Transport proteins determine drug sensitivity and resistance in a protozoan parasite, Trypanosoma brucei

Drug resistance in pathogenic protozoa is very often caused by changes to the ‘transportome’ of the parasites. In Trypanosoma brucei, several transporters have been implicated in uptake of the main classes of drugs, diamidines and melaminophenyl arsenicals. The resistance mechanism had been thought to be due to loss of a transporter known to carry both types of agents: the aminopurine transporter P2, encoded by the gene TbAT1. However, although loss of P2 activity is well-documented as the cause of resistance to the veterinary diamidine diminazene aceturate (Berenil®), cross-resistance between the human-use arsenical melarsoprol and the diamidine pentamidine (MPXR) is the result of loss of a separate High Affinity Pentamidine Transporter (HAPT1). A genome-wide RNAi library screen for resistance to pentamidine, published in 2012, gave the key to the genetic identity of HAPT1 by linking the phenomenon to a locus that contains the closely related T. brucei aquaglyceroporin genes TbAQP2 and TbAQP3. Further analysis determined that knockdown of only one pore, TbAQP2, produced the MPXR phenotype. TbAQP2 is an unconventional aquaglyceroporin with unique residues in the “selectivity region” of the pore, and it was found that in several MPXR lab strains the WT gene was either absent or replaced by a chimeric protein, recombined with parts of TbAQP3. Importantly, wild-type AQP2 was also absent in field isolates of T. b. gambiense, correlating with the outcome of melarsoprol treatment. Expression of a wild-type copy of TbAQP2 in even the most resistant strain completely reversed MPXR and re-introduced HAPT1 function and transport kinetics. Expression of TbAQP2 in Leishmania mexicana introduced a pentamidine transport activity indistinguishable from HAPT1. Although TbAQP2 has been shown to function as a classical aquaglyceroporin it is now clear that it is also a high affinity drug transporter, HAPT1. We discuss here a possible structural rationale for this remarkable ability

The drugs of sleeping sickness: their mechanisms of action and resistance, and a brief history

With the incidence of sleeping sickness in decline and genuine progress being made towards the WHO goal of eliminating sleeping sickness as a major public health concern, this is a good moment to evaluate the drugs that ‘got the job done’: their development, their limitations and the resistance that the parasites developed against them. This retrospective looks back on the remarkable story of chemotherapy against trypanosomiasis, a story that goes back to the very origins and conception of chemotherapy in the first years of the 20 century and is still not finished today

Drug resistance in protozoan parasites

As with all other anti-infectives (antibiotics, anti-viral drugs, and anthelminthics), the limited arsenal of anti-protozoal drugs is being depleted by a combination of two factors: increasing drug resistance and the failure to replace old and often shamefully inadequate drugs, including those compromised by (cross)-resistance, through the development of new anti-parasitics. Both factors are equally to blame: a leaking bathtub may have plenty of water if the tap is left open; if not, it will soon be empty. Here, I will reflect on the factors that contribute to the drug resistance emergency that is unfolding around us, specifically resistance in protozoan parasites

The ever unfolding story of cAMP signaling in trypanosomatids: vive la difference!

Kinetoplastids are unicellular, eukaryotic, flagellated protozoans containing the eponymous kinetoplast. Within this order, the family of trypanosomatids are responsible for some of the most serious human diseases, including Chagas disease (Trypanosoma cruzi), sleeping sickness (Trypanosoma brucei spp.), and leishmaniasis (Leishmania spp). Although cAMP is produced during the life cycle stages of these parasites, its signaling pathways are very different from those of mammals. The absence of G-protein-coupled receptors, the presence of structurally different adenylyl cyclases, the paucity of known cAMP effector proteins and the stringent need for regulation of cAMP in the small kinetoplastid cells all suggest a significantly different biochemical pathway and likely cell biology. However, each of the main kinetoplastid parasites express four class 1-type cyclic nucleotide-specific phosphodiesterases (PDEA-D), which have highly similar catalytic domains to that of human PDEs. To date, only TbrPDEB, expressed as two slightly different isoforms TbrPDEB1 and B2, has been found to be essential when ablated. Although the genomes contain reasonably well conserved genes for catalytic and regulatory domains of protein kinase A, these have been shown to have varied structural and functional roles in the different species. Recent discovery of a role of cAMP/AMP metabolism in a quorum-sensing signaling pathway in T. brucei, and the identification of downstream cAMP Response Proteins (CARPs) whose expression levels correlate with sensitivity to PDE inhibitors, suggests a complex signaling cascade. The interplay between the roles of these novel CARPs and the quorum-sensing signaling pathway on cell division and differentiation makes for intriguing cell biology and a new paradigm in cAMP signal transduction, as well as potential targets for trypanosomatid-specific cAMP pathway-based therapeutics

