Genetically encoded calcium indicators (GECIs) are proteins used to monitor calcium ion (Ca2+) changes in living cells. They can be targeted to the cytosol or to organelles for continuous measurements in vivo. In this chapter, we present an overview of the methods that our lab used to detect Ca2+ changes in Trypanosoma cruzi elicited by stimulation of Ca2+ channels in the plasma membrane or in intracellular organelles, including selection of adequate GECIs, their transfection and selection, and their use for microscopic and fluorometric analysis.

Inorganic polyphosphate (polyP) is a linear polymer composed of three to several hundred orthophosphate units linked by high-energy phosphoanhydride bonds and is found in both prokaryotes and eukaryotes. In Trypanosoma cruzi, the causative agent of Chagas disease, polyP plays important roles in osmoregulation and persistence within host tissues and is synthesized by a polyP polymerase known as the vacuolar transporter chaperone (VTC) complex. This complex, localized to acidocalcisomes, is composed of Vtc1 and the catalytic subunit Vtc4. Using CRISPR/Cas9-mediated genome editing, we generated Vtc1 knockout (Vtc1-KO), Vtc4 single knockout (Vtc4-SKO), and conditional knockout lines for both subunits (Vtc1-CKO and Vtc4-CKO). Analysis of these mutants revealed essential roles for Vtc1 and Vtc4 in parasite proliferation, differentiation, and egress from mammalian host cells. Moreover, co-immunoprecipitation and proteomic analyses identified a novel component of the complex, termed TcVtc6, which associates with Vtc1 and Vtc4, forms part of the VTC complex, and is involved in polyP synthesis in Trypanosoma cruzi. IMPORTANCE:Chagas disease affects millions of people across the Americas and remains a major unmet medical challenge. Here, we investigate the essentiality and molecular composition of the vacuolar transporter chaperone (VTC) complex in Trypanosoma cruzi, the causative agent of the disease. We identify a previously unrecognized component of this complex, which we term TcVtc6, and show that it is involved in polyphosphate synthesis. Functional analyses reveal that the VTC complex is indispensable for parasite differentiation and host cell egress, two processes critical for infectivity. Although the VTC complex is conserved in trypanosomatids, apicomplexans, fungi, and algae, it is absent from mammalian cells. This evolutionary divergence, together with the essential role of the pathway in infectious stages of T. cruzi, highlights the VTC complex as a promising and selective therapeutic target for the treatment of Chagas disease.

Trypanosoma cruzi, the causative agent of Chagas disease, is a parasitic protist that affects millions of people worldwide. Currently there are no fully effective drugs or vaccines available. Contact of T. cruzi infective forms with their host cells or with the extracellular matrix increases their intracellular Ca2+ concentration suggesting a mechano-transduction process. We report here that T. cruzi possesses two distinct mechanosensitive Piezo channels, named TcPiezo1 and TcPiezo2, with different subcellular localizations but similarly essential for normal proliferation, differentiation, and infectivity. While TcPiezo1 localizes to the plasma membrane, TcPiezo2 localizes to the lysosomes. Downregulation of TcPiezo1 expression by a novel ligand-regulated hammerhead ribozyme (HHR) significantly inhibited Ca2+ entry in cells expressing a genetically encoded Ca2+ indicator while downregulation of TcPiezo2 expression inhibited Ca2+ release from lysosomes, which are now identified as novel acidic Ca2+ stores in trypanosomes. The channels are activated by contact with extracellular matrix and by hypoosmotic stress. The results establish the essentiality of Piezo channels for the life cycle and Ca2+ homeostasis of T. cruzi and a novel lysosomal localization for a Piezo channel in eukaryotes.

