cAMP signalling in the kinetoplastid protozoa.

Several species of kinetoplastid protozoa cause major human infectious diseases. Trypanosoma cruzi is responsible for the fatal Chagas disease in large parts of South America, the various species of Leishmania cause a number of different human diseases with millions of patients world-wide, and the African trypanosome Trypanosoma brucei is the agent of human sleeping sickness, a disastrously re-emerging epidemic of fatal infections in Sub-Saharan Africa. Chemotherapy of all of these infections is in a very unsatisfactory state. cAMP signalling pathways in humans have provided interesting drug targets for a number of clinical conditions, from asthma to impotency. Similarly, cAMP signalling in kinetoplastids might offer useful targets for the development of novel antiparasitic drugs, which makes their exploration an urgent need. Current knowledge suggests that cAMP signalling proceeds along very similar pathways in all kinetoplastid pathogens (T. cruzi, the Leishmanias and T. brucei). Their adenylyl cyclases are structurally very different from the human enzymes and appear to function as enzyme-linked cell surface receptors. They might represent the major sensory apparatus of the kinetoplastids, guiding much of their environmental sensing and host/parasite interaction. The cAMP-specific phosphodiesterases of the kinetoplastids are rather similar to those of human cells and might function in similar ways. Essentially nothing is known on downstream effectors of cAMP in the kinetoplastids. Homologues of protein kinase A and its regulatory subunits have been identified, but their biochemical properties seem to be disctinct from that of mammalian protein kinase A.

The regulatory subunit of a cGMP-regulated protein kinase A of Trypanosoma brucei.

This study reports the identification and characterization of the regulatory subunit, TbRSU, of protein kinase A of the parasitic protozoon Trypanosoma brucei. TbRSU is coded for by a single copy gene. The protein contains an unusually long N-terminal domain, the pseudosubstrate site involved in binding and inactivation of the catalytic subunit, and two C-terminally located, closely spaced cyclic nucleotide binding domains. Immunoprecipitation of TbRSU coprecipitates a protein kinase activity with the characteristics of protein kinase A: it phosphorylates a protein kinase specific substrate, and it is strongly inhibited by a synthetic protein kinase inhibitor peptide. Unexpectedly, this kinase activity could not be stimulated by cAMP, but by cGMP only. Binding studies with recombinant cyclic nucleotide binding domains of TbRSU confirmed that both domains bind cGMP with Kd values in the lower micromolar range, and that up to a 100-fold excess of cAMP does not compete with cGMP binding.

Genetic variants of the TbAT1 adenosine transporter from African trypanosomes in relapse infections following melarsoprol therapy.

We have analyzed the TbAT1 gene, which codes for the P2 adenosine transporter, from Trypanosoma brucei field isolates to investigate a possible link between the presence of mutations in this gene and melarsoprol treatment failure. Of 65 T. b. gambiense isolates analyzed from a focus in north-western Uganda with high treatment failure rates following melarsoprol therapy, 38 had a mutated TbAT1. Unexpectedly, all individual isolates contained the same set of nine mutations in their TbAT1 genes. Of these, five point mutations resulted in amino acid substitutions, one resulted in the deletion of an entire codon, and three were silent point mutations. Eight of these mutations had previously been reported in a laboratory-derived Cymelarsan-resistant T. b. brucei clone. Identical sets of mutations were also found in a drug-resistant T.b.rhodesiense isolate from south-eastern Uganda and in a T.b.gambiense isolate from a relapsing patient from northern Angola. A deletion of the TbAT1 gene was found in a single T. b. gambiense isolate from a relapsing patient from northern Angola. The data presented demonstrate the surprising finding that trypanosomes from individual relapse patients of one area, as well as from geographically distant localities, contain an identical set of point mutations in the transporter gene TbAT1. They further demonstrate that many isolates from relapse patients contained the wild-type TbAT1 genes, suggesting that melarsoprol refractoriness is not solely due to a mutational inactivation of TbAT1.

Drug resistance in Trypanosoma brucei spp., the causative agents of sleeping sickness in man and nagana in cattle.