Stability of earned value management: Do project characteristics influence the stability moment of the cost and schedule performance index

Stability of the Cost Performance Index (CPI) and Schedule Performance Index (SPI(t)) refers to the moment in the project life cycle at which the CPI and SPI(t) are accurate and constant. For a project manager a reliable CPI and SPI(t) is essential for taking corrective actions in time to keep the project on budget, planning and scope. The focus of this paper lies on identifying project characteristics which in uence this mo- ment of CPI and SPI(t) in the project life cycle. Both existing theories from earlier academic research and newly identi ed project characteristics are tested by using empirical data from nine projects executed by an engineering and consultancy company in the Nether- lands. It is found that some project characteristics in uence the moment of CPI and SPI(t) in the project lifecycle whereas other do not. The results of this paper contribute to the body of knowledge on EVM and might provide valuable information to project managers who consider to use EVM in their projects. The results of this research also point out new areas to explore the understanding of the stability of CPI and SPI(t)

Trypanosoma brucei methylthioadenosine phosphorylase protects the parasite from the antitrypanosomal effect of deoxyadenosine

Trypanosoma brucei causes African sleeping sickness for which no vaccine exists and available treatments are of limited use due to their high toxicity or lack of efficacy. T. brucei cultivated in the presence of deoxyadenosine accumulates high levels of dATP in an adenosine kinase-dependent process and dies within a few hours. Here we show that T. brucei treated with 1 mM deoxyadenosine accumulates higher dATP levels than mammalian cells but that this effect diminishes quickly as the concentration of the deoxynucleoside decreases. Radioactive tracer studies showed that the parasites are partially protected against lower concentrations of deoxyadenosine by the ability to cleave it and use the adenine for ATP synthesis. T. brucei methylthioadenosine phosphorylase (TbMTAP) was found to be responsible for the cleavage as indicated by the phosphate dependence of deoxyadenosine cleavage in T. brucei cell extracts and increased deoxyadenosine sensitivity in TbMTAP knockdown cells. Recombinant TbMTAP exhibited higher turnover number (kcat) and Km values for deoxyadenosine than for the regular substrate, methylthioadenosine. One of the reaction products, adenine, inhibited the enzyme, which might explain why TbMTAP-mediated protection is less efficient at higher deoxyadenosine concentrations. Consequently, T. brucei grown in the presence of adenine demonstrated increased sensitivity to deoxyadenosine. For deoxyadenosine/adenosine analogues to remain intact and be active against the parasite, they need to either be resistant to TbMTAP-mediated cleavage, which is the case with the three known antitrypanosomal agents adenine arabinoside, tubercidin, and cordycepin, or they need to be combined with TbMTAP inhibitors

Genotypic status of the TbAT1/P2 adenosine transporter of Trypanosoma brucei gambiense isolates from northwestern Uganda following melarsoprol withdrawal

Human African trypanosomiasis (HAT) manifests as a chronic infection caused by <i>Trypanosoma brucei gambiense</i>, or as a more acute form due to <i>T. b. rhodesiense</i>. Both manifestations occur in Uganda and melarsoprol use against the former was jeopardised in the 1990s as reports of reduced efficacy increased to the point where it was dismissed as first-line treatment at some treatment centers. Previous work to elucidate possible mechanisms leading to melarsoprol resistance pointed to a P2 type adenosine transporter known to mediate melarsoprol uptake and previously shown to be mutated in significant numbers of patients not responding to the drug. Our present findings indicate that there is a low prevalence of mutants in foci where melarsoprol relapses are infrequent. In addition we observe that at the Omugo focus where the drug was withdrawn as first line over 6 years ago, the mutant alleles have disappeared, suggesting that drug pressure is responsible for fuelling their spread. Thus constant monitoring for mutants could play a key role in cost-effective HAT management by identifying which foci can still use the less logistically demanding melarsoprol as opposed to the alternative drug eflornithine. What is required now is a simple method for identifying such mutants at the point of care, enabling practitioners to make informed prescriptions at first diagnosis

Finite temperature molecular dynamics study of unstable stacking fault free energies in silicon

We calculate the free energies of unstable stacking fault (USF) configurations on the glide and shuffle slip planes in silicon as a function of temperature, using the recently developed Environment Dependent Interatomic Potential (EDIP). We employ the molecular dynamics (MD) adiabatic switching method with appropriate periodic boundary conditions and restrictions to atomic motion that guarantee stability and include volume relaxation of the USF configurations perpendicular to the slip plane. Our MD results using the EDIP model agree fairly well with earlier first-principles estimates for the transition from shuffle to glide plane dominance as a function of temperature. We use these results to make contact to brittle-ductile transition models.Comment: 6 pages revtex, 4 figs, 16 refs, to appear in Phys. Rev.