Screening the Leishmania major genome database 30 with amino acid sequences of class I PDE catalytic domains identified five genes that are predicted to code for class I PDEs (Fig. 1). Chromosome 18 contains the single copy gene LmjPDEA (GeneDB identification number: LmjF18.1090) that is predicted to code for a polypeptide of 631 amino acids. Its catalytic domain comprises amino acids 384 – 609 (as defined by the conserved domain database), with no other functional domains discernible. The open reading frame of LmjPDEA was amplified from genomic DNA and sequenced and its sequence was submitted to GenBank under the accession number AY462262.

Chromosome 15 contains a locus containing two tandemly arranged PDE genes, LmjPDEB1 and LmjPDEB2 (Fig 1). The open reading frames of both genes were amplified from genomic DNA and were sequenced and the sequences have been submitted to GenBank under the following accession numbers: LmjPDEB1: AY462264, and LmjPDEB2: AY462263.

The sequencing data, in conjunction with Southern blot and PCR analyses demonstrated that the current version of the L. major database (version of July 15, 2005) contains an assembly error in this region, in that it represents only a single PDE gene that is a chimera between LmjPDEB1 and LmjPDEB2. The genomic organization of the LmjPDEB locus now presented in Fig 1B closely corresponds to that found for the homologous PDE genes in the T. brucei and the T. cruzi genomes. In T. brucei, TbrPDEB1 (old designation: TbPDE2C; Tb09.160.3590) and TbrPDEB2 (old designation: TbPDE2B; Tb09.160.3630) are also tandemly arranged on chromosome 9, with 2379 bp between the two open reading frames, followed by a gene for the small non-histone protein NHP2/RS6 (Fig 1C). In T. cruzi, the two homologues are similarly arranged on chromosome 3, in the sequence TcrPDEB1 (Tc00.1047053508277.100) followed by TcrPDEB2 (old designation TcPDE1; GenBank accession number AAP49573; 24) and by NHP2/RS6.

Expression of the two genes in L. major promastigotes was analyzed by Northern blot analysis and by RT-PCR. When Northern blots of total RNA were hybridized with the respective probes, stable mRNA was detected for both of them (Fig. 2, panel A). Similar results were also obtained using total RNA of L. mexicana promastigotes and amastigotes (Fig. 2, panels B and C), suggesting that the two genes are similarly expressed in both life cycle stages. The 5'-termini of the respective mRNAs in L. major promastigotes were analyzed by RT-PCR amplification using a common, mini-exon specific forward primer and individual gene-specific reverse primers for each gene. For LmjPDEB1, a single type of mRNA was found, with an unusually long 5'-UTR region of 403 nucleotides. For LmjPDB2, two splice variants were identified, with 5'-UTR regions of 273 and 233 nucleotides, respectively (Fig. 3).

The polypeptide chain of LmjPDEA consists of 631 amino acids without any recognizable functional domain, except for the class I PDE catalytic domain at the C-terminus (amino acids 384 – 609).

The open reading frames of LmjPDEB1 (2820 bp) and LmjPDEB2 (2790 bp) code for proteins of 940 and 930 amino acids, respectively. The two share an overall 82 % sequence identity, with a markedly uneven distribution along the polypeptides. The first 230 amino acids are highly diverged (only 34 % amino acid sequence identity), whereas the remainder of the sequence is almost completely conserved (96.6 % sequence identity). The only exception is a stretch of 24 amino acids in the catalytic domain that are entirely dissimilar between LmjPDEB1 and LmjPDEB2 (Fig. 4). Interestingly, similar stretches of divergence within the catalytic domains are also found, at identical positions, in the respective homologues of T. brucei and T. cruzi. LmjPDEB1 and LmjPDEB2 both contain two closely spaced GAF domains 31 in their N-terminal regions (LmjPDEB1: GAF-A amino acids 250-398, GAF-B 422 – 567; LmjPDEB2: GAF-A: 240 – 388, GAF-B: 412 -557). Between the two GAF domains, a putative protein kinase A phosphorylation site is located (KKKS; LmjPDEB1 402 – 404; LmjPDEB2 392 – 395). This is the only protein kinase A site that is also conserved, and found at an identical position, in the T. brucei and T. cruzi homologues.

