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1. Genomic Occupancy of the Bromodomain Protein Bdf3 Is Dynamic during Differentiation of African Trypanosomes from Bloodstream to Procyclic Forms

   https://doi.org/10.1128/msphere.00023-22  

   Ashby, Ethan; Paddock, Lucinda; Betts, Hannah L.; Liao, Jingwen; Miller, Geneva; Porter, Anya; Rollosson, Lindsey M.; Saada, Carrie; Tang, Eric; Wade, Serenity J.; et al (June 2022, mSphere)

   Phillips, Margaret (Ed.)

   ABSTRACT Trypanosoma brucei , the causative agent of human and animal African trypanosomiasis, cycles between a mammalian host and a tsetse fly vector. The parasite undergoes huge changes in morphology and metabolism during adaptation to each host environment. These changes are reflected in the different transcriptomes of parasites living in each host. However, it remains unclear whether chromatin-interacting proteins help mediate these changes. Bromodomain proteins localize to transcription start sites in bloodstream parasites, but whether the localization of bromodomain proteins changes as parasites differentiate from bloodstream to insect stages remains unknown. To address this question, we performed cleavage under target and release using nuclease (CUT&RUN) against bromodomain protein 3 (Bdf3) in parasites differentiating from bloodstream to insect forms. We found that Bdf3 occupancy at most loci increased at 3 h following onset of differentiation and decreased thereafter. A number of sites with increased bromodomain protein occupancy lie proximal to genes with altered transcript levels during differentiation, such as procyclins, procyclin-associated genes, and invariant surface glycoproteins. Most Bdf3-occupied sites are observed throughout differentiation. However, one site appears de novo during differentiation and lies proximal to the procyclin gene locus housing genes essential for remodeling surface proteins following transition to the insect stage. These studies indicate that occupancy of chromatin-interacting proteins is dynamic during life cycle stage transitions and provide the groundwork for future studies on the effects of changes in bromodomain protein occupancy. Additionally, the adaptation of CUT&RUN for Trypanosoma brucei provides other researchers with an alternative to chromatin immunoprecipitation (ChIP). IMPORTANCE The parasite Trypanosoma brucei is the causative agent of human and animal African trypanosomiasis (sleeping sickness). Trypanosomiasis, which affects humans and cattle, is fatal if untreated. Existing drugs have significant side effects. Thus, these parasites impose a significant human and economic burden in sub-Saharan Africa, where trypanosomiasis is endemic. T. brucei cycles between the mammalian host and a tsetse fly vector, and parasites undergo huge changes in morphology and metabolism to adapt to different hosts. Here, we show that DNA-interacting bromodomain protein 3 (Bdf3) shows changes in occupancy at its binding sites as parasites transition from the bloodstream to the insect stage. Additionally, a new binding site appears near the locus responsible for remodeling of parasite surface proteins during transition to the insect stage. Understanding the mechanisms behind host adaptation is important for understanding the life cycle of the parasite. 

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2. Plasmodium falciparum Calcium-Dependent Protein Kinase 4 is Critical for Male Gametogenesis and Transmission to the Mosquito Vector

   https://doi.org/10.1128/mBio.02575-21  

   Kumar, Sudhir; Haile, Meseret T.; Hoopmann, Michael R.; Tran, Linh T.; Michaels, Samantha A.; Morrone, Seamus R.; Ojo, Kayode K.; Reynolds, Laura M.; Kusebauch, Ulrike; Vaughan, Ashley M.; et al (December 2021, mBio)

   Weiss, Louis M. (Ed.)

   ABSTRACT Gametocytes of the malaria parasite Plasmodium are taken up by the mosquito vector with an infectious blood meal, representing a critical stage for parasite transmission. Calcium-independent protein kinases (CDPKs) play key roles in calcium-mediated signaling across the complex life cycle of the parasite. We sought to understand their role in human parasite transmission from the host to the mosquito vector and thus investigated the role of the human-infective parasite Plasmodium falciparum CDPK4 in the parasite life cycle. P. falciparum cdpk4 − parasites created by targeted gene deletion showed no effect in blood stage development or gametocyte development. However, cdpk4 − parasites showed a severe defect in male gametogenesis and the emergence of flagellated male gametes. To understand the molecular underpinnings of this defect, we performed mass spectrometry-based phosphoproteomic analyses of wild-type and Plasmodium falciparum cdpk4 − late gametocyte stages to identify key CDPK4-mediated phosphorylation events that may be important for the regulation of male gametogenesis. We further employed in vitro assays to identify these putative substrates of Plasmodium falciparum CDPK4. This indicated that CDPK4 regulates male gametogenesis by directly or indirectly controlling key essential events, such as DNA replication, mRNA translation, and cell motility. Taken together, our work demonstrates that PfCDPK4 is a central kinase that regulates exflagellation and thereby is critical for parasite transmission to the mosquito vector. IMPORTANCE Transmission of the malaria parasite to the mosquito vector is critical for the completion of the sexual stage of the parasite life cycle and is dependent on the release of male gametes from the gametocyte body inside the mosquito midgut. In the present study, we demonstrate that PfCDPK4 is critical for male gametogenesis and is involved in phosphorylation of proteins essential for male gamete emergence. Targeting PfCDPK4 and its substrates may provide insights into achieving effective malaria transmission-blocking strategies. 

