Bruno Kilunga Kubata

A metacaspase of Trypanosoma brucei causes loss of respiration competence and clonal death in the yeast Saccharomyces cerevisiae

Metacaspases constitute a new group of cysteine proteases homologous to caspases. Heterologous expression of Trypanosoma brucei metacaspase TbMCA4 in the budding yeast Saccharomyces cerevisiae resulted in growth inhibition, mitochondrial dysfunction and clonal death. The metacaspase orthologue of yeast, ScMCA1 (YOR197w), exhibited genetic interaction with WWM1 (YFL010c), which encodes a small WW domain protein. WWM1 overexpression resulted in growth arrest and clonal death, which was suppressed by concomitant overexpression of ScMCA1. GFP‐fusion reporters of WWM1, ScMCA1 and TbMCA4 localized to the nucleus. Taken together, we suggest that metacaspases may play a role in nuclear function controlling cellular proliferation coupled to mitochondrial biogenesis.

Identification of a Novel Prostaglandin F2α Synthase in Trypanosoma brucei

Members of the genus Trypanosoma cause African trypanosomiasis in humans and animals in Africa. Infection of mammals by African trypanosomes is characterized by an upregulation of prostaglandin (PG) production in the plasma and cerebrospinal fluid. These metabolites of arachidonic acid (AA) may, in part, be responsible for symptoms such as fever, headache, immunosuppression, deep muscle hyperaesthesia, miscarriage, ovarian dysfunction, sleepiness, and other symptoms observed in patients with chronic African trypanosomiasis. Here, we show that the protozoan parasite T. brucei is involved in PG production and that it produces PGs enzymatically from AA and its metabolite, PGH2. Among all PGs synthesized, PGF2α was the major prostanoid produced by trypanosome lysates. We have purified a novel T. brucei PGF2α synthase (TbPGFS) and cloned its cDNA. Phylogenetic analysis and molecular properties revealed that TbPGFS is completely distinct from mammalian PGF synthases. We also found that TbPGFS mRNA expression and TbPGFS activity were high in the early logarithmic growth phase and low during the stationary phase. The characterization of TbPGFS and its gene in T. brucei provides a basis for the molecular analysis of the role of parasite-derived PGF2α in the physiology of the parasite and the pathogenesis of African trypanosomiasis.

Antichagasic Activity of Komaroviquinone Is Due to Generation of Reactive Oxygen Species Catalyzed by Trypanosoma cruzi Old Yellow Enzyme

ABSTRACT A novel potent trypanocidal diterpene, komaroviquinone, was reduced by Trypanosoma cruzi old yellow enzyme (TcOYE) to its semiquinone radical. The reductase activity in trypanosome lysates was completely immunoabsorbed by anti-TcOYE antibody. Since TcOYE is expressed throughout the T. cruzi life cycle, komaroviquinone is an interesting candidate for developing new antichagasic drugs.

Late Stage Infection in Sleeping Sickness

At the turn of the 19th century, trypanosomes were identified as the causative agent of sleeping sickness and their presence within the cerebrospinal fluid of late stage sleeping sickness patients was described. However, no definitive proof of how the parasites reach the brain has been presented so far. Analyzing electron micrographs prepared from rodent brains more than 20 days after infection, we present here conclusive evidence that the parasites first enter the brain via the choroid plexus from where they penetrate the epithelial cell layer to reach the ventricular system. Adversely, no trypanosomes were observed within the parenchyma outside blood vessels. We also show that brain infection depends on the formation of long slender trypanosomes and that the cerebrospinal fluid as well as the stroma of the choroid plexus is a hostile environment for the survival of trypanosomes, which enter the pial space including the Virchow-Robin space via the subarachnoid space to escape degradation. Our data suggest that trypanosomes do not intend to colonize the brain but reside near or within the glia limitans, from where they can re-populate blood vessels and disrupt the sleep wake cycles.

Cyclical Appearance of African Trypanosomes in the Cerebrospinal Fluid: New Insights in How Trypanosomes Enter the CNS

It is textbook knowledge that human infective forms of Trypanosoma brucei, the causative agent of sleeping sickness, enter the brain across the blood-brain barrier after an initial phase of weeks (rhodesiense) or months (gambiense) in blood. Based on our results using an animal model, both statements seem questionable. As we and others have shown, the first infection relevant crossing of the blood brain border occurs via the choroid plexus, i.e. via the blood-CSF barrier. In addition, counting trypanosomes in blood-free CSF obtained by an atlanto-occipital access revealed a cyclical infection in CSF that was directly correlated to the trypanosome density in blood infection. We also obtained conclusive evidence of organ infiltration, since parasites were detected in tissues outside the blood vessels in heart, spleen, liver, eye, testis, epididymis, and especially between the cell layers of the pia mater including the Virchow-Robin space. Interestingly, in all organs except pia mater, heart and testis, trypanosomes showed either a more or less degraded appearance of cell integrity by loss of the surface coat (VSG), loss of the microtubular cytoskeleton and loss of the intracellular content, or where taken up by phagocytes and degraded intracellularly within lysosomes. This is also true for trypanosomes placed intrathecally into the brain parenchyma using a stereotactic device. We propose a different model of brain infection that is in accordance with our observations and with well-established facts about the development of sleeping sickness.

Novel Characteristics of Trypanosoma brucei Guanosine 5'-monophosphate Reductase Distinct from Host Animals.

