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Apicomplexan parasites cause persistent mortality and morbidity worldwide through diseases including malaria, toxoplasmosis, and cryptosporidiosis. Ca2+ signaling pathways have been repurposed in these eukaryotic pathogens to regulate parasite-specific cellular processes governing the transition between the replicative and lytic phases of the infectious cycle. Despite the presence of conserved Ca2+-responsive proteins, little is known about how specific signaling elements interact to impact pathogenesis. We mapped the Ca2+-responsive proteome of the model apicomplexan T. gondii via time-resolved phosphoproteomics and thermal proteome profiling. The waves of phosphoregulation following PKG activation and stimulated Ca2+ release corroborate known physiological changes but identify specific proteins operating in these pathways. Thermal profiling of parasite extracts identified many expected Ca2+-responsive proteins, such as parasite Ca2+-dependent protein kinases. Our approach also identified numerous Ca2+-responsive proteins that are not predicted to bind Ca2+, yet are critical components of the parasite signaling network. We characterized protein phosphatase 1 (PP1) as a Ca2+-responsive enzyme that relocalized to the parasite apex upon Ca2+ store release. Conditional depletion of PP1 revealed that the phosphatase regulates Ca2+ uptake to promote parasite motility. PP1 may thus be partly responsible for Ca2+-regulated serine/threonine phosphatase activity in apicomplexan parasites.

Funder: U.S. Department of Health & Human Services | National Institutes of Health (NIH), Funder: U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID), Two-pore channels (TPCs) are a ubiquitous family of cation channels that localize to acidic organelles in animals and plants to regulate numerous Ca2+-dependent events. Little is known about TPCs in unicellular organisms despite their ancient origins. Here, we characterize a TPC from Toxoplasma gondii, the causative agent of toxoplasmosis. TgTPC is a member of a novel clad of TPCs in Apicomplexa, distinct from previously identified TPCs and only present in coccidians. We show that TgTPC localizes not to acidic organelles but to the apicoplast, a non-photosynthetic plastid found in most apicomplexan parasites. Conditional silencing of TgTPC resulted in progressive loss of apicoplast integrity, severely affecting growth and the lytic cycle. Isolation of TPC null mutants revealed a selective role for TPCs in replication independent of apicoplast loss that required conserved residues within the pore-lining region. Using a genetically-encoded Ca2+ indicator targeted to the apicoplast, we show that Ca2+ signals deriving from the ER but not from the extracellular space are selectively transmitted to the lumen. Deletion of the TgTPC gene caused reduced apicoplast Ca2+ uptake and membrane contact site formation between the apicoplast and the ER. Fundamental roles for TPCs in maintaining organelle integrity, inter-organelle communication and growth emerge.

The tuberculosis drug candidate SQ109 targets the trehalose monomycolate transporter MmpL3 in Mycobacterium tuberculosis and also has activity against other pathogens. We found related proteins in 22 protozoa, including Trypanosoma cruzi and Entamoeba histolytica, as well as in archaea and other bacteria, including the fatty acid transporter, FarE. We show these proteins, alpha-MMPL proteins, adopt similar structures to that of MsMmpL3 having two sets (P1 and P2) of conserved active site/H+-transporter Asp, Tyr and Phe residues in “pentad” motifs (DYxxF) that can bind to the SQ109 ethylenediamine and adamantyl moieties. Based on structural comparisons with MsMmpL3, we find that there are superimposable transmembrane and H+-transporter structures in much larger proteins, beta-MMPLs, found in apicomplexan parasites, fungi, plants and animals. They also contain double “pentad” motifs in which the P1 Asp is totally conserved, but the P2 Asp may also be a Glu, and the P2 Phe seen in the alpha-MMPLs is a His that H-bonds to the P1 and P2 Asp/Glu residues. There are also 5 conserved Ser/Thr residues that extend the H-bond/H+-transporter network with 2 interacting directly with the P1 Asp, and the His. We propose that all MMPL proteins are involved in proton motive force-mediated lipid (phospholipid, glycolipid, sterol, fatty acid) transport, and that SQ109 may target some pathogens directly, by binding to the P1/P2 motifs. Overall, the results are of general interest since they indicate that there are two major classes of lipid transporters: alpha-MMPL proteins found in many bacteria and protozoa, and much larger, beta-MMPL proteins, found in fungi, apicomplexa, plants and animals and, in some cases, they are potential drug targets.

