TheTrypanosoma bruceicAMP phosphodiesterases TbrPDEBl and TbrPDEB2: flagellar enzymes that are essential for parasite virulence

Abstract

Cyclic nucleotide specific phosphodiesterases (PDEs) are pivotal regulators of cellular signaling. They are also important drug targets. Besides catalytic activity and substrate specificity, their subcellular localization and interaction with other cell components are also functionally important. In contrast to the mammalian PDEs, the significance of PDEs in protozoal pathogens remains mostly unknown. The genome of Trypanosoma brucei, the causative agent of human sleeping sickness, codes for five different PDEs. Two of these, TbrPDEB1 and TbrPDEB2, are closely similar, cAMP-specific PDEs containing two GAF-domains in their N-terminal regions. Despite their similarity, these two PDEs exhibit different subcellular localizations. TbrPDEB1 is located in the flagellum, whereas TbrPDEB2 is distributed between flagellum and cytoplasm. RNAi against the two mRNAs revealed that the two enzymes can complement each other but that a simultaneous ablation of both leads to cell death in bloodstream form trypanosomes. RNAi against TbrPDEB1 and TbrPDEB2 also functions in vivo where it completely prevents infection and eliminates ongoing infections. Our data demonstrate that TbrPDEB1 and TbrPDEB2 are essential for virulence, making them valuable potential targets for new PDE-inhibitor based trypanocidal drugs. Furthermore, they are compatible with the notion that the flagellum of T. brucei is an important site of cAMP signaling.

The flagellum of Trypanosoma brucei: New tricks from an old dog

“…There is precedence for flagellar adenylate kinases as the central pair component cpc1 of C. reinhardtii contains an adenylate kinase domain (Zhang and Mitchell, 2004). Two additional PFR-associated proteins involved in nucleotide metabolism, TbrPDEB1 and TbrPDEB2, were recently identified (Zoraghi and Seebeck, 2002;Oberholzer et al, 2007). These cAMP phosphodiesterases localize to the flagellum, though the majority of TbrPDEB2 localizes to punctuate spots in the cytoplasm (Oberholzer et al, 2007).…”

“…Two additional PFR-associated proteins involved in nucleotide metabolism, TbrPDEB1 and TbrPDEB2, were recently identified (Zoraghi and Seebeck, 2002;Oberholzer et al, 2007). These cAMP phosphodiesterases localize to the flagellum, though the majority of TbrPDEB2 localizes to punctuate spots in the cytoplasm (Oberholzer et al, 2007). Knockdown of flagellar ADKs or PDEBs did not impair growth of procyclic cells, but simultaneous ablation of PDEB1 and 2 was found to be lethal in bloodstream-form parasites (Zoraghi and Seebeck, 2002;Ginger et al, 2005;Oberholzer et al, 2007).…”

“…These cAMP phosphodiesterases localize to the flagellum, though the majority of TbrPDEB2 localizes to punctuate spots in the cytoplasm (Oberholzer et al, 2007). Knockdown of flagellar ADKs or PDEBs did not impair growth of procyclic cells, but simultaneous ablation of PDEB1 and 2 was found to be lethal in bloodstream-form parasites (Zoraghi and Seebeck, 2002;Ginger et al, 2005;Oberholzer et al, 2007). The motility phenotypes of PDEB knockdown mutants have not been described.…”

“…ADK-A Adenylate Kinase (Pullen et al, 2004;Ginger et al, 2005) ADK-B Adenylate Kinase (Pullen et al, 2004;Ginger et al, 2005) PDEB1 cAMP Homeostasis (Oberholzer et al, 2007) PDEB2 cAMP Homeostasis (Oberholzer et al, 2007) PFR1 PFR Structural Protein (Deflorin et al, 1994;Durand-Dubief et al, 2003) PFR2 PFR Structural Protein (Schlaeppi et al, 1989;Bastin et al, 1998) Tryp-CaM Ca 2+ binding (Ridgley et al, 2000) 5.20 PFR component (Woodward et al, 1994) Flagellum Attachment Zone While many additional novel flagellar proteins have been identified through comparative genomics (Baron et al, 2007b) and proteomics (Broadhead et al, 2006), and have been linked to flagellar functions by mutant or RNAi analyses, their precise functions and locations are unknown. For a comprehensive inventory of flagellar proteins in a variety of organisms, the reader is referred to the ciliome database: www.ciliome.com (Inglis et al, 2006 …”

In Vitro and in Vivo Antitrypanosomal Activitiy of Two Microbial Metabolites, KS-505a and Alazopeptin

“…With regard to Trypanosoma brucei species, Zoraghi et al [15], Rascón et al [16], and Zoraghi et al [17] recently reported that T. brucei PDEs (TbPDE2 family: TbPDE2A, TbPDE2B and TbPDE2C) were found to be essential for bloodstream form trypanosome proliferation. Furthermore, Oberholzer et al, reported that TbPDE2B and TbPDE2C, as flagellar enzymes, were found to be essential for parasite virulence [18]. They also suggested that the TbPDE2 family might be considered as new molecular targets for chemotherapeutic antitrypanosomal drugs [15ϳ18].…”