Drug resistance in pathogenic trypanosomes threatens successful control of fatal sleeping sickness in man and hinders economic livestock production in sub-Saharan Africa. We report on the occurrence and development of drug resistance, and discuss the genetic basis of such resistance in Trypanosoma brucei. Understanding these mechanisms at the molecular level will enable improved management of existing drugs and provide valuable clues to the development of new trypanocides.

Melarsoprol refractory T. b. gambiense from Omugo, north-western Uganda.

Culture adapted T. b. gambiense isolated from Northwest Uganda were exposed to 0.001-0.14 microg/ml melarsoprol or 1.56-100 microg/ml DL-alpha-difluoromethylornithine (DFMO). Minimum inhibitory concentrations (MICs) of each drug were scored for each isolate after a period of 10 days drug exposure. The results indicate that T. b. gambiense isolates from Northwest Uganda had elevated MIC values for melarsoprol ranging from 0.009 to 0.072 microg/ml as compared with T. b. gambiense isolates from Cote d'Ivoire with MIC values ranging from 0.001 to 0.018 microg/ml or with T. b. rhodesiense from Southeast Uganda with MIC values from 0.001 to 0.009 microg/ml. All MIC values obtained fell below expected peak melarsoprol concentrations in serum of treated patients. However, it may not be possible to maintain constant drug concentrations in serum of patients as was the case in our in vitro experiments. Importantly, the MIC of 0.072 microg/ml exhibited by one of the isolates from Northwest Uganda was above levels attainable in CSF indicating that this isolate would probably not be eliminated from CSF of treated patients. PCR amplification of the gene encoding the P2-like adenosine transporter followed by restriction digestion with Sfa NI enzyme revealed presence of fragments previously observed in a trypanosome clone with laboratory-induced arsenic resistance. From our findings it appears that reduced drug susceptibility may be one factor for the frequent relapses of sleeping sickness after melarsoprol treatment occurring in Northwest Uganda.

cAMP signalling in Trypanosoma brucei.

Cyclic AMP was the first second messenger to be identified. After five decades of research, much is currently known about its biological functions and clinical implications. Several components of the cAMP signalling pathways, such as the G-protein coupled receptors and the phosphodiesterases, have become sensitive and specific drug targets for a host of clinical applications. Surprisingly, very little effort has been invested so far into the study of cAMP signalling in parasites, and its significance in host/parasite interaction. Our laboratory has embarked on a study of cAMP signalling in Trypanosoma brucei. A newly identified adenylyl cyclase, GRESAG4.4B, a member of a small family of closely related genes, is being used as a model molecule for investigating the mechanisms which control cyclase activity in the T. brucei cell. On the other hand, a number of genes for different families of cAMP-specific phosphodiesterases have been identified and characterised. One enzyme, TbPDE1, is coded for by a single-copy gene. Knock-outs of this gene display an almost normal phenotype in culture, indicating that TbPDE1 is not an essential enzyme under culture conditions. A second phosphodiesterase which is being studied in detail, TbPDE2A, is clearly different from TbPDE1, and it is coded for by a member of a small gene family containing about six similar, but non-identical genes. TbPDE2A, as TbPDE1, is specific for cAMP. In its N-terminal, it contains a GAF domain which may represent an allosteric cGMP-binding site. The other members of the TbPDE2 family all exhibit strongly conserved catalytic domains, but vary widely in their N-terminal regulatory domains. With regard to downstream signalling by the cAMP generated through the interplay of adenylyl cyclases and phosphodiesterases, we have recently identified a single-copy gene (TbRSU1) which codes for a putative regulatory subunit of the cAMP-regulated protein kinase A. This protein exhibits considerable similarity with its mammalian counterparts. Immunoprecipitation co-precipitates a protein kinase activity with the characteristics of protein kinase A.

Spontaneous dimerization and leucine-zipper induced activation of the recombinant catalytic domain of a new adenylyl cyclase of Trypanosoma brucei, GRESAG4.4B.