Bisphosphonates are the most commonly prescribed drugs for the treatment of osteoporosis and other bone illnesses. Some of them have also shown antiparasitic activity. In search of improving the pharmacological profile of commercial bisphosphonates, our group had previously developed first row transition metal complexes with N-containing bisphosphonates (NBPs). In this work, we extended our studies to heteroleptic palladium–NBP complexes including DNA intercalating polypyridyl co-ligands (NN) with the aim of obtaining potential multi-target species. Complexes of the formula [Pd(NBP)₂(NN)]·2NaCl·xH₂O with NBP = alendronate (ale) or pamidronate (pam) and NN = 1,10 phenanthroline (phen) or 2,2′-bipyridine (bpy) were synthesized and fully characterized. All the obtained compounds were much more active in vitro against T. cruzi (amastigote form) than the corresponding NBP ligands. In addition, complexes were nontoxic to mammalian cells up to 50–100 µM. Compounds with phen as ligand were 15 times more active than their bpy analogous. Related to the potential mechanism of action, all complexes were potent inhibitors of two parasitic enzymes of the isoprenoid biosynthetic pathway. No correlation between the anti-T. cruzi activity and the enzymatic inhibition results was observed. On the contrary, the high antiparasitic activity of phen-containing complexes could be related to their ability to interact with DNA in an intercalative-like mode. These rationally designed compounds are good candidates for further studies and good leaders for future drug developments. Four new palladium heteroleptic complexes with N-containing commercial bisphosphonates and DNA intercalating polypyridyl co-ligands were synthesized and fully characterized. All complexes displayed high anti-T. cruzi activity which could be related to the inhibition of the parasitic farnesyl diphosphate synthase enzyme but mainly to their ability to interact DNA., Centro de Química Inorgánica

Toxoplasma gondii , an obligate intracellular parasite, is capable of invading virtually any nucleated cell. Active invasion by the parasite is a precise process and intracellular parasites reside and replicate inside a parasitophorous vacuole (PV). The membrane of the PV functions as a molecular sieve allowing for its ionic milieu to be in equilibrium with the host cytosol. The parasite would thus be exposed to the ionic fluctuations of the host cytoplasm, such as increases in host cytosolic Ca2+ during Ca2+ signaling events. Ca2+ is a universal signaling molecule and both the host cell and T. gondii will utilize Ca2+ signaling to stimulate diverse cellular functions. Using T. gondii tachyzoites and host cells expressing genetically encoded Ca2+ indicators, we demonstrate that host Ca2+ signaling impacts intracellular Ca2+ levels of the parasite. We propose that Ca2+ influx derived from host Ca2+ signaling into the parasite is utilized to maintain the intracellular Ca2+ stores replenished as the parasite replicates within the low Ca2+ environment of the host cell. Intracellular Ca2+ store(s) release is needed to reach the initiation spike in Ca2+ that meets the threshold of Ca2+ needed to initiate the tightly coordinated process of egress. Post activation of the signal and subsequent microneme secretion, would cause breakdown of the host cell and allow for extracellular Ca2+ influx, causing a second, larger spike in Ca2+ and activation of the glideosome, the main motility machinery. To directly deliver precise concentrations of ions needed for egress, we used patch-clamped infected host cells and monitored parasite egress. In doing so, we discovered an alternative role for K+ in timing parasite egress. This is the first study using whole-cell patch clamp to study the role of ions such as K+ and Ca2+ in T. gondii egress.