Inositol phosphates are involved in a myriad of biological roles and activities such as Ca2+ signaling, phosphate homeostasis, energy metabolism, and disease pathogenicity. In Saccharomyces cerevisiae, synthesis of inositol phosphates occurs through the phosphoinositide phospholipase C (PLC)-catalyzed hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol and further IP3 phosphorylation by additional kinases that leads to the formation of highly phosphorylated inositol derivatives, known as inositol pyrophosphates. Inositol-tetrakisphosphate 1-kinase (ITPK1) is an enzyme that mediates a PLC-independent inositol polyphosphate synthesis through phosphorylation of inositol monophosphates and other intermediates in the cytosol. In this work, we identified and characterized a Trypanosoma cruzi ITPK1 (TcITPK1) homolog. The ability of TcITPK1 to act as the mediator for this alternative pathway was established through plc1Δ and plc1Δ isc1Δ yeast complementation assays and SAX-HPLC analyses of radioactively labeled inositol. TcITPK1 localizes to the cytosol, and knockout attempts of TcITPK1 revealed that only one allele was replaced by the DNA donor cassette at the specific locus, suggesting that null alleles may have lethal effects in epimastigotes. Ablation of T. cruzi phosphoinositide phospholipase C 1 (TcPI-PLC1) affected the synthesis of IP3 from glucose 6-phosphate but did not affect the synthesis of inositol polyphosphates, while ablation of inositol phosphosphingolipid phospholipase (TcISC1) affected the synthesis of inositol polyphosphates, thus revealing that the PLC-independent pathway using either glucose 6-phosphate or inositol phosphoceramide is involved in the synthesis of inositol polyphosphates, while the PLC-dependent pathway is involved in IP3 formation needed for Ca2+ signaling. Millions of people are infected with Trypanosoma cruzi, and the current treatment is not satisfactory. Inositol pyrophosphates have been established as important signaling molecules. Our work demonstrates the presence of a phospholipase C-independent pathway for the synthesis of inositol pyrophosphates in T. cruzi. Furthermore, we demonstrate that this pathway starts with the synthesis of inositol monophosphates from glucose 6-phosphate or from inositol phosphoceramide, linking it to carbohydrate and sphingolipid metabolism. The essentiality of the pathway for the survival of T. cruzi infective stages makes it an ideal drug target for treating American trypanosomiasis.

Trypanosoma cruzi , the causative agent of Chagas disease, relies on complex gene regulatory mechanisms to adapt to its diverse host environments. Although this parasite lacks canonical transcriptional control, epigenetic regulation plays a pivotal role in modulating gene expression. Bromodomain-containing factors (BDFs), known for recognizing acetylated lysines on histones, have emerged as key regulators of chromatin structure and gene activity. Among the eight predicted BDFs in T. cruzi, Tc BDF6 is part of a TINTIN-like complex with Tc MRGx and Tc MRGBP, homologous to components of the NuA4/TIP60 chromatin remodeling complex. To investigate the function of Tc BDF6, we generated knockout (KO) parasites using CRISPR/Cas9 gene editing. While BDF6-deficient epimastigotes did not exhibit very significative growth differences, the resulting metacyclic trypomastigotes displayed drastically reduced infectivity. Strikingly, once inside host cells, Tc BDF6 deficient parasites differentiated into amastigotes but failed to replicate. This intracellular arrest was partially reversed by episomal re-expression of Tc BDF6. Consistently, BDF6 KO parasites also exhibited impaired infectivity, a defect that was also rescued in the add-back line. Our findings highlight Tc BDF6 as a critical regulator of intracellular parasite development, operating in stages beyond epimastigotes where epigenetic plasticity is essential for host adaptation. The unique, stage-specific phenotype of BDF6 knockouts underscores its functional importance and suggests that bromodomains may represent novel therapeutic targets against T. cruzi . Author Summary To survive in the dramatically different environments of its life cycle, Trypanosoma cruzi , the parasite that causes Chagas disease, must finely tune its gene expression. Unlike many organisms, T. cruzi lacks classical transcriptional regulation and instead relies on epigenetic mechanisms. Among the proteins involved in this regulation are bromodomain containing factors, which interpret chemical marks on chromatin. In this study, we focused on Tc BDF6, a bromodomain protein thought to be part of a chromatin remodeling complex. Using CRISPR/Cas9, we disrupted the TcBDF6 gene and discovered that, while the mutant parasites could still form infective stages, they failed to replicate inside host cells. Reintroducing TcBDF6 , it was rescued this defect, confirming its critical role in intracellular development. Our findings highlight the importance of epigenetic control in parasite survival and suggest that bromodomain proteins could be valuable targets for future treatments against Chagas disease. ### Competing Interest Statement The authors have declared no competing interest.

