Novel Trypanocidal Inhibitors that Block Glycosome Biogenesis by Targeting PEX3–PEX19 Interaction

Human pathogenic trypanosomatid parasites harbor a unique form of peroxisomes termed glycosomes that are essential for parasite viability. We and others previously identified and characterized the essential Trypanosoma brucei ortholog TbPEX3, which is the membrane-docking factor for the cytosolic receptor PEX19 bound to the glycosomal membrane proteins. Knockdown of TbPEX3 expression leads to mislocalization of glycosomal membrane and matrix proteins, and subsequent cell death. As an early step in glycosome biogenesis, the PEX3–PEX19 interaction is an attractive drug target. We established a high-throughput assay for TbPEX3–TbPEX19 interaction and screened a compound library for small-molecule inhibitors. Hits from the screen were further validated using an in vitro ELISA assay. We identified three compounds, which exhibit significant trypanocidal activity but show no apparent toxicity to human cells. Furthermore, we show that these compounds lead to mislocalization of glycosomal proteins, which is toxic to the trypanosomes. Moreover, NMR-based experiments indicate that the inhibitors bind to PEX3. The inhibitors interfering with glycosomal biogenesis by targeting the TbPEX3–TbPEX19 interaction serve as starting points for further optimization and anti-trypanosomal drug development.

m6A RNA methylation facilitates pre-mRNA 3’-end formation and is essential for viability of Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular parasite that can cause serious opportunistic disease in the immunocompromised or through congenital infection. To progress through its life cycle, Toxoplasma relies on multiple layers of gene regulation that includes an array of transcription and epigenetic factors. Over the last decade, the modification of mRNA has emerged as another important layer of gene regulation called epitranscriptomics. Here, we report that epitranscriptomics machinery exists in Toxoplasma, namely the methylation of adenosines (m6A) in mRNA transcripts. We identified novel components of the m6A methyltransferase complex and determined the distribution of m6A marks within the parasite transcriptome. m6A mapping revealed the modification to be preferentially located near the 3’-boundary of mRNAs. Knockdown of the m6A writer components METTL3 and WTAP resulted in diminished m6A marks and a complete arrest of parasite replication. Furthermore, we examined the two proteins in Toxoplasma that possess YTH domains, which bind m6A marks, and showed them to be integral members of the cleavage and polyadenylation machinery that catalyzes the 3’-end processing of pre-mRNAs. Loss of METTL3, WTAP, or YTH1 led to a defect in transcript 3’-end formation. Together, these findings establish that the m6A epitranscriptome is essential for parasite viability by contributing to the processing of mRNA 3’-ends.

An SR protein is essential for activating DNA repair in malaria parasites

Plasmodium falciparum, the parasite responsible for the deadliest form of human malaria, replicates within the erythrocytes of its host where it encounters numerous pressures that cause extensive DNA damage, which must be repaired efficiently to ensure parasite survival. Malaria parasites, which lost the NHEJ pathway for repairing DNA double strand breaks, have evolved unique mechanisms that enable them to robustly maintain genome integrity under such harsh conditions. However, the nature of these adaptations is unknown. We show that a highly conserved RNA splicing factor, PfSR1, plays an unexpected and crucial role in DNA repair in malaria parasites. Using an inducible and reversible system to manipulate PfSR1 expression, we demonstrate that this protein is recruited to foci of DNA damage. While loss of PfSR1 does not impair parasite viability, the protein is essential for parasites recovery from DNA damaging agents or exposure to artemisinin, the first line antimalarial drug, demonstrating its necessity for DNA repair. These findings provide key insights into the evolution of DNA repair pathways in malaria parasites as well as the parasite's ability to recover from antimalarial treatment.

