by TbrPDEB1 and TbrPDEB2. These genes were genetically confirmed as essential, and a high-affinity inhibitor, CpdA, displays potent antitrypanosomal activity. To identify effectors of the elevated cAMP levels resulting from CpdA action and, consequently, potential sites for adaptations giving resistance to PDE inhibitors, resistance to the drug was induced. Selection of mutagenized trypanosomes resulted in resistance to CpdA as well as cross-resistance to membrane-permeable cAMP analogues but not to currently used trypanocidal drugs. Resistance was not due to changes in cAMP levels or in PDEB genes. A second approach, a genome-wide RNA interference (RNAi) library screen, returned four genes giving resistance to CpdA upon knockdown. Validation by independent RNAi strategies confirmed resistance to CpdA and suggested a role for the identified cAMP Response Proteins (CARPs) in cAMP action. CARP1 is unique to kinetoplastid parasites and has predicted cyclic nucleotide binding-like domains, and RNAi repression resulted in >100-fold resistance. CARP2 and CARP4 are hypothetical conserved proteins associated with the eukaryotic flagellar proteome or with flagellar function, with an orthologue of CARP4 implicated in human disease. CARP3 is a hypothetical protein, unique to Trypanosoma. CARP1 to CARP4 likely represent components of a novel cAMP signaling pathway in the parasite. As cAMP metabolism is validated as a drug target in Trypanosoma brucei, cAMP effectors highly divergent from the mammalian host, such as CARP1, lend themselves to further pharmacological development.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:De Koning, Professor Harry and Gould, Matthew and Horn, Mr David and Munday, Dr Jane
Authors: Gould, M.K., Bachmaier, S., Ali, J.A.M., Alsford, S., Tagoe, D.N.A., Munday, J.C., Schnaufer, A.C., Horn, D., Boshart, M., and De Koning, H.
College/School:College of Medical Veterinary and Life Sciences > Institute of Infection Immunity and Inflammation
Journal Name:Antimicrobial Agents and Chemotherapy
Publisher:American Society for Microbiology
ISSN:0066-4804
ISSN (Online):1098-6596
Copyright Holders:Copyright © 2013 The Authors
First Published:First published in Antimicrobial Agents and Chemotherapy 57(10):4882-4893
Publisher Policy:Reproduced under a Creative Commons License

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
371793The Wellcome Centre for Molecular Parasitology ( Core Support )Andrew WatersWellcome Trust (WELLCOME)073990/Z/03/BIII - PARASITOLOGY