The Trypanosoma brucei cAMP phosphodiesterases TbrPDEB1 and TbrPDEB2: flagellar enzymes that are essential for parasite virulence

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.--Oberholzer, M., Marti, G., Baresic, M., Kunz, S., Hemphill, A., Seebeck, T. The Trypanosoma brucei cAMP phosphodiesterases TbrPDEB1 and TbrPDEB2: flagellar enzymes that are essential for parasite virulence.

Cyclic nucleotide specific phosphodiesterases of Leishmania major

Background Leishmania represent a complex of important human pathogens that belong to the systematic order of the kinetoplastida. They are transmitted between their human and mammalian hosts by different bloodsucking sandfly vectors. In their hosts, the Leishmania undergo several differentiation steps, and their coordination and optimization crucially depend on numerous interactions between the parasites and the physiological environment presented by the fly and human hosts. Little is still known about the signalling networks involved in these functions. In an attempt to better understand the role of cyclic nucleotide signalling in Leishmania differentiation and host-parasite interaction, we here present an initial study on the cyclic nucleotide-specific phosphodiesterases of Leishmania major. Results This paper presents the identification of three class I cyclic-nucleotide-specific phosphodiesterases (PDEs) from L. major, PDEs whose catalytic domains exhibit considerable sequence conservation with, among other, all eleven human PDE families. In contrast to other protozoa such as Dictyostelium, or fungi such as Saccharomyces cerevisiae, Candida ssp or Neurospora, no genes for class II PDEs were found in the Leishmania genomes. LmjPDEA contains a class I catalytic domain at the C-terminus of the polypeptide, with no other discernible functional domains elsewhere. LmjPDEB1 and LmjPDEB2 are coded for by closely related, tandemly linked genes on chromosome 15. Both PDEs contain two GAF domains in their N-terminal region, and their almost identical catalytic domains are located at the C-terminus of the polypeptide. LmjPDEA, LmjPDEB1 and LmjPDEB2 were further characterized by functional complementation in a PDE-deficient S. cerevisiae strain. All three enzymes conferred complementation, demonstrating that all three can hydrolyze cAMP. Recombinant LmjPDEB1 and LmjPDEB2 were shown to be cAMP-specific, with Km values in the low micromolar range. Several PDE inhibitors were found to be active against these PDEs in vitro, and to inhibit cell proliferation. Conclusion The genome of L. major contains only PDE genes that are predicted to code for class I PDEs, and none for class II PDEs. This is more similar to what is found in higher eukaryotes than it is to the situation in Dictyostelium or the fungi that concomitantly express class I and class II PDEs. Functional complementation demonstrated that LmjPDEA, LmjPDEB1 and LmjPDEB2 are capable of hydrolyzing cAMP. In vitro studies with recombinant LmjPDEB1 and LmjPDEB2 confirmed this, and they demonstrated that both are completely cAMP-specific. Both enzymes are inhibited by several commercially available PDE inhibitors. The observation that these inhibitors also interfere with cell growth in culture indicates that inhibition of the PDEs is fatal for the cell, suggesting an important role of cAMP signalling for the maintenance of cellular integrity and proliferation.

Crystal structure of the Leishmania major phosphodiesterase LmjPDEB1 and insight into the design of the parasite-selective inhibitors

Human leishmaniasis is a major public health problem in many countries, but chemotherapy is in an unsatisfactory state. Leishmania major phosphodiesterases (LmjPDEs) have been shown to play important roles in cell proliferation and apoptosis of the parasite. Thus LmjPDE inhibitors may potentially represent a novel class of drugs for the treatment of leishmaniasis. Reported here are the kinetic characterization of the LmjPDEB1 catalytic domain and its crystal structure as a complex with 3-isobutyl-1-methylxanthine (IBMX) at 1.55 A resolution. The structure of LmjPDEB1 is similar to that of human PDEs. IBMX stacks against the conserved phenylalanine and forms a hydrogen bond with the invariant glutamine, in a pattern common to most inhibitors bound to human PDEs. However, an extensive structural comparison reveals subtle, but significant differences between the active sites of LmjPDEB1 and human PDEs. In addition, a pocket next to the inhibitor binding site is found to be unique to LmjPDEB1. This pocket is isolated by two gating residues in human PDE families, but constitutes a natural expansion of the inhibitor binding pocket in LmjPDEB1. The structure particularity might be useful for the development of parasite-selective inhibitors for the treatment of leishmaniasis.