The translational challenge in Chagas disease drug development.

Chagas disease is a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi. There is an urgent need for safe, effective, and accessible new treatments since the currently approved drugs have serious limitations. Drug development for Chagas disease has historically been hampered by the complexity of the disease, critical knowledge gaps, and lack of coordinated R&D efforts. This review covers some of the translational challenges associated with the progression of new chemical entities from preclinical to clinical phases of development, and discusses how recent technological advances might allow the research community to answer key questions relevant to the disease and to overcome hurdles in R&D for Chagas disease.

The translational challenge in Chagas disease drug development

Chagas disease is a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi. There is an urgent need for safe, effective, and accessible new treatments since the currently approved drugs have serious limitations. Drug development for Chagas disease has historically been hampered by the complexity of the disease, critical knowledge gaps, and lack of coordinated R&D efforts. This review covers some of the translational challenges associated with the progression of new chemical entities from preclinical to clinical phases of development, and discusses how recent technological advances might allow the research community to answer key questions relevant to the disease and to overcome hurdles in R&D for Chagas disease.

Cyclophilin 19 secreted in the host cell cytosol by Trypanosoma cruzi promotes ROS production required for parasite growth.

Infection by Trypanosoma cruzi, the protozoan parasite that causes Chagas disease, depends on reactive oxygen species (ROS), which has been described to induce parasite proliferation in mammalian host cells. It is unknown how the parasite manages to increase host ROS levels. Here, we found that intracellular T. cruzi forms release in the host cytosol its major cyclophilin of 19 kDa (TcCyp19). Parasites depleted of TcCyp19 by using CRISPR/Cas9 gene replacement proliferate inefficiently and fail to increase ROS, compared to wild type parasites or parasites with restored TcCyp19 gene expression. Expression of TcCyp19 in L6 rat myoblast increased ROS levels and restored the proliferation of TcCyp19 depleted parasites. These events could also be inhibited by cyclosporin A, (a cyclophilin inhibitor), and by polyethylene glycol-linked to antioxidant enzymes. TcCyp19 was found more concentrated in the membrane leading edges of the host cells in regions that also accumulate phosphorylated p47phox , as observed to the endogenous cyclophilin A, suggesting some mechanisms involved with the translocation process of the regulatory subunit p47phox in the activation of the NADPH oxidase enzymatic complex. We concluded that cyclophilin released in the host cell cytosol by T. cruzi mediates the increase of ROS, required to boost parasite proliferation in mammalian hosts.

EIF2α phosphorylation is regulated in intracellular amastigotes for the generation of infective Trypanosoma cruzi trypomastigote forms.

Trypanosomatids regulate gene expression mainly at the post-transcriptional level through processing, exporting and stabilising mRNA and control of translation. In most eukaryotes, protein synthesis is regulated by phosphorylation of eukaryotic initiation factor 2 (eIF2) at serine 51. Phosphorylation halts overall translation by decreasing availability of initiator tRNAmet to form translating ribosomes. In trypanosomatids, the N-terminus of eIF2α is extended with threonine 169 the homologous phosphorylated residue. Here, we evaluated whether eIF2α phosphorylation varies during the Trypanosoma cruzi life cycle, the etiological agent of Chagas' disease. Total levels of eIF2α are diminished in infective and non-replicative trypomastigotes compared with proliferative forms from the intestine of the insect vector or amastigotes from mammalian cells, consistent with decreased protein synthesis reported in infective forms. eIF2α phosphorylation increases in proliferative intracellular forms prior to differentiation into trypomastigotes. Parasites overexpressing eIF2αT169A or with an endogenous CRISPR/Cas9-generated eIF2αT169A mutation were created and analysis revealed alterations to the proteome, largely unrelated to the presence of µORF in epimastigotes. eIF2αT169A mutant parasites produced fewer trypomastigotes with lower infectivity than wild type, with increased levels of sialylated mucins and oligomannose glycoproteins, and decreased galactofuranose epitopes and the surface protease GP63 on the cell surface. We conclude that eIF2α expression and phosphorylation levels affect proteins relevant for intracellular progression of T. cruzi.

GCN2-Like Kinase Modulates Stress Granule Formation During Nutritional Stress in Trypanosoma cruzi.