The metabolic pathway of purine nucleotides in parasitic protozoa is a potent drug target for treatment of parasitemia. Guanosine 5’-monophosphate reductase (GMPR), which catalyzes the deamination of guanosine 5’-monophosphate (GMP) to inosine 5’-monophosphate (IMP), plays an important role in the interconversion of purine nucleotides to maintain the intracellular balance of their concentration. However, only a few studies on protozoan GMPR have been reported at present. Herein, we identified the GMPR in Trypanosoma brucei, a causative protozoan parasite of African trypanosomiasis, and found that the GMPR proteins were consistently localized to glycosomes in T. brucei bloodstream forms. We characterized its recombinant protein to investigate the enzymatic differences between GMPRs of T. brucei and its host animals. T. brucei GMPR was distinct in having an insertion of a tandem repeat of the cystathionine β-synthase (CBS) domain, which was absent in mammalian and bacterial GMPRs. The recombinant protein of T. brucei GMPR catalyzed the conversion of GMP to IMP in the presence of NADPH, and showed apparent affinities for both GMP and NADPH different from those of its mammalian counterparts. Interestingly, the addition of monovalent cations such as K+ and NH4+ to the enzymatic reaction increased the GMPR activity of T. brucei, whereas none of the mammalian GMPR’s was affected by these cations. The monophosphate form of the purine nucleoside analog ribavirin inhibited T. brucei GMPR activity, though mammalian GMPRs showed no or only a little inhibition by it. These results suggest that the mechanism of the GMPR reaction in T. brucei is distinct from that in the host organisms. Finally, we demonstrated the inhibitory effect of ribavirin on the proliferation of trypanosomes in a dose-dependent manner, suggesting the availability of ribavirin to develop a new therapeutic agent against African trypanosomiasis.

Identification of a Novel Prostaglandin F2{alpha} Synthase in Trypanosoma brucei

Members of the genus Trypanosoma cause African trypanosomiasis in humans and animals in Africa. Infection of mammals by African trypanosomes is characterized by an upregulation of prostaglandin (PG) production in the plasma and cerebrospinal fluid. These metabolites of arachidonic acid (AA) may, in part, be responsible for symptoms such as fever, headache, immunosuppression, deep muscle hyperaesthesia, miscarriage, ovarian dysfunction, sleepiness, and other symptoms observed in patients with chronic African trypanosomiasis. Here, we show that the protozoan parasite T. brucei is involved in PG production and that it produces PGs enzymatically from AA and its metabolite, PGH2. Among all PGs synthesized, PGF2α was the major prostanoid produced by trypanosome lysates. We have purified a novel T. brucei PGF2α synthase (TbPGFS) and cloned its cDNA. Phylogenetic analysis and molecular properties revealed that TbPGFS is completely distinct from mammalian PGF synthases. We also found that TbPGFS mRNA expression and TbPGFS activity were high in the early logarithmic growth phase and low during the stationary phase. The characterization of TbPGFS and its gene in T. brucei provides a basis for the molecular analysis of the role of parasite-derived PGF2α in the physiology of the parasite and the pathogenesis of African trypanosomiasis.

Enzymatic formation of prostaglandin D2, E2, and F2α in the parasitic protozoan Trypanosoma brucei

Abstract African trypanosomiasis (sleeping sickness in humans or nagana in domestic animals) is caused by infection with African trypanosomes and is characterized by a marked increase in prostaglandin (PG) production. Although PGs are known to cause pathogenesis of African trypanosomiasis, little is known about the mechanism of up-regulation of PG production during this disease. In the present study, we investigated the metabolism of arachidonic acid in African trypanosomes and found that by using EIA and gas chromatography mass spectrometry (GC-MS), Trypanosoma brucei produces and secretes PGD2, E2, and F2α. Interestingly, PGs were differentially produced by the different life stages of the parasite. PGF2α was the most abundant PG produced by the cell lysates. Moreover, PG synthase activities were recovered in the cytosolic fraction and were abolished by heating (100 °C, 5 min). Prostaglandin (PG) formation in African trypanosomes is catalyzed by enzymatic systems, which are markedly different from their mammalian counterparts that are localized in the microsomal fraction. The release of PGs from live trypanosomes into the culture media indicates that T. brucei may well produce PGs that are, in part, responsible for the symptoms observed in patients with African trypanosomiasis.

Identification of a Novel Prostaglandin F2αSynthase inTrypanosoma brucei

Members of the genus Trypanosoma cause African trypanosomiasis in humans and animals in Africa. Infection of mammals by African trypanosomes is characterized by an upregulation of prostaglandin (PG) production in the plasma and cerebrospinal fluid. These metabolites of arachidonic acid (AA) may, in part, be responsible for symptoms such as fever, headache, immunosuppression, deep muscle hyperaesthesia, miscarriage, ovarian dysfunction, sleepiness, and other symptoms observed in patients with chronic African trypanosomiasis. Here, we show that the protozoan parasite T. brucei is involved in PG production and that it produces PGs enzymatically from AA and its metabolite, PGH2. Among all PGs synthesized, PGF2α was the major prostanoid produced by trypanosome lysates. We have purified a novel T. brucei PGF2α synthase (TbPGFS) and cloned its cDNA. Phylogenetic analysis and molecular properties revealed that TbPGFS is completely distinct from mammalian PGF synthases. We also found that TbPGFS mRNA expression and TbPGFS activity were high in the early logarithmic growth phase and low during the stationary phase. The characterization of TbPGFS and its gene in T. brucei provides a basis for the molecular analysis of the role of parasite-derived PGF2α in the physiology of the parasite and the pathogenesis of African trypanosomiasis.