Lopinavir and Nelfinavir Induce the Accumulation of Crystalloid Lipid Inclusions within the Reservosomes of Trypanosoma cruzi and Inhibit Both Aspartyl-Type Peptidase and Cruzipain Activities Detected in These Crucial Organelles

Several research groups have explored the repositioning of human immunodeficiency virus aspartyl peptidase inhibitors (HIV-PIs) on opportunistic infections caused by bacteria, fungi and protozoa. In Trypanosoma cruzi, HIV-PIs have a high impact on parasite viability, and one of the main alterations promoted by this treatment is the imbalance in the parasite’s lipid metabolism. However, the reasons behind this phenomenon are unknown. In the present work, we observed by transmission electron microscopy (TEM) that the treatment of T. cruzi epimastigotes with the HIV-PIs lopinavir and nelfinavir induced a huge accumulation of crystalloid-shaped lipids within the reservosomes, most of them deforming these key organelles. As previously reported, those structures are characteristic of lipid inclusions formed mostly of cholesterol and cholesterol-esters. The fractionation of nontreated epimastigotes generated two distinct fractions enriched in reservosomes: one mostly composed of lipid inclusion-containing reservosomes (Fraction B1) and one where lipid inclusions were much less abundant (Fraction B2). Interestingly, the extract of Fraction B2 presented enzymatic activity related to aspartyl-type peptidases 3.5 times higher than that found in the extract obtained from Fraction B1. The cleavage of cathepsin D substrate by this class of peptidases was strongly impaired by pepstatin A, a prototypical aspartyl PI, and the HIV-PIs lopinavir and nelfinavir. In addition, both HIV-PIs also inhibited (to a lesser extent) the cruzipain activity present in reservosomes. Finally, our work provides new evidence concerning the presence and supposed participation of aspartyl peptidases in T. cruzi, even as it adds new information about the mechanisms behind the alterations promoted by lopinavir and nelfinavir in the protozoan.

Oxidative Stress as a Possible Target in the Treatment of Toxoplasmosis: Perspectives and Ambiguities

Toxoplasma gondii is an apicomplexan parasite causing toxoplasmosis, a common disease, which is most typically asymptomatic. However, toxoplasmosis can be severe and even fatal in immunocompromised patients and fetuses. Available treatment options are limited, so there is a strong impetus to develop novel therapeutics. This review focuses on the role of oxidative stress in the pathophysiology and treatment of T. gondii infection. Chemical compounds that modify redox status can reduce the parasite viability and thus be potential anti-Toxoplasma drugs. On the other hand, oxidative stress caused by the activation of the inflammatory response may have some deleterious consequences in host cells. In this respect, the potential use of natural antioxidants is worth considering, including melatonin and some vitamins, as possible novel anti-Toxoplasma therapeutics. Results of in vitro and animal studies are promising. However, supplementation with some antioxidants was found to promote the increase in parasitemia, and the disease was then characterized by a milder course. Undoubtedly, research in this area may have a significant impact on the future prospects of toxoplasmosis therapy.

The Trypanosoma brucei subpellicular microtubule array is organized into functionally discrete subdomains defined by microtubule associated proteins

Microtubules are inherently dynamic cytoskeletal polymers whose length and organization can be altered to perform essential functions in eukaryotic cells, such as providing tracks for intracellular trafficking and forming the mitotic spindle. Microtubules can be bundled to create more stable structures that collectively propagate force, such as in the flagellar axoneme, which provides motility. The subpellicular microtubule array of the protist parasite Trypanosoma brucei, the causative agent of African sleeping sickness, is a remarkable example of a highly specialized microtubule bundle. It is comprised of a single layer of microtubules that are crosslinked to each other and to the overlying plasma membrane. The array microtubules appear to be highly stable and remain intact throughout the cell cycle, but very little is known about the pathways that tune microtubule properties in trypanosomatids. Here, we show that the subpellicular microtubule array is organized into subdomains that consist of differentially localized array-associated proteins at the array posterior, middle, and anterior. The array-associated protein PAVE1 stabilizes array microtubules at the cell posterior and is essential for maintaining its tapered shape. PAVE1 and the newly identified protein PAVE2 form a complex that binds directly to the microtubule lattice, demonstrating that they are a true kinetoplastid-specific MAP. TbAIR9, which localizes to the entirety of the subpellicular array, is necessary for maintaining the localization of array-associated proteins within their respective subdomains of the array. The arrangement of proteins within the array likely tunes the local properties of array microtubules and creates the asymmetric shape of the cell, which is essential for parasite viability.