Diverse microbial ecosystems underpin life in the sea. Among these microbes are many unicellular eukaryotes that span the diversity of the eukaryotic tree of life. However, genetic tractability has been limited to a few species, which do not represent eukaryotic diversity or environmentally relevant taxa. Here, we report on the development of genetic tools in a range of protists primarily from marine environments. We present evidence for foreign DNA delivery and expression in 13 species never before transformed and for advancement of tools for eight other species, as well as potential reasons for why transformation of yet another 17 species tested was not achieved. Our resource in genetic manipulation will provide insights into the ancestral eukaryotic lifeforms, general eukaryote cell biology, protein diversification and the evolution of cellular pathways., This collaborative effort was supported by the Gordon and Betty Moore Foundation EMS Program of the Marine Microbiology Initiative (grant nos. GBMF4972 and 4972.01 to F.-Y.B.; GBMF4970 and 4970.01 to M.A. and A.Z.W.; GBMF3788 to A.Z.W.; GBMF 4968 and 4968.01 to H.C.; GBMF4984 to V.H.; GBMF4974 and 4974.01 to C. Brownlee; GBMF4964 to Y. Hirakawa; GBMF4961 to T. Mock; GBMF4958 to P.S.; GBMF4957 to A.T.; GBMF4960 to G.J.S.; GBMF4979 to K.C.; GBMF4982 and 4982.01 to J.L.C.; GBMF4964 to P.J.K.; GBMF4981 to P.v.D.; GBMF5006 to A.E.A.; GBMF4986 to C.M.; GBMF4962 to J.A.F.R.; GBMF4980 and 4980.01 to S.L.; GBMF 4977 and 4977.01 to R.F.W.; GBMF4962.01 to C.H.S.; GBMF4985 to J.M.; GBMF4976 and 4976.01 to C.H.; GBMF4963 and 4963.01 to V.E.; GBMF5007 to C.L.D.; GBMF4983 and 4983.01 to J.L.; GBMF4975 and 4975.01 to A.D.T.; GBMF4973 and 4973.01 to I.R.-T. and GBMF4965 to N.K.), by The Leverhulme Trust (RPG-2017-364) to T. Mock and A. Hopes, and by ERD funds (16_019/0000759) from the Czech Ministry of Education to J.L.

Fluctuations of the cytosolic calcium ion (Ca2+) concentration regulate a variety of cellular functions in all eukaryotes. Cells express a sophisticated set of mechanisms to balance the cytosolic Ca2+ levels and the signals that elevate Ca2+ in the cytosol are compensated by mechanisms that reduce it. Alterations in Ca2+-dependent homeostatic mechanisms are the cause of many prominent diseases in humans, such as heart failure or neuronal death.The genetic tractability of Toxoplasma gondii and the availability of genetic tools enabled the use of Genetically Encoded Calcium Indicators (GECIs) expressed in the cytoplasm, which started a new era in the studies of Toxoplasma calcium signaling. It was finally possible to see Ca2+ oscillations prior to exit of the parasite from host cells. Years after Endo et al showed that ionophores triggered egress, the assumption that oscillations occur prior to egress from host cells has been validated by experiments using GECIs. GECIs allowed the visualization of specific Ca2+ signals in live intracellular parasites and to distinguish these signals from host cell calcium fluctuations. In this chapter we present an overview describing "tried and true" methods of our lab who pioneered the first use of GECI's in Toxoplasma, including GECI choice, methodology for transfection and selection of ideal clones, their characterization, and the use of GECI-expressing parasites for fluorometric and microscopic analysis.

1, Preparation of Terrific Broth 800 mL distilled H2O 12g Tryptone 24g Yeast extract 4 mL Glycerol Bring volume up to 900 mL with distilled H2O Autoclave and cool to room temperature Bring volume to 1000 mL with 100 mL filter sterilized solution containing 0.17M KH2PO4(2.314g – Potassium phosphate monobasic) 0.72M K2HPO4(16.43g – Potassium phosphate dibasic trihydrate) 2, Cells Rosetta-gami 2 cells expressing SaCas9 on pET32/Lic vector. Protein Expression Grow 10 mL starter culture overnight at 37°C (LB is fine) Inoculate 1 mL in 100 mL of Terrific Broth the next morning O.D. should be between 0.2 and 0.5 after 3 hours – if higher discard culture When O.D. is between 0.5 and 0.7 induce with IPTG and move o 18°C – grow overnight The next morning spin down and purify 3, Protein Purification Binding Buffer - 1 L - pH 8.0 Elution Buffer - 200 mL - pH 8.0 Lysis buffer 40 mL biding buffer 1 EDTA-free Protease Inhibitor pellet 300 mL P8849 500 mL lysozyme 10 mL DNase 10 mL RNase - Resuspend the pellet in 12 ml Lysis buffer - 1 hour on ice at 4°C - 80 seconds 30% sonication in 10 mL aliquotes (10 seconds on, 10 seconds off) - 12,000 rpm 20 minutes 4°C - Filter the supernatant with 0.45 uM filter to remove all bacterial - Purify the SaCas9 from the filtered supernatant by using a commercial His column (by following the manufacturer’s manual)