The catalytic domains of the leishmanial PDEs are highly conserved, and they all contain the signature sequence [HD(LIVMFY)XHX(AG)XXNX(LIVMFY)] that characterizes them as class I PDEs 18. The amino acid sequence identity between the catalytic domains of LmjPDEB1 and LmjPDEB2 and the corresponding regions of the human PDEs 1 – 11 varies between 41.7% (to HsPDE3A) to 48.9% (to HsPDE10A). Based on the publicly available 3D-structures of several human PDE catalytic domains 32, sixteen amino acid residues are absolutely conserved between all human PDEs (Table 1). In only two of these positions, some of the leishmanial PDEs exhibit amino acid replacements, both in positions that are not directly located in the active site. A conserved alanine residue (A⁴⁶⁶ in HsPDE4B) is replaced by G⁷⁴¹, G⁷³¹ and Y⁶⁷³ in LmjPDEB1, LmjPDEB2 and LmjPDEC, respectively. In addition, a conserved histidine residue (H⁴⁷⁸ in HsPDE4B) is replaced by leucine (L⁴⁵⁹ in LmjPDEA) or methionine (M⁶⁸⁵ in LmjPDEC). Of the two residues that confer selectivity for cAMP over cGMP, Q⁴⁴³ and N⁵⁶⁷ in HsPDE4B 33, the glutamine residue is conserved in all leishmanial PDEs. In the position corresponding to N⁵⁶⁷, an asparagine residue is maintained in all leishmanial PDEs except for LmjPDEC. Here the corresponding position is taken by an alanine residue (A⁷⁷⁰ in LmjPDEC). This suggests that LmjPDEC might represent a dual-substrate PDE, as has already been experimentally demonstrated for its T. cruzi homologue, TcrPDEC 22. While the catalytic domains of all five L. major PDEs share a high degree of similarity, the two catalytic domains of LmjPDEB1 and LmjPDEB2 are completely identical, with the exception of a stretch of 24 amino acids that comprises the predicted helices 12 and 13. Similar non-conserved stretches of comparable length and at identical locations are also observed in the homologues of T. brucei and T. cruzi (Fig 4).

Deletion of the two PDE genes ScPDE1 and ScPDE2 from the S. cerevisiae genome renders the mutants highly sensitive to stress conditions such as a heat-shock. Heterologous complementation of this heat-shock phenotype has proven to be a highly sensitive validation procedure for suspected PDE genes 1934. The full-size open reading frames of LmjPDEA, LmjPDEB1 and LmjPDEB2 were cloned into the yeast expression vector pLT1 19 and expressed in the PDE-deficient S. cerevisiae strain PP5 35. Transformants were patched and tested for heat shock resistance (Fig 5). All three PDE genes restored heat shock resistance to the indicator strain, though with different efficiencies. LmjPDEB1 and LmjPDEB2 completely restored the wild-type phenotype, whereas complementation by LmjPDEA was much weaker, as seen at short incubation times after the heat shock. Patches expressing LmjPDEA had only grown weakly when observed after 18 h post-heat-shock incubation at 30°C (Fig. 5B), while patches expressing LmjPDEB1 or LmjPDEB2 already showed vigorous growth after this time. When observed after 36 h of incubation, all three recombinant strains had grown to a similar extent (Fig. 5D). The results of these complementation experiments established that all three genes code for functional PDEs, and they demonstrate that these PDEs can use cAMP as their substrate.

Soluble cell lysates were prepared from yeast strains expressing each of the three PDEs and were assayed for PDE activity. Lysates prepared from the LmjPDEA expressing strain consistently showed no measurable PDE activity. While the reason for this is still unclear and might reflect a trivial technical problem, the observation is in line with the finding that LmjPDEA complements the PDE-deficient yeast strain PP5 much less efficiently than do the two other PDE genes (see Fig. 5). In contrast to LmjPDEA, lysates from yeast strains expressing LmjPDEB1 and LmjPDEB2 showed strong PDE activities. The two enzymes exhibit K_m values of 1 and 7 μM for cAMP, respectively, well within the range of other class I PDEs (Table 2). The apparent K_m for cAMP (0.97 ± 0.09 μM) was not altered by the presence of a 100-fold excess of cGMP (Fig 6A and 6B), nor was V_max (2.56 ± 0.71 nmol/mg lysate/15 min), indicating that LmjPDEB1 and LmjPDEB2 are specific for cAMP, and that their activity is not modulated by cGMP. A 50-fold excess of the reaction product 5'-AMP also did not alter the K_m (Fig 6C), indicating that the PDEs are not subject to marked product inhibition. For all parameters determined, LmjPDEB1 and LmjPDEB2 behaved very similarly, indicating that the small stretch of sequence divergence between the two catalytic domains does not markedly alter their functionality. Also, the divergent N-terminal regions do not seem to affect the kinetics under in vitro conditions.

As an initial survey, a number of commercially available inhibitors that are frequently used in cell biological studies were tested at a 100-fold excess over substrate (100 μM inhibitor vs 1 μM cAMP; Fig. 6D), using extracts from yeast cells expressing the recombinant PDEs. Most of the inhibitors tested (cilostamide, zaprinast, etazolate, dipyridamole, Ro-20-1724, rolipram, isobutyl-methylxanthine (IBMX), 8-methoxymethyl-IBMX, trequinsin, papaverine, milrinone, pentoxifylline, and erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) were essentially inactive, even at the high concentration used in these experiments. Only dipyridamole (IC₅₀ = 29 μM), trequinsin (IC₅₀ = 96 μM) and etazolate caused significant inhibition.

