Mode of action studies confirm on-target engagement of lysyl-tRNA synthetase inhibitor and lead to new selection marker for Cryptosporidium.

Introduction: Cryptosporidiosis is a leading cause of diarrheal-associated morbidity and mortality, predominantly affecting children under 5 years old in low-and-middle-income countries. There is no effective treatment and no vaccine. New therapeutics are emerging from drug discovery efforts. It is critical that mode of action studies are performed alongside drug discovery to ensure the best clinical outcomes. Unfortunately, technology to identify and validate drug targets for Cryptosporidium is severely lacking. Methods: We used C. parvum lysyl-tRNA synthetase (CpKRS) and DDD01510706 as a target-compound pair to develop both chemical and genetic tools for mode of action studies for Cryptosporidium. We adapted thermal proteome profiling (TPP) for Cryptosporidium, an unbiased approach for target identification. Results: Using TPP we identified the molecular target of DDD01510706 and confirm that it is CpKRS. Genetic tools confirm that CpKRS is expressed throughout the life cycle and that this target is essential for parasite survival. Parasites genetically modified to over-express CpKRS or parasites with a mutation at the compound-binding site are resistant to treatment with DDD01510706. We leveraged these mutations to generate a second drug selection marker for genetic modification of Cryptosporidium, KRSR. This second selection marker is interchangeable with the original selection marker, NeoR, and expands the range of reverse genetic approaches available to study parasite biology. Due to the sexual nature of the Cryptosporidium life cycle, parental strains containing different drug selection markers can be crossed in vivo. Discussion: Selection with both drug markers produces highly efficient genetic crosses (>99% hybrid progeny), paving the way for forward genetics approaches in Cryptosporidium.

Ethanolaminephosphate cytidylyltransferase is essential for survival, lipid homeostasis and stress tolerance in Leishmania major.

Glycerophospholipids including phosphatidylethanolamine (PE) and phosphatidylcholine (PC) are vital components of biological membranes. Trypanosomatid parasites of the genus Leishmania can acquire PE and PC via de novo synthesis and the uptake/remodeling of host lipids. In this study, we investigated the ethanolaminephosphate cytidylyltransferase (EPCT) in Leishmania major, which is the causative agent for cutaneous leishmaniasis. EPCT is a key enzyme in the ethanolamine branch of the Kennedy pathway which is responsible for the de novo synthesis of PE. Our results demonstrate that L. major EPCT is a cytosolic protein capable of catalyzing the formation of CDP-ethanolamine from ethanolamine-phosphate and cytidine triphosphate. Genetic manipulation experiments indicate that EPCT is essential in both the promastigote and amastigote stages of L. major as the chromosomal null mutants cannot survive without the episomal expression of EPCT. This differs from our previous findings on the choline branch of the Kennedy pathway (responsible for PC synthesis) which is required only in promastigotes but not amastigotes. While episomal EPCT expression does not affect promastigote proliferation under normal conditions, it leads to reduced production of ethanolamine plasmalogen or plasmenylethanolamine, the dominant PE subtype in Leishmania. In addition, parasites with episomal EPCT exhibit heightened sensitivity to acidic pH and starvation stress, and significant reduction in virulence. In summary, our investigation demonstrates that proper regulation of EPCT expression is crucial for PE synthesis, stress response, and survival of Leishmania parasites throughout their life cycle.

A One Health Approach to Tackle Cryptosporidiosis.

Cryptosporidiosis is a significant diarrhoeal disease in both people and animals across the world and is caused by several species of the protozoan parasite Cryptosporidium. Recent research has highlighted the longer-term consequences of the disease for malnourished children, involving growth stunting and cognitive deficits, and significant growth and production losses for livestock. There are no vaccines currently available to prevent the disease and few treatment options in either humans or animals, which has been a significant limiting factor in disease control to date. A One Health approach to tackle zoonotic cryptosporidiosis looking at new advances in veterinary, public, and environmental health research may offer several advantages and new options to help control the disease.