In this study, we describe the isolation and characterization of a new adenylyl cyclase from Trypanosoma brucei and its activation by dimerization of the catalytic domain. In agreement with the current nomenclature of trypanosomal adenylyl cyclases, this new gene is termed GRESAG4.4B. The complete ORF of the GRESAG4.4B gene encodes a protein of 1291 amino acids. Its predicted protein structure is consistent with the structure of other trypanosomal cyclases, and with the cyclases of L. donovani. GRESAG 4.4B is constitutively expressed during the life cycle of trypanosomes. GRESAG4.4B is a member of a gene family, which contains at least six members, which are all clustered on chromosome IV. The catalytic domain of GRESAG4.4B is able to dimerize spontaneously. However, these spontaneously formed, stable dimers only show minimal enzymatic activity. The addition of a leucine zipper (LZ) derived from the S. cerevisiae GCN 4 gene to the N-terminus of the catalytic domain of GRESAG4.4B strongly activated its enzymatic activity. The LZ appears to enforce a distinct conformation of the dimer, which leads to an increased enzymatic activity, and thus may mimic the effect of ligand-induced dimerization of adenylyl cyclase in vivo.

Characterization of TbPDE2A, a novel cyclic nucleotide-specific phosphodiesterase from the protozoan parasite Trypanosoma brucei.

This study reports the identification and characterization of a cAMP-specific phosphodiesterase from the parasitic hemoflagellate Trypanosoma brucei. TbPDE2A is a class I phosphodiesterase. Its catalytic domain exhibits 30-40% sequence identity with those of all 11 mammalian phosphodiesterase (PDE) families, as well as with PDE2 from Saccharomyces cerevisiae, dunce from Drosophila melanogaster, and regA from Dictyostelium discoideum. The overall structure of TbPDE2A resembles that of human PDE11A in that its N-terminal region contains a single GAF domain. This domain is very similar to those of the mammalian PDE2, -5, -6, -10, and -11, where it constitutes a potential cGMP binding site. TbPDE2A can be expressed in S. cerevisiae, and it complements an S. cerevisiae PDE deletion strain. Recombinant TbPDE2A is specific for cAMP, with a K(m) of approximately 2 micrometer. It is entirely resistant to the nonselective PDE inhibitor 3-isobutyl-1-methylxanthine, but it is sensitive to trequinsin, dipyridamole, sildenafil, and ethaverine with IC(50) values of 5.4, 5.9, 9.4, and 14.2 micrometer, respectively. All four compounds inhibit proliferation of bloodstream form trypanosomes in culture, indicating that TbPDE2A is an essential enzyme.

Drug resistance in Trypanosoma brucei spp.; the causative agents of sleeping sickness in man and nagana in cattle

Drug resistance in pathogenic trypanosomes threatens successful control of fatal sleeping sickness in man and hinders economic livestock production in sub-Saharan Africa. We report on the occurrence and development of drug resistance; and discuss the genetic basis of such resistance in Trypanosoma brucei. Understanding these mechanisms at the molecular level will enable improved management of existing drugs and provide valuable clues to the development of new trypanocides

Nucleoside diphosphate kinase of Trypanosoma brucei.

Nucleoside diphosphate kinase (NDPK) is a highly conserved, multifunctional enzyme. Its originally described function is the phosphorylation of nucleoside diphosphates to the corresponding triphosphates, using ATP as the phosphate donor and a high-energy phosphorylated histidine residue as the reaction intermediate. More recently, a host of additional functions of NDPK have been discovered. Some of these correlate with the capacity of NDPK to transphosphorylate other proteins, in a manner reminiscent of bacterial two-component systems. Other functions may be mediated by direct DNA-binding of NDPK. This study describes the identification of NDPK from the parasitic protozoon Trypanosoma brucei. The genome of this major disease agent contains a single gene for NDPK. The predicted amino acid sequence of the trypanosomal enzyme is highly conserved with respect to all other species. The protein is constitutively expressed and is present in procyclic and in bloodstream forms. Immunofluorescence and immuno-electron microscopy demonstrate that trypanosomal NDPK (TbNDPK) is predominantly localized in the cell nucleus. Histidine phosphorylation of TbNDPK is essentially resistant to the experimental compound LY266500, a potent inhibitor of histidine phosphorylation of trypanosomal succinyl coenzyme A synthase.