The Trypanosoma brucei group of parasites causes Nagana in cattle and human African trypanosomiasis, or sleeping sickness, in humans. Current drugs against these parasites have severe toxicity, vaccines are not available, and development of drug resistance makes finding new chemotherapeutic targets imperative. Ion channels, which are involved in several biological processes, are targets of many therapeutically useful agents, and they remain significantly underexplored as therapeutic targets in parasites. Here, we report the presence of a voltage gated Ca2+ channel (VGCC, TbCav), which is localized in the flagellar plasma membrane (PM) of T. brucei and is essential for proliferation of both bloodstream (BSF) and procyclic forms (PCF) of the parasite. TbCaV is a single subunit channel capable of transporting Ca2+ when expressed in mutant yeast lacking PM Ca2+ channels or in HEK293T cells. Through the virtual screening of a commercial chemical library using dynamic ensembles of various conformations of TbCav and associated docking analyses, several inhibitors of TbCav were discovered. As pharmacological validation of the essential roles of TbCav, these compounds were shown to inhibit T. brucei growth with the most potent agent, N-(7-nitro-2,1,3-benzoxadiazol-4-yl) acetamide (NBD-A), exhibiting an EC50 of 25 ± 3 nM and no cytotoxicity in Vero cells possessing related channels. Thus, such studies constitute pharmacological validation of TbCav as a viable therapeutic target of T. brucei.

As a continuation of the project aimed at searching for new chemotherapeuticagents against Chagas disease or American trypanosomiasis, new selenocyanate derivatives are designed, synthesized, and biologically evaluated against the clinically more relevant dividing amastigote form of Trypanosoma cruzi , the etiologic agent of this illness. Furthermore, as all the title compounds are fluorine‐containing molecules, it seemed to be reasonable to explore the role of fluorine atoms in the aromatic system and to determine the optimal position at the terminal phenoxy group, and therefore, various regioisomers are prepared. The conformationally restricted selenocyates structurally related to WC‐9Se exhibited improved antiparasitic activity compared to the lead drugs, Out to be extremely potent inhibitors of T. cruzi growth. In particular, (±)‐5‐(3‐fluorophenoxy)‐2‐(selenocyanatomethyl)−2,3‐dihydrobenzofuran exhibited an EC 50 value of 0.032 µM, which resulted in the most potent selenocyanate developed in the laboratory. The presence of the fluorine atom together with the rigidity of the molecules are beneficial for the anti‐ T. cruzi effect. The resulting antiparasitic activity provides further insight into the role of the selenocyanate group in its effective and putative anti‐ T. cruzi action.

Trypanosoma cruzi is the etiologic agent of Chagas disease, an infection that can lead to the development of cardiac fibrosis, which is characterized by the deposition of extracellular matrix (ECM) components in the interstitial region of the myocardium. The parasite itself can induce myofibroblast differentiation of cardiac fibroblast in vitro, leading to increased expression of ECM. Inorganic polyphosphate (polyP) is a linear polymer of orthophosphate that can also induce myofibroblast differentiation and deposition of ECM components and is highly abundant in T. cruzi. PolyP can modify proteins post-translationally by non-enzymatic polyphosphorylation of lysine residues of poly-acidic, serine-(S) and lysine (K)-rich (PASK) motifs. In this work, we used a bioinformatics screen and identified the presence of PASK domains in several surface proteins of T. cruzi. We also detected polyP in the external surface of its different life cycle stages and confirmed the stimulation of host cell fibrosis by trypomastigote infection. However, we were not able to detect significant secretion of the polymer or activation of transforming growth factor beta (TGF-β), an important factor for the generation of fibrosis by inorganic polyP- or trypomastigote-conditioned medium.