Genetic ablation of purine salvage in Cryptosporidium parvum reveals nucleotide uptake from the host cell.

The apicomplexan parasite Cryptosporidium is a leading global cause of severe diarrheal disease and an important contributor to early-childhood mortality. Waterborne outbreaks occur frequently, even in countries with advanced water treatment capabilities, and there is currently no fully effective treatment. Nucleotide pathways are attractive targets for antimicrobial development, and several laboratories are designing inhibitors of these enzymes as potential treatment for Cryptosporidium infections. Here we take advantage of newly available molecular genetics for Cryptosporidium parvum to investigate nucleotide biosynthesis by directed gene ablation. Surprisingly, we found that the parasite tolerates the loss of classical targets including dihydrofolate reductase-thymidylate synthase (DHFR-TS) and inosine monophosphate dehydrogenase (IMPDH). We show that thymidine kinase provides a route to thymidine monophosphate in the absence of DHFR-TS. In contrast, only a single pathway has been identified for C. parvum purine nucleotide salvage. Nonetheless, multiple enzymes in the purine pathway, as well as the adenosine transporter, can be ablated. The resulting mutants are viable under normal conditions but are hypersensitive to inhibition of purine nucleotide synthesis in their host cell. Cryptosporidium might use as-yet undiscovered purine transporters and salvage enzymes; however, genetic and pharmacological experiments led us to conclude that Cryptosporidium imports purine nucleotides from the host cell. The potential for ATP uptake from the host has significant impact on our understanding of parasite energy metabolism given that Cryptosporidium lacks oxidative phosphorylation and glycolytic enzymes are not constitutively expressed throughout the parasite life cycle.

Phosphatidylcholine synthesis through cholinephosphate cytidylyltransferase is dispensable in Leishmania major.

Phosphatidylcholine (PC) is a major cell membrane constituent and precursor of important second messengers. In Leishmania parasites, PC synthesis can occur via the choline branch of the Kennedy pathway, the N-methylation of phosphatidylethanolamine (PE), or the remodeling of exogenous phospholipids. To investigate the role of de novo PC synthesis in Leishmania major, we focused on the cholinephosphate cytidylyltransferase (CPCT) which catalyzes the formation of CDP-choline, a key intermediate in the choline branch of the Kennedy pathway. Without CPCT, L. major parasites cannot incorporate choline into PC, yet the CPCT-null mutants contain similar levels of PC and PE as wild type parasites. Loss of CPCT does not affect the growth of parasites in complete medium or their virulence in mice. These results suggest that other mechanisms of PC synthesis can compensate the loss of CPCT. Importantly, CPCT-null parasites exhibited severe growth defects when ethanolamine and exogenous lipids became limited or when they were co-cultured with certain bacteria that are known to be members of sandfly midgut microbiota. These findings suggest that Leishmania employ multiple PC synthesis pathways to utilize a diverse pool of nutrients, which may be crucial for their survival and development in the sandfly.

Generating and Maintaining Transgenic Cryptosporidium parvum Parasites.

The apicomplexan parasite Cryptosporidium is a leading cause of diarrheal disease and an important contributor to overall global child mortality. We currently lack effective treatment and immune prophylaxis. Recent advances now permit genetic modification of this important pathogen. We expect this to produce rapid advances in fundamental as well as translational research on cryptosporidiosis. Here we outline genetic engineering for Cryptosporidium in sufficient detail to establish transfection in any laboratory that requires access to this key technology. This chapter details the conceptual design consideration, as well as the experimental steps required to transfect, select, and isolate transgenic parasites. We also provide detail on key in vitro and in vivo assays to detect, validate, and quantify genetically modified Cryptosporidium parasites. © 2017 by John Wiley & Sons, Inc.

Plasmenylethanolamine synthesis in Leishmania major.