Infection of organotypic slice cultures from rat central nervous tissue with Trypanosoma brucei brucei.

We recently described a new procedure to grow nervous tissue as organotypic culture. The main feature of these slice cultures is to maintain a well preserved, three-dimensional organisation of the central nervous tissue. As these cultures can be kept for several weeks (up to three months), we have used this in vitro approach to study the complex interactions between host tissue and parasites during late stages of cerebral African trypanosomiasis. Light and electron microscopical studies, as well as electrophysiological recordings demonstrate that the structure and function of the nervous tissue is not severely affected even after several weeks of trypanosome infection. The presence of a large number of parasites does not seem to be deleterious to neuronal survival. Secondly, most of the trypanosomes are located around the periphery of the nervous tissue, but many of them also penetrate into the nervous parenchyma. Thirdly, trypanosomes with well-conserved morphology are found within the cytoplasm of glial cells, which in some cases were identified as astrocytes. These "intracellular parasites" seem to actively invade the target cells. Our study demonstrates that the presence of proliferating trypanosomes does not per se interfere with the neural activity of CNS tissues. Secondly, it provides, to the best of our knowledge, the first in vitro demonstration of intracellular forms of African trypanosomes.

Cyclic AMP signaling in trypanosomatids.

Curative interference with signal transduction pathways is a spectacularly successful concept in many domains of modern pharmacology; indeed, the 'wonder drug' Viagra is but a humble inhibitor of a cyclic GMP (cGMP)-specific phosphodiesterase and, thus, interferes with cGMP-signaling in a strategic organ. In fact, about half of the 100 most successful drugs currently on the market act through modulating cellular signal transduction. Despite these encouraging findings, signal transduction pathways as potential drug targets in trypanosomatids have remained largely unexplored. However, what little is known indicates that adenylyl cyclases of trypanosomatids, and probably other enzymes of the cyclic nucleotide signaling pathways, are significantly different from their mammalian counterparts. Here, Christina Naula and Thomas Seebeck summarize what is known about cAMP signal transduction in trypanosomatids.

Histidine-phosphorylation of succinyl CoA synthetase from Trypanosoma brucei.

The insect form of Trypanosoma brucei depends on respiration for its energy requirements. It contains a fully functional mitochondrion with a complete citric acid cycle. Most of its enyzmes have been characterized to date. The current study presents the characterization of the histidine phosphorylation activity of one of the few remaining enzymes, succinyl CoA synthetase. The trypanosomal enyzme was identified by partial purification, followed by direct protein sequencing. It is rapidly phosphorylated, presumably through auto-phosphorylation, using either ATP or GTP as phosphate donors. The phosphorylation occurs exclusively on histidine residues. The histidine-bound phosphate can be donated to suitable phosphate acceptors in a rapid reaction. This phosphotransfer reaction is highly nucleotide selective, as only ADP, but none of the other nucleoside-diphosphates tested, can be used as a phosphate acceptor.

Inhibition of succinyl CoA synthetase histidine-phosphorylation in Trypanosoma brucei by an inhibitor of bacterial two-component systems.

Recent drug screenings for new antibacterial drugs directed against histidine phospho-relay signalling pathways in bacteria have resulted in compounds which potently inhibit the histidine kinase activity of bacterial two-component systems. The present study demonstrates that one of these compounds, LY266500, is also a potent inhibitor of histidine phosphorylation in the unicellular eukaryotic parasite Trypanosoma brucei, both in vitro and in whole cells. In vitro, it inhibits histidine phosphorylation of mitochondrial succinyl CoA synthetase. LY26650 does not interfere with the phosphotransfer from the histidine-phosphorylated protein to ADP. In standardized cell culture tests, LY266500 potently inhibits the proliferation of the human pathogens T. brucei rhodesiense and Leishmania donovani. Since the inhibitory activity in vivo is life-cycle stage specific and correlates well with the mitochondrial activity in the different stages, the effect of LY266500 is most likely due to its specific inhibition of the mitochondrial succinyl CoA synthetase.