Dipyridamole, etazolate and trequinsin were further tested for their effect on cell proliferation (Fig. 7). Promastigote cultures of L. major or amastigote cultures of L. infantum were grown in the presence of various concentrations of inhibitors, or in the presence of 1 % (v/v) dimethylsulfoxide as a control. All three compounds were strongly inhibitory (IC_{50 dipyridamole} = 44.7 ± 12.2 μM; IC_{50 etazolate} = 57.5 ± 27.0 μM; IC_{50 trequinsin} = 43.6 ± 3.7 μM for promastigotes; and IC_{50 trequinsin} = 10.2 ± 4.2 μM for amastigotes, respectively). All three inhibitors are markedly more potent than the frequently used, wide-spectrum PDE-inhibitor isobutyl-methyl-xanthine (1.03 ± 0.67 mM). The inhibitory effects were independent of cell density and were not reduced upon prolonged incubation of the culturesThese observations indicate that the inhibitors are metabolically stable, and that the PDEs may be of similar importance for proliferation of both promastigotes and amastigotes in culture.

³H-labeled cAMP was purchased from Hartmann Analytics, Braunschweig, Germany (Cat. Nr MT 616, 15-50 Ci/mmol). After purification by ion-exchange chromatography on QAE-Sephadex A25, it was stored frozen at -20°C. Oligonucleotide primers were purchased either from Microsynth, Balgach, Switzerland, or from Qiagen. DNA sequencing was done using the ABI Prism BigDye Terminator v3.0 Cycle Sequencing Ready Reaction Kit (Applied Biosystems). Sequences were analyzed on an ABI 3100 instrument at the Computational and Molecular Population Genetics Lab, University of Bern.

Leishmania major MHRO/IR/75/ER or LV39 promastigote forms were cultivated at 27°C in SDM medium containing 5% heat-inactivated foetal bovine serum 37. The MHRO/IR/75/ER isolate was originally recovered from a patient in Iran, and was obtained through Nicolas Fasel, Dept. of Biochemistry, University of Lausanne. Cell proliferation assays were done in 5 ml cultures to which drugs dissolved in DMSO were added. The final DMSO concentration was 1 % in all cases, and appropriate DMSO controls were always included. At various times, 150 ml aliquots were withdrawn and absorbance was measured at 600 nm in a microtiter plate reader. The correlation between OD₆₀₀ and cell number was strictly linear over at least the range between 3 × 10⁵ and 4 × 10⁷ cells/ml.

The Leishmania genome database 30 was searched with the amino acid sequences of the cAMP-specific PDEs from Trypanosoma brucei TbrPDEA (old nomenclature: TbPDE1; GenBank accession number AAL58095) and TbrPDEB1 (old nomenclature: TbPDE2C; GenBank accession number AAK33016). The genes to be characterized in this study were named LmjPDEA, LmjPDEB1and LmjPDEB2, in accordance with the recently proposed unified nomenclature 36. Based on the database sequences, PCR primers were designed to amplify the open reading frames of LmjPDEA, LmjPDEB1 and LmjPDEB2. Forward primers for LmjPDEA, LmjPDEB1 and LmjPDEB2, respectively were LmPDEAup1 (5'-GTG

GTCGA

GTCGA

GTCGAC

CTA

CTA

Genomic DNA was isolated from L. major promastigotes, digested with the appropriate restriction enzymes, separated on 0.8 % agarose gels and transferred to a positively charged nylon membrane (Roche). Digoxigenin-labelled DNA probes were generated using the PCR DIG probe synthesis kit (Roche). Probes were amplified with the following primers: LmPDEAup5 (5'-TAACCACCCGAAGGAGTACG-3') and LmPDEAlo5 (5'-CTCGGCTACCTGAGAGTTGG-3'), resulting in a fragment of 449 bp specific for LmjPDEA; LmPDEB2up6 (5'-CGTCGGACTGGTACATCCTT-3') and LmPDEB2lo6 (5'-GTTTGCGATCACCATGTACG-3') produced a 312 bp fragment specific for LmjPDEB1; LmPDEBup5 (5'-CTGCATTCTGAGCCGTTACA-3') and LmPDEBlo5 (5'-AATGGTAACGGTCGTCTTCG-3') produced a 394 bp fragment specific for LmjPDEB2. A hybridization probe recognizing both genes, LmjPDEB1 and LmjPDEB2, was amplified by PCR with the primers LmPDEBup6 (5'-CCTGCAGCGTAACAGCATTA-3') and LmPDEBlo6 (5'-GCGAGTCCGTCTTCAGGTAG-3'; fragment length 476 bp). In order to achieve a minimal hybridization background, the DNA templates for the PCR reactions were excised from the respective plasmid vectors by digestion with BamHI and SalI and were purified by gel extraction (QIA quick Gel Extraction Kit, Qiagen). Blots were prehybridized for 6 h in DIG Easy Hyb buffer (Roche) and were hybridized at 42°C overnight in the same buffer containing 20 ng/ml of DIG-labelled probe. High stringency washes were done in 0.5 × SSC, 0.1 % SDS at 68°C twice for 15 min. Hybridization signals were detected with an alkaline phosphatase-conjugated anti-DIG antibody (Roche) and the CDP-Star substrate (Roche) and were visualized on a LAS-1000 Image Reader (Fuji).

