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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.
Highlights
Human African trypanosomiasis (HAT, or sleeping sickness) is a potentially lethal parasitic disease caused by two subspecies of Trypanosoma brucei, T. brucei rhodesiense and T. brucei gambiense, which have distinct geographical distributions [1]
In order to further dissect the mode of action of CpdA in T. brucei brucei, as well as to identify potential modes of resistance to tetrahydrophthalazinones, cells resistant to CpdA were selected
The cyclic AMP (cAMP) level in the wild-type and resistant R0.8 populations increase indicating that resistance is not caused by mutated PDEs or adapted PDE expression
Summary
Human African trypanosomiasis (HAT, or sleeping sickness) is a potentially lethal parasitic disease caused by two subspecies of Trypanosoma brucei, T. brucei rhodesiense and T. brucei gambiense, which have distinct geographical distributions [1]. In T. brucei brucei, a crucial role of cyclase activity, encoded by the most abundant ESAG4 gene product and probably other members of the large family of adenylate cyclases, is to produce extracellular cAMP as part of the parasite’s ability to subvert the host innate immunity upon infection [14]. Simultaneous RNA interference (RNAi) knockdown of both PDEB genes in T. brucei brucei bloodstream forms generated an uncontrolled and sustained increase in cAMP concentration, resulting in cytokinesis defects producing multinuclear and multiflagellated cells that eventually die [18]. A similar impaired-cytokinesis phenotype is produced by repression of adenylate cyclase activity [21] This apparent paradox suggests that fine-tuning of cAMP levels plays a role in regulation of cell division, with extreme or deregulated concentrations in either direction being detrimental (see discussion in reference 21)
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