Phosphorylation of a major GPI-anchored surface protein of Trypanosoma brucei during transport to the plasma membrane.

The surface coat of procyclic forms of Trypanosoma brucei consists of related, internally repetitive glycoproteins known as EP and GPEET procyclins. Previously we showed that the extracellular domain of GPEET is phosphorylated. We now show that phosphorylation of this glycosylphosphatidylinositol-anchored surface protein can be induced in vitro using a procyclic membrane extract. Using antibodies that recognize either the phosphorylated or unphosphorylated form of GPEET, we analyzed their expression during differentiation of bloodstream forms to procyclic forms. Unphosphorylated GPEET, together with EP, was detected in cell lysates 2-4 hours after initiating differentiation whereas phosphorylated GPEET only appeared after 24 hours. Surface expression of EP and both forms of GPEET occurred after 24-48 hours and correlated with the detection of phosphorylated GPEET on immuno-blots. Electron micrographs showed that unphosphorylated GPEET was predominantly in the flagellar pocket whereas the phosphorylated form was distributed over the cell surface. In contrast, expression of a membrane-bound human placental alkaline phosphatase in procyclic forms caused the accumulation of dephosphorylated GPEET on the cell surface, while the phosphorylated form was restricted to the flagellar pocket. A GPEET-Fc fusion protein, which was retained intracellularly, was not phosphorylated. We propose that unphosphorylated GPEET procyclin is transported to a location close to or at the cell surface, most probably the flagellar pocket, where it becomes phosphorylated. To the best of our knowledge, this study represents the first localization of phosphorylated and unphosphorylated forms of a GPI-anchored protein within a cell.

The Trypanosoma brucei autoantigen I/6 is an internally repetitive cytoskeletal protein.

Self-reactive host antibodies were shown earlier to exhibit strong and specific cross-reactivity to a particular trypanosomal antigen, protein I/6. The current study presents the molecular characterization of protein I/6. The major structural component of the cell body cytoskeleton of Trypanosoma brucei is a cagelike array of tightly connected microtubules which is in close contact to the overlaying cell membrane. Many of the unususal properties of the cytoskeleton of trypanosomes are due to the proteins associated with these microtubules. Protein I/6 was now shown to be a microtubule-associated protein, and it may be involved in crosslinking microtubules. Protein I/6 is coded for by a single gene, representing an exception rather than the rule for trypanosomal gene organization. From this single gene, two distinct mRNAs are generated through differential splicing. They differ in their polyadenylation sites, but both code for an identical polypeptide sequence of 33 kDa. Protein I/6 contains a non-functional EF-hand calcium binding domain and a domain of six tandemly arranged repeat units of eight amino acids length.

Identification of a profilin homologue in Trypanosoma brucei by complementation screening.

Using genetic complementation in Saccharomyces cerevisiae, we have isolated a Trypanosoma brucei gene encoding profilin. Overexpression of trypanosome profilin suppresses defects that are associated with the loss of the C-terminal domain of the adenylyl cyclase-associated protein in S. cerevisiae. Similarly, the T. brucei gene complements a profilin-deletion mutant of S. cerevisiae. The full-length cDNA clone isolated contains an open reading frame of 150 amino acids, with a predicted molecular mass of 16.1 kDa. The gene appears to be present at single copy and is expressed at approximately equal levels in both mammalian and insect forms of the parasite.

Trypanosome variant surface glycoproteins are recognized by self-reactive antibodies in uninfected hosts.

The variant surface glycoproteins (VSGs) of African trypanosomes form a dense surface coat on the bloodstream parasites. VSGs are immunodominant antigens that stimulate a rapid antibody response in trypanosome-infected individuals. In the present study, we examined VSG-specific antibodies present not only in sera from infected individuals but also in sera from individuals that had never been exposed to trypanosomes. Native antibodies against different VSGs were detected in sera from uninfected mice, bovines, and humans; the antibodies were revealed to be exclusively directed against variable determinants of the antigens. Further experimentation demonstrated that such native antibodies immunoreact with cellular components of mice and thus are most likely produced by the self-reactive B-cell compartment of the murine immune system.