Total RNA from L. major promastigotes or L. mexicana amastigotes was denatured in 50 % (v/v) DMSO, 4 % (v/v) deionised glyoxal and 10 mM sodium phosphate, pH 6.85, for 5 min at 50°C and separated on a 1.2 % agarose gel in 10 mM sodium phosphate. RNA was transferred to positively charged nylon membranes (Roche) by capillary force. Prehybridization and hybridization with the DIG-labelled probes were done as described above, but at a hybridisation temperature of 50°C. High stringency washes and hybridisation signal detection were done as described above. A hybridization probe specific for α-tubulin was used to normalize all blots.

The leishmanial PDEs were expressed in the PDE-deficient S. cerevisiae strain PP5 (MATa leu2-3 leu2-112 ura3-52 his3-532 his4 cam pde1::ura3 pde2::HIS3), a gift of John Colicelli (UCLA). The hemagglutinin-tagged, full-length PDE genes were excised from the cloning vectors by digestion with BamHI and SalI, purified by gel extraction and introduced into the yeast expression vector pLT1 19. The pLT1 vector contains a strong TEF 2 promotor, followed by an optimized Kozak sequence, the start codon and a SalI site (5'-CTAAACATG

GTCGAC

The heat-shock assay to detect complementation of the PDE-deficient phenotype of the S. cerevisiae strain PP5 was done exactly as described 19. Single yeast colonies were patched onto YPD plates prewarmed to 55°C, and were incubated for another 15 min at 55°C. Plates were then cooled to room temperature and were incubated at 30°C for 18 – 36 h.

Yeast cell lysis was performed as described by Kunz et al 19 with minor modifications. Briefly, yeast cells grown to mid-log to end-log phase in SC-leu medium were collected, resuspended in the original volume of prewarmed YPD medium, and incubated for an additional 3.5 h at 30°C to maximize protein expression. Cells were then harvested and washed twice in H₂O, and the washed cell pellet was stored overnight at -70°C. For preparing the lysate, the cells were thawed on ice and suspended in ice-cold extraction buffer (50 mM Hepes pH 7.5, 100 mM NaCl, 1 × Complete^® protease inhibitor cocktail without EDTA (Roche)). Cells were lysed by grinding with glass beads (0.45 – 0.50 mm) in 2 ml Sarstedt tubes, using a FastPrep FP120 cell disruptor (3 × 45 s at setting 4). The subsequent centrifugation steps were done exactly as described. To the resulting supernatant, glycerol was added to a final concentration of 15 % (v/v), aliquots were snap-frozen in liquid nitrogen and were stored at -70°C.

PDE activity was determined in 50 mM HEPES, pH 7.5, 0.5 mM EDTA, 10 mM MgCl₂, 50 mg/ml BSA in a final assay volume of 100 μl. Each assay contained 50'000 cpm ³H-labelled cAMP, with unlabeled cAMP added to adjust the desired total substrate concentration. Reactions were run at 30°C and were linear for at least 60 min. The standard reaction time was set to 15 min, and the amount of enzyme was always chosen so that no more than 15 % of the substrate was hydrolyzed. Inhibitor studies were done at a cAMP concentration of 1 μM. Inhibitors were dissolved in DMSO, but the final DMSO concentration in the assays never exceeded 1%. Control reactions with DMSO alone were always included. Reactions were stopped by the addition of 25 μl of 0.5 N HCl. For the subsequent dephosphorylation of the AMP, the stopped reactions were neutralized with 20 μl 1 M Tris base, followed by the addition of 10 μl of calf intestinal alkaline phosphatase (Roche Diagnostics; 1 unit/10 μl). The dephosphorylation reactions were incubated for 15 min at 37°C and were then applied to 1 ml columns of QAE-Sephadex A25 in 30 mM ammonium formiate, pH 6.0. The ³H-adenosine formed during the reaction was eluted with 1.6 ml of 30 mM ammonium formiate, pH 6.0 and was collected into 3.5 ml water-miscible scintillation fluid (Packard Ultima Flo). Assays were always run in triplicates, and at least three independent experiments were performed in every case. Data were analyzed using the GraphPad Prism software package.