Toxoplasma gondii is an obligate intracellular parasite and replicates inside a parasitophorous vacuole (PV) within the host cell. The membrane of the PV (PVM) contains pores that permits for equilibration of ions and small molecules between the host cytosol and the PV lumen. Ca(2+) signaling is universal and both T. gondii and its mammalian host cell utilize Ca(2+) signals to stimulate diverse cellular functions. Egress of T. gondii from host cells is an essential step for the infection cycle of T. gondii and a cytosolic Ca(2+) increase initiates a Ca(2+) signaling cascade that culminates in the stimulation of motility and egress. In this work, we demonstrate that intracellular T. gondii tachyzoites are able to take up Ca(2+) from the host cytoplasm during host signaling events. Both intracellular and extracellular Ca(2+) sources are important in reaching a threshold of cytosolic Ca(2+) needed for successful egress. Two peaks of Ca(2+) were observed in single parasites that egressed with the second peak resulting from Ca(2+) entry. We patched infected host cells to allow the delivery of precise concentrations of Ca(2+) for stimulation of motility and egress. Using this approach of patching infected host cells allowed to determine that increasing the host cytosolic Ca(2+) to a specific concentration can trigger egress, which is further accelerated by diminishing the concentration of potassium (K(+)).

Author(s): Faktorova, Drahomira; Nisbet, R Ellen R; Fernandez Robledo, Jose A; Casacuberta, Elena; Sudek, Lisa; Allen, Andrew E; Ares, Manuel; Areste, Cristina; Balestreri, Cecilia; Barbrook, Adrian C; Beardslee, Patrick; Bender, Sara; Booth, David S; Bouget, Francois-Yves; Bowler, Chris; Breglia, Susana A; Brownlee, Colin; Burger, Gertraud; Cerutti, Heriberto; Cesaroni, Rachele; Chiurillo, Miguel A; Clemente, Thomas; Coles, Duncan B; Collier, Jackie L; Cooney, Elizabeth C; Coyne, Kathryn; Docampo, Roberto; Dupont, Christopher L; Edgcomb, Virginia; Einarsson, Elin; Elustondo, Pia A; Federici, Fernan; Freire-Beneitez, Veronica; Freyria, Nastasia J; Fukuda, Kodai; Garcia, Paulo A; Girguis, Peter R; Gomaa, Fatma; Gornik, Sebastian G; Guo, Jian; Hampl, Vladimir; Hanawa, Yutaka; Haro-Contreras, Esteban R; Hehenberger, Elisabeth; Highfield, Andrea; Hirakawa, Yoshihisa; Hopes, Amanda; Howe, Christopher J; Hu, Ian; Ibanez, Jorge; Irwin, Nicholas AT; Ishii, Yuu; Janowicz, Natalia Ewa; Jones, Adam C; Kachale, Ambar; Fujimura-Kamada, Konomi; Kaur, Binnypreet; Kaye, Jonathan Z; Kazana, Eleanna; Keeling, Patrick J; King, Nicole; Klobutcher, Lawrence A; Lander, Noelia; Lassadi, Imen; Li, Zhuhong; Lin, Senjie; Lozano, Jean-Claude; Luan, Fulei; Maruyama, Shinichiro; Matute, Tamara; Miceli, Cristina; Minagawa, Jun; Moosburner, Mark; Najle, Sebastian R; Nanjappa, Deepak; Nimmo, Isabel C; Noble, Luke; Novak Vanclova, Anna MG; Nowacki, Mariusz; Nunez, Isaac; Pain, Arnab; Piersanti, Angela; Pucciarelli, Sandra; Pyrih, Jan; Rest, Joshua S | Abstract: An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Using FACS to sort fluorescent Bodo cells Introduction: Fluorescence-activated cell sorting (FACS) is a powerful method to isolate cells expressing fluorescent proteins. It is a routine technique in our lab to enrich, and subclone cells that express exogenously introduced fluorescent reporter proteins. We developed this protocol to isolate Bodo saltans that express a fluorescent protein. Unlike our previous work to isolate T. gondii, or T cruzi, there are two major obstacles to the application of this technology to B. saltans. The first