Ethanolamine glycerophospholipids are ubiquitous cell membrane components. Trypanosomatid parasites of the genus Leishmania synthesize the majority of their ethanolamine glycerophospholipids as 1-O-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine or plasmenylethanolamine (PME) through the Kennedy pathway. PME is a subtype of ether phospholipids also known as ethanolamine plasmalogen whose functions are not well characterized. In this study, we investigated the role of PME synthesis in Leishmania major through the characterization of an ethanolamine phosphotransferase (EPT) mutant. EPT-null parasites are largely devoid of PME and fully viable in regular medium but fail to proliferate in the absence of fetal bovine serum. They exhibit significant abnormalities in the synthesis and localization of GPI-anchored surface molecules. EPT-null mutants also show attenuated virulence in BALB/c mice. Furthermore, in addition to PME synthesis, ethanolamine also contributes to the production of phosphatidylcholine, the most abundant class of lipids in Leishmania. Together, these findings suggest that ethanolamine production is likely required for Leishmania promastigotes to generate bulk phospholipids, to handle stress, and to control the expression of membrane bound virulence factors.

Genetic modification of the diarrhoeal pathogen Cryptosporidium parvum.

Recent studies into the global causes of severe diarrhoea in young children have identified the protozoan parasite Cryptosporidium as the second most important diarrhoeal pathogen after rotavirus. Diarrhoeal disease is estimated to be responsible for 10.5% of overall child mortality. Cryptosporidium is also an opportunistic pathogen in the contexts of human immunodeficiency virus (HIV)-caused AIDS and organ transplantation. There is no vaccine and only a single approved drug that provides no benefit for those in gravest danger: malnourished children and immunocompromised patients. Cryptosporidiosis drug and vaccine development is limited by the poor tractability of the parasite, which includes a lack of systems for continuous culture, facile animal models, and molecular genetic tools. Here we describe an experimental framework to genetically modify this important human pathogen. We established and optimized transfection of C. parvum sporozoites in tissue culture. To isolate stable transgenics we developed a mouse model that delivers sporozoites directly into the intestine, a Cryptosporidium clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system, and in vivo selection for aminoglycoside resistance. We derived reporter parasites suitable for in vitro and in vivo drug screening, and we evaluated the basis of drug susceptibility by gene knockout. We anticipate that the ability to genetically engineer this parasite will be transformative for Cryptosporidium research. Genetic reporters will provide quantitative correlates for disease, cure and protection, and the role of parasite genes in these processes is now open to rigorous investigation.

Leishmania parasites possess a platelet-activating factor acetylhydrolase important for virulence.

Leishmania parasites are intracellular protozoans capable of salvaging and remodeling lipids from the host. To understand the role of lipid metabolism in Leishmania virulence, it is necessary to characterize the enzymes involved in the uptake and turnover of phospholipids. This study focuses on a putative phospholipase A2 (PLA2)/platelet-activating factor acetylhydrolase (PAF-AH) in Leishmania major. In mammals, PAF-AH is a subgroup of PLA2 catalyzing the hydrolysis/inactivation of platelet-activating factor (PAF), a potent mediator of many leukocyte functions. By immunofluorescence microscopy, L. major PLA2/PAF-AH is predominantly localized in the ER. While wild type L. major parasites are able to hydrolyze PAF, this activity is completely absent in the PLA2/PAF-AH-null mutants. Meanwhile, deletion of PLA2/PAF-AH had no significant effect on the turnover of common glycerophospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylglycerol. PLA2/PAF-AH is not required for the growth of L. major parasites in culture, or the production of GPI-anchored virulence factors. Nonetheless, it does play a key role in the mammalian host as the PLA2/PAF-AH null mutants exhibit attenuated virulence in BALB/c mice. In conclusion, these data suggest that Leishmania parasites possess a functional PAF-AH and the degradation of PAF or PAF-like lipids is an important step in infection.