Acidocalcisomes of Trypanosoma brucei are membrane-bounded organelles characterized by their acidity and high content of polyphosphate and cations, like calcium and magnesium. They have important roles in cation and phosphorus storage, osmoregulation, autophagy initiation, calcium signaling, and virulence. Acidocalcisomes of T. brucei possess several membrane transporters, pumps, and channels, some of which were identified by proteomic and immunofluorescence analyses and validated as acidocalcisome proteins by their colocalization with the acidocalcisome marker vacuolar proton pyrophosphatase (VP1). Here, we report that a set of membrane transporters and enzymes, which were proposed to be present in acidocalcisomes by the morphological appearance of tagged proteins, colocalize with VP1, validating their character as acidocalcisome proteins. IMPORTANCE:Acidocalcisomes are acidic organelles rich in polyphosphate and calcium present in a variety of eukaryotes and important for osmoregulation and calcium signaling. Several proteins were postulated to localize to acidocalcisomes based on their morphological characteristics. We provide validation of the localization of ten10 acidocalcisome proteins by their co-localization with enzymatic markers. These findings reveal the roles of acidocalcisomes in the storage of toxic metals, and the presence of enzymes involved in palmitoylation and polyphosphate metabolism.

SUMMARY Acidocalcisomes are organelles conserved during evolution and closely related to the so-called volutin granules of bacteria and archaea, to the acidocalcisome-like vacuoles of yeasts, and to the lysosome-related organelles of animal species. All these organelles have in common their acidity and high content of polyphosphate and calcium. They are characterized by a variety of functions from storage of phosphorus and calcium to roles in Ca 2+ signaling, osmoregulation, blood coagulation, and inflammation. They interact with other organelles through membrane contact sites or by fusion, and have several enzymes, pumps, transporters, and channels.

Trypanosoma brucei is the causative agent of African trypanosomiasis, a deadly disease that affects humans and cattle. There are very few drugs to treat it, and there is evidence of mounting resistance, raising the need for new drug development. Here, we report the presence of a phosphoinositide phospholipase C (TbPI-PLC-like), containing an X and a PDZ domain, that is similar to the previously characterized TbPI-PLC1. TbPI-PLC-like only possesses the X catalytic domain and does not have the EF-hand, Y, and C2 domains, having instead a PDZ domain. Recombinant TbPI-PLC-like does not hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP2) and does not modulate TbPI-PLC1 activity in vitro. TbPI-PLC-like shows a plasma membrane and intracellular localization in permeabilized cells and a surface localization in non-permeabilized cells. Surprisingly, knockdown of TbPI-PLC-like expression by RNAi significantly affected proliferation of both procyclic and bloodstream trypomastigotes. This is in contrast with the lack of effect of downregulation of expression of TbPI-PLC1.

• Schistosomiasis is a devastating neglected helminthic disease. • Praziquantel (PZQ) is the most important drug against schistosomiasis. • Previous work identified a TRP channel of the melastatin type as a PZQ target. • Molecular studies now reveal the basis for varied PZQ sensitivity of different helminths .

Biomolecular condensation underlies the biogenesis of an expanding array of membraneless assemblies, including stress granules (SGs), which form under a variety of cellular stresses. Advances have been made in understanding the molecular grammar of a few scaffold proteins that make up these phases, but how the partitioning of hundreds of SG proteins is regulated remains largely unresolved. While investigating the rules that govern the condensation of ataxin-2, an SG protein implicated in neurodegenerative disease, we unexpectedly identified a short 14 aa sequence that acts as a condensation switch and is conserved across the eukaryote lineage. We identify poly(A)-binding proteins as unconventional RNA-dependent chaperones that control this regulatory switch. Our results uncover a hierarchy of cis and trans interactions that fine-tune ataxin-2 condensation and reveal an unexpected molecular function for ancient poly(A)-binding proteins as regulators of biomolecular condensate proteins. These findings may inspire approaches to therapeutically target aberrant phases in disease.