The integrated stress response in eukaryotic cells is an orchestrated pathway that leads to eukaryotic Initiation Factor 2 alpha subunit (eIF2α) phosphorylation at ser51 and ultimately activates pathways to mitigate cellular damages. Three putative kinases (Tck1, Tck2, and Tck3) are found in the Trypanosoma cruzi genome, the flagellated parasite that causes Chagas disease. These kinases present similarities to other eukaryotic eIF2α kinases, exhibiting a typical insertion loop in the kinase domain of the protein. We found that this insertion loop is conserved among kinase 1 of several T. cruzi strains but differs among various Kinetoplastidae species, suggesting unique roles. Kinase 1 is orthologous of GCN2 of several eukaryotes, which have been implicated in the eIF2α ser51 phosphorylation in situations that mainly affects the nutrients levels. Therefore, we further investigated the responses to nutritional stress of T. cruzi devoid of TcK1 generated by CRISPR/Cas9 gene replacement. In nutrient-rich conditions, replicative T. cruzi epimastigotes depleted of TcK1 proliferate as wild type cells but showed increased levels of polysomes relative to monosomes. Upon nutritional deprivation, the polysomes decreased more than in TcK1 depleted line. However, eIF2α is still phosphorylated in TcK1 depleted line, as in wild type parasites. eIF2α phosphorylation increased at longer incubations times, but KO parasites showed less accumulation of ribonucleoprotein granules containing ATP-dependent RNA helicase involved in mRNA turnover (DHH1) and Poly-A binding protein (PABP1). Additionally, the formation of metacyclic-trypomastigotes is increased in the absence of Tck1 compared to controls. These metacyclics, as well as tissue culture trypomastigotes derived from the TcK1 knockout line, were less infective to mammalian host cells, although replicated faster inside mammalian cells. These results indicate that GCN2-like kinase in T. cruzi affects stress granule formation, independently of eIF2α phosphorylation upon nutrient deprivation. It also modulates the fate of the parasites during differentiation, invasion, and intracellular proliferation.

Polyphenol-Rich Diets Exacerbate AMPK-Mediated Autophagy, Decreasing Proliferation of Mosquito Midgut Microbiota, and Extending Vector Lifespan.

BACKGROUND: Mosquitoes feed on plant-derived fluids such as nectar and sap and are exposed to bioactive molecules found in this dietary source. However, the role of such molecules on mosquito vectorial capacity is unknown. Weather has been recognized as a major determinant of the spread of dengue, and plants under abiotic stress increase their production of polyphenols. RESULTS: Here, we show that including polyphenols in mosquito meals promoted the activation of AMP-dependent protein kinase (AMPK). AMPK positively regulated midgut autophagy leading to a decrease in bacterial proliferation and an increase in vector lifespan. Suppression of AMPK activity resulted in a 6-fold increase in midgut microbiota. Similarly, inhibition of polyphenol-induced autophagy induced an 8-fold increase in bacterial proliferation. Mosquitoes maintained on the polyphenol diet were readily infected by dengue virus. CONCLUSION: The present findings uncover a new direct route by which exacerbation of autophagy through activation of the AMPK pathway leads to a more efficient control of mosquito midgut microbiota and increases the average mosquito lifespan. Our results suggest for the first time that the polyphenol content and availability of the surrounding vegetation may increase the population of mosquitoes prone to infection with arboviruses.

Genome of Rhodnius prolixus, an insect vector of Chagas disease, reveals unique adaptations to hematophagy and parasite infection.

Rhodnius prolixus not only has served as a model organism for the study of insect physiology, but also is a major vector of Chagas disease, an illness that affects approximately seven million people worldwide. We sequenced the genome of R. prolixus, generated assembled sequences covering 95% of the genome (∼ 702 Mb), including 15,456 putative protein-coding genes, and completed comprehensive genomic analyses of this obligate blood-feeding insect. Although immune-deficiency (IMD)-mediated immune responses were observed, R. prolixus putatively lacks key components of the IMD pathway, suggesting a reorganization of the canonical immune signaling network. Although both Toll and IMD effectors controlled intestinal microbiota, neither affected Trypanosoma cruzi, the causal agent of Chagas disease, implying the existence of evasion or tolerance mechanisms. R. prolixus has experienced an extensive loss of selenoprotein genes, with its repertoire reduced to only two proteins, one of which is a selenocysteine-based glutathione peroxidase, the first found in insects. The genome contained actively transcribed, horizontally transferred genes from Wolbachia sp., which showed evidence of codon use evolution toward the insect use pattern. Comparative protein analyses revealed many lineage-specific expansions and putative gene absences in R. prolixus, including tandem expansions of genes related to chemoreception, feeding, and digestion that possibly contributed to the evolution of a blood-feeding lifestyle. The genome assembly and these associated analyses provide critical information on the physiology and evolution of this important vector species and should be instrumental for the development of innovative disease control methods.