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3. Enolase Inhibitors as Early Lead Therapeutics against Trypanosoma brucei

   https://doi.org/10.3390/pathogens12111290  

   Roster, Colm P.; LaVigne, Danielle; Milanes, Jillian E.; Knight, Emily; Anderson, Heidi D.; Pizarro, Sabrina; Harding, Elijah M.; Morris, Meredith T.; Yan, Victoria C.; Pham, Cong-Dat; et al (November 2023, Pathogens)

   Glucose metabolism is critical for the African trypanosome, Trypanosoma brucei, serving as the lone source of ATP production for the bloodstream form (BSF) parasite in the glucose-rich environment of the host blood. Recently, phosphonate inhibitors of human enolase (ENO), the enzyme responsible for the interconversion of 2-phosphoglycerate (2-PG) to phosphoenolpyruvate (PEP) in glycolysis or PEP to 2-PG in gluconeogenesis, have been developed for the treatment of glioblastoma multiforme (GBM). Here, we have tested these agents against T. brucei ENO (TbENO) and found the compounds to be potent enzyme inhibitors and trypanocides. For example, (1-hydroxy-2-oxopyrrolidin-3-yl) phosphonic acid (deoxy-SF2312) was a potent enzyme inhibitor (IC50 value of 0.60 ± 0.23 µM), while a six-membered ring-bearing phosphonate, (1-hydroxy-2-oxopiperidin-3-yl) phosphonic acid (HEX), was less potent (IC50 value of 2.1 ± 1.1 µM). An analog with a larger seven-membered ring, (1-hydroxy-2-oxoazepan-3-yl) phosphonic acid (HEPTA), was not active. Molecular docking simulations revealed that deoxy-SF2312 and HEX had binding affinities of −6.8 and −7.5 kcal/mol, respectively, while the larger HEPTA did not bind as well, with a binding of affinity of −4.8 kcal/mol. None of these compounds were toxic to BSF parasites; however, modification of enzyme-active phosphonates through the addition of pivaloyloxymethyl (POM) groups improved activity against T. brucei, with POM-modified (1,5-dihydroxy-2-oxopyrrolidin-3-yl) phosphonic acid (POMSF) and POMHEX having EC50 values of 0.45 ± 0.10 and 0.61 ± 0.08 µM, respectively. These findings suggest that HEX is a promising lead against T. brucei and that further development of prodrug HEX analogs is warranted. 

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4. An intrinsic oscillator drives the blood stage cycle of the malaria parasite Plasmodium falciparum

   https://doi.org/10.1126/science.aba4357  

   Smith, Lauren M.; Motta, Francis C.; Chopra, Garima; Moch, J. Kathleen; Nerem, Robert R.; Cummins, Bree; Roche, Kimberly E.; Kelliher, Christina M.; Leman, Adam R.; Harer, John; et al (May 2020, Science)

   The blood stage of the infection of the malaria parasite Plasmodium falciparum exhibits a 48-hour developmental cycle that culminates in the synchronous release of parasites from red blood cells, which triggers 48-hour fever cycles in the host. This cycle could be driven extrinsically by host circadian processes or by a parasite-intrinsic oscillator. To distinguish between these hypotheses, we examine the P. falciparum cycle in an in vitro culture system and show that the parasite has molecular signatures associated with circadian and cell cycle oscillators. Each of the four strains examined has a different period, which indicates strain-intrinsic period control. Finally, we demonstrate that parasites have low cell-to-cell variance in cycle period, on par with a circadian oscillator. We conclude that an intrinsic oscillator maintains Plasmodium ’s rhythmic life cycle. 

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5. Evaluating host diet effects on microparasites by measuring the stoichiometry of infrapopulations one cell at a time

   https://doi.org/10.1002/ece3.11645  

   Narr, Charlotte_F; Binger, Scott; Sedlacek, Erin; Anderson, Bianca; Shoemaker, Grace; Stanley, Adrienne; Stokoski, Madison; Hall, Ed (July 2024, Ecology and Evolution)

   Abstract Progress in the field of ecological stoichiometry has demonstrated that the outcome of ecological interactions can often be predicted a priori based on the nutrient ratios (e.g., carbon: nitrogen: phosphorus, C:N:P) of interacting organisms. However, the challenges of accurately measuring the nutrient content of active parasites within hosts has limited our ability to rigorously apply ecological stoichiometry to host–parasite systems. Traditional nutrient analyses require high parasite biomasses, often preventing individual‐level analyses. This prevents researchers from estimating variation in the nutrient content of individual parasites within a single host infrapopulation, a critical factor that could define how the ecology of the parasite affects the host–parasite interaction. Here, we explain how energy dispersive technology, a technique currently used to measure the elemental content of free‐living microbes, can be adapted for parasitic microbial infrapopulations. We demonstrate the power of accurately quantifying the biomass stoichiometry of individual microbial parasites sampled directly from individual hosts. Using this approach, we show that the stoichiometric composition of two microbial parasites capable of infecting the same host are stoichiometrically distinct and respond to host diet quality differently. We also demonstrate that characteristics of the stoichiometric trait distributions of these infrapopulations were important predictors of host fecundity, a proxy for virulence in this system, and better predictors of parasite load than the mean parasite stoichiometry or our parasite and diet treatments alone. EDS provides a rigorous tool for applying ecological stoichiometry to host–parasite systems and enables researchers to explore the nutritional physiology of host–parasite interactions at a scale that is more relevant to the ecology and evolution of the system than traditional nutrient analyses. Here we demonstrate that this level of resolution provides useful insights into the diet‐dependent physiology of microbial parasites and their hosts. We anticipate that this improved level of resolution has the potential to elucidate a range of eco–evo interactions in host–parasite systems that were previously unobservable. 

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