The Role of the Histone Methyltransferase PfSET10 in Antigenic Variation by Malaria Parasites: a Cautionary Tale

ABSTRACT The virulence of the malaria parasite Plasmodium falciparum is due in large part to its ability to avoid immune destruction through antigenic variation. This results from changes in expression within the multicopy var gene family that encodes the surface antigen P. falciparum erythrocyte protein one (PfEMP1). Understanding the mechanisms underlying this process has been a high-profile research focus for many years. The histone methyltransferase PfSET10 was previously identified as a key enzyme required both for parasite viability and for regulating var gene expression, thus making it a prominent target for developing antimalarial intervention strategies and the subject of considerable research focus. Here, however, we show that disruption of the gene encoding PfSET10 is not lethal and has no effect on var gene expression, in sharp contrast with previously published reports. The contradictory findings highlight the importance of reevaluating previous conclusions when new technologies become available and suggest the possibility of a previously unappreciated plasticity in epigenetic gene regulation in P. falciparum. IMPORTANCE The identification of specific epigenetic regulatory proteins in infectious organisms has become a high-profile research topic and a focus for several drug development initiatives. However, studies that define specific roles for different epigenetic modifiers occasionally report differing results, and we similarly provide evidence regarding the histone methyltransferase PfSET10 that is in stark contrast with previously published results. We believe that the conflicting results, rather than suggesting erroneous conclusions, instead reflect the importance of revisiting previous conclusions using newly developed methodologies, as well as caution in interpreting seemingly contrary results in fields that are known to display considerable plasticity, for example metabolism and epigenetics.

Parasite Viability as a Superior Measure of Antimalarial Drug Activity in Humans

Background Artemisinin derivatives are the leading class of antimalarial drugs due to their rapid onset of action and rapid clearance of circulating parasites. The parasite clearance half-life measures the rate of loss of parasites from blood after treatment, and this is currently used to assess antimalarial activity of novel agents and to monitor resistance. However, a number of recent studies have challenged the use of parasite clearance to measure drug activity, arguing that many circulating parasites may be nonviable. Methods Plasmodium falciparum–infected subjects (n = 10) in a malaria volunteer infection study were administered a single dose of artesunate (2 mg/kg). Circulating parasite concentration was assessed by means of quantitative polymerase chain reaction (qPCR). Parasite viability after artesunate administration was estimated by mathematical modeling of the ex vivo growth of parasites collected from subjects. Results We showed that in artemisinin-sensitive infection, viable parasites declined to <0.1% of baseline within 8 hours after artesunate administration, while the total number of circulating parasites measured with quantitative polymerase chain reaction remained unchanged. In artemisinin-resistant infections over the same interval, viable parasites declined to 51.4% (standard error of the mean, 4.6%) of baseline. Conclusions These results demonstrate that in vivo drug activity of artesunate is faster than is indicated by the parasite clearance half-life.

The Trypanosoma brucei Cytoskeletal Protein KHARON Associates with Partner Proteins to Mediate Both Cytokinesis and Trafficking of Flagellar Membrane Proteins

ABSTRACTIn the African trypanosome Trypanosoma brucei, the cytoskeletal protein TbKHARON is required for trafficking of a putative Ca2+ channel to the flagellar membrane, and it is essential for parasite viability in both the mammalian stage bloodstream forms and the tsetse fly procyclic forms. This protein is located at the base of the flagellum, in the pellicular cytoskeleton, and in the mitotic spindle in both life cycle forms, and it likely serves multiple functions for these parasites. To begin to deconvolve the functions of KHARON, we have investigated partners associated with this protein and their roles in parasite biology. One KHARON associated protein, TbKHAP1, is a close interaction partner that can be crosslinked to KHARON by formaldehyde and pulled down in a molecular complex, and it colocalizes with TbKHARON in the basal body at the base of the flagellum. Knockdown of TbKHAP1 mRNA has similar phenotypes to knockdown of its partner TbKHARON, impairing trafficking of the Ca2+ channel to the flagellar membrane and blocking cytokinesis, implying that the TbKHARON/TbKHAP1 complex mediates trafficking of flagellar membrane proteins. Two other KHAPs, TbKHAP2 and TbKHAP3, are in close proximity to TbKHARON, but knockdown of their mRNAs does not affect trafficking of the Ca2+ channel. Two different flagellar membrane proteins, which are extruded from the flagellar membrane into extracellular vesicles, are also dependent upon TbKHARON for flagellar trafficking. These studies confirm that TbKHARON acts in complexes with other proteins to carry out various biological functions, and that some partners are involved in the core activity of targeting membrane proteins to the flagellum.