Perimicrovillar membrane assembly: the fate of phospholipids synthesised by the midgut of Rhodnius prolixus.

In this study, we describe the fate of fatty acids that are incorporated from the lumen by the posterior midgut epithelium of Rhodnius prolixus and the biosynthesis of lipids. We also demonstrate that neutral lipids (NL) are transferred to the haemolymphatic lipophorin (Lp) and that phospholipids remain in the tissue in which they are organised into perimicrovillar membranes (PMMs). 3H-palmitic acid added at the luminal side of isolated midguts of R. prolixus females was readily absorbed and was used to synthesise phospholipids (80%) and NL (20%). The highest incorporation of 3H-palmitic acid was on the first day after a blood meal. The amounts of diacylglycerol (DG) and triacylglycerol synthesised by the tissue decreased in the presence of Lp in the incubation medium. The metabolic fates of 3H-lipids synthesised by the posterior midgut were followed and it was observed that DG was the major lipid released to Lp particles. However, the majority of phospholipids were not transferred to Lp, but remained in the tissue. The phospholipids that were synthesised and accumulated in the posterior midgut were found to be associated with Rhodnius luminal contents as structural components of PMMs.

Perimicrovillar membrane assembly: the fate of phospholipids synthesised by the midgut of Rhodnius prolixus

In this study, we describe the fate of fatty acids that are incorporated from the lumen by the posterior midgut epithelium of Rhodnius prolixus and the biosynthesis of lipids. We also demonstrate that neutral lipids (NL) are transferred to the haemolymphatic lipophorin (Lp) and that phospholipids remain in the tissue in which they are organised into perimicrovillar membranes (PMMs). 3H-palmitic acid added at the luminal side of isolated midguts of R. prolixus females was readily absorbed and was used to synthesise phospholipids (80%) and NL (20%). The highest incorporation of 3H-palmitic acid was on the first day after a blood meal. The amounts of diacylglycerol (DG) and triacylglycerol synthesised by the tissue decreased in the presence of Lp in the incubation medium. The metabolic fates of 3H-lipids synthesised by the posterior midgut were followed and it was observed that DG was the major lipid released to Lp particles. However, the majority of phospholipids were not transferred to Lp, but remained in the tissue. The phospholipids that were synthesised and accumulated in the posterior midgut were found to be associated with Rhodnius luminal contents as structural components of PMMs.

The major insect lipoprotein is a lipid source to mosquito stages of malaria parasite.

Once mosquito midgut barrier was crossed malaria parasite faces a extensive metabolic developmental program in order to ensure its transmission. In the hemolymph of the mosquito the dynamics of lipid metabolism is conducted by a major lipoprotein, lipophorin (Lp). It was recently shown that Lp is engaged in the mosquito immune response to parasite infection. However, it is not clear if Lp is uptaken by the parasite. Here, we show that oocysts are able to uptake mosquito Lp. The uptake of FITC-labeled Lp was demonstrated in midgut-associated oocysts. Alternatively, to confirm Lp incorporation by oocysts we have conducted another set of experiments with iodinated Lp ((125)I-Lp). Oocysts were able to incorporate (125)I-Lp and the process is both time and temperature dependent. This set of results indicated that no matter oocysts are attached to mosquito midgut wall they bear a lipid sequestering machinery from its surroundings. Phospholipid transfer to sporozoites was also demonstrated. In conclusion, these results demonstrate for the first time that malaria parasite undergoes lipid uptake while in the invertebrate host.

Extracellular lipid droplets promote hemozoin crystallization in the gut of the blood fluke Schistosoma mansoni.

Hemozoin (Hz) is a heme crystal produced upon hemoglobin digestion as the main mechanism of heme disposal in several hematophagous organisms. Here, we show that, in the helminth Schistosoma mansoni, Hz formation occurs in extracellular lipid droplets (LDs). Transmission electron microscopy of adult worms revealed the presence of numerous electron-lucent round structures similar to LDs in gut lumen, where multicrystalline Hz assemblies were found associated to their surfaces. Female regurgitates promoted Hz formation in vitro in reactions partially inhibited by boiling. Fractionation of regurgitates showed that Hz crystallization activity was essentially concentrated on lower density fractions, which have small amounts of pre-formed Hz crystals, suggesting that hydrophilic-hydrophobic interfaces, and not Hz itself, play a key catalytic role in Hz formation in S. mansoni. Thus, these data demonstrate that LDs present in the gut lumen of S. mansoni support Hz formation possibly by allowing association of heme to the lipid-water interface of these structures.