obstacle is that B. saltans needs bacteria as food in the culture so there are multiple organisms in the medium. We have to determine which cells are B. saltans, and which are feeder bacteria. The second problem is that the osmolarity of the solution used for standard FACS is not appropriate for B. saltans. B. saltans lives in low osmolarity solutions. We needed to determine a proper solution in which B. saltans can survive, and if it works with the FACS system. Step 1. Determine a FACS solution appropriate for B. saltans. The solution that runs in a flow cytometer is called sheath fluid. Our FACS facility has its standard sheath fluid. In order to choose a proper sheath fluid for B. saltans we needed to make sure that: 1) the cells can survive in this sheath fluid for a significant time, 2) the sheath fluid can still support FACS since there is a requirement for the presence of ions for FACS to work. We tested serial dilutions of the standard sheath fluid. We found that B. saltans dies almost instantly in standard sheath fluid, but can live for at least 20 minutes in 50% sheath fluid. We also determined that the minimal sheath fluid needed for FACS is 40% of the standard sheath fluid. So we decided to use this diluted sheath fluid. Step 2. Determine which is the population of B. saltans. Wild type cultures are passed through a 10 nm Nylon filter to remove big residues in the culture medium, and filtered cells are collected by centrifugation at 1,200 x g for 5 minutes. The cells are further washed three times by centrifugation at 1,200 x g for 5 minutes, and twice at 1,200 x g for 5 minutes. The washing step is to get rid of most bacteria in the culture medium. After the last wash, the cells are resuspended in culture medium. We sort the cells by size and shape. There are usually two major distinct populations. We sort each of the populations into a collection tube. After the sorting, the cells are checked by microscopy. We confirm that the population 1 is B. saltans, and that the cells can survive the FACS sorting when we use 40% of standard sheath fluid as our sorting solution. Fig 1: R1 is B. saltans population. We previously used MoFlo XDP Sorter From Beckman Coulter, now we are upgrading to Beckman Coulter MoFlo Astrios EQ version. In the SSC Vs FSC dot plot, R1 represents B. saltans cells. Step 3. Sorting of B. saltans transfected with a reporter gene. 1, 24 hours after electroporation, B. saltans (wild type and transfected) are filtered through a 10 nm Nylon membrane. The cells are collected by centrifugation at 1,000 x g for 5 minutes. 2, Cells are washed twice with autoclaved distilled water. 3, After the final wash, cells are resuspended in ~1-1.5 ml medium. We also prepare a collection tube with 2 ml fresh culture medium. 4, After the sorter has been set up, we first run the wild type control cells to set up the parameters for sorting. In the SSC Vc FSC dot plot, the B. saltans population is chosen. Then in the fluorescence plot, we use DsRed Vs GFP dot plot to set the gate. There are no cells in the gated area for wild type cells. 5, After the parameters are set, we run the transfected B. saltans. A small population of fluorescent cells should appear in the fluorescence plot. This is the population we want to sort into a collection tube. 6, After the sorting is done, cells are transferred to the collection tube and then to a T25 flask, adding 4 ml culture medium and a small colony of bacteria taken from a plate. 7, Cells are cultured for further analysis. Fig. 2. Representative sorting of B. saltans