SARCOCYSTIS NEURONA: INVESTIGATION OF HOST CELL INTERACTIONS THAT CONTRIBUTE TO PARASITE SURVIVAL

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1012262
• Project Start Date: Feb 15, 2017
• Project End Date: Sep 30, 2021
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF KENTUCKY
500 S LIMESTONE 109 KINKEAD HALL
LEXINGTON,KY 40526-0001

Performing Department
Veterinary Science

Non Technical Summary
Given the economic importance of EPM to the horse industry, more effective methods for diagnosis, treatment, and prevention are greatly needed for this significant neurologic disease. Improvements in these methods are partially hampered, however, by a lack of information about interactions between intracellular Sarcocystis neurona and its host cell. The proposed research will generate valuable data to help understain S. neurona's ability to invade cells and survive as an intracellular pathogen. Collectively, the results obtained by these studies will give greater insight into the biology of this coccidian parasite while producing information and reagents that can be exploited for the development of improved diagnostic assays for EPM, novel chemotherapeutics, and protective vaccines.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
313	4050	1103	100%

Knowledge Area
313 - Internal Parasites in Animals;

Subject Of Investigation
4050 - Protozoa;

Field Of Science
1103 - Other microbiology;

Keywords

equine parasite

intracellular pathogen

neurologic disease

coccidia

apicomplexa

Goals / Objectives
Apicomplexans are obligate intracellular parasites that must be able to invade into their host cell and then modify the intracellular environment to allow them to survive and propagate. Not surprisingly, the members of this phylum share a number of traits that allow them these capabilities. Conversely, this is also a diverse group of pathogens that have different host cell specificities, different life cycles, and inhabit different intracellular niches. Consequently, each parasite species has evolved unique characteristics that support their particular life style.Sarcocystis neurona is closely-related to the well-studied T. gondii, so it is anticipated that these two apicomplexan species will share many of the traits related to parasite-host cell interactions. However, S. neurona has some similarity to the distantly-related Theileria spp., in that both reside free in the host cell cytoplasm, which is profoundly different than the PV that surrounds T. gondii and many other apicomplexans. Therefore, it is anticipated that S. neurona will possess a number of traits that are quite distinct from its close relative T. gondii. To date, little is known about the parasite-host cell interactions of S. neurona. The following objectives will be pursued to help reduce this knowledge gap:Examine motility, invasion into the host cell, and establishment of the initial intracellular niche by S. neurona merozoites. Investigate protein secretion by S. neurona during invasion and intracellular growth.

Project Methods
Objective 1A) Confirm that S. neurona motility is actin-based. Toxoplasma gondii motility and host cell invasion is known to be dependent on the actin-myosin motor of the parasite. To confirm that S. neurona motility is actin-based, gliding assays will be performed in the presence or absence of cytochalasin D (CytD), which inhibits actin filament polymerization and paralyzes the parasite.B) Confirm that S. neurona microneme secretion and motility/invasion are dependent on intracellular calcium. Recently, we have shown that bumped-kinase inhibitors (BKIs) designed to target apicomplexan CDPK1 inhibit S. neurona both in vitro and in vivo, thus suggesting that motility and invasion by this parasite is regulated by calcium. To further document that S. neurona merozoite motility and invasion is calcium dependent, gliding assays as described above (Objective 1A) will be conducted in the presence of absence of BAPTA-AM, a chelator of calcium that is membrane permeant. Additionally, host cell invasion assays will be conducted with parasites that have been pre-treated with BAPTA-AM. To aid these invasion assays, an S. neurona clone that expresses YFP will be used. C) Establish the events during and after invasion that lead to "naked" S. neurona in the host cell cytoplasm. Intracellular T. gondii are bounded by a PV that is formed from the host cell surface membrane during invasion by the parasite, while intracellular S. neurona merozoites and developing schizonts are located free in the host cell cytoplasm. We hypothesize that S. neurona is surrounded initially by a vacuole derived from the host cell, like T. gondii but then the vacuole is lysed to release the parasite into the host cell cytoplasm, as observed for Theileria spp. To assess whether S. neurona merozoites invade into a host-derived vacuole that is subsequently lysed, pulse invasions will be conducted and the internalized merozoites will be examined for host cell membrane markers. Briefly, culture-derived S. neurona merozoites will be incubated in a low volume of medium and for a short period (2 minutes) with host cell monolayers seeded onto coverslips. The cultures will be washed extensively with PBS, and then either fixed immediately (T0) or returned to medium for a defined period before fixation (e.g., 2 min, 5 min, 10 min, 30 min, 1 hr). The host cells will either be pre-loaded with the lipid label DiIC16 or the coverslips will be labeled with antibodies against the GPI-anchored host cell surface proteins Sca-1 or CD55. It is anticipated that intracellular merozoites at early time points (e.g., T0, T2) will be surrounded by the DiIC16 and Sca-1/CD55 label, indicating that the parasite invades initially into a PV derived from the host cell. Soon after invasion (e.g., T5 and later), it is expected that the host membrane labels surrounding the parasite will disappear, indicating dissolution of the PVM.Investigate protein secretion by S. neurona during invasion and intracellular growth (Objective 2).A) Clone and characterize representative ROP protein orthologues that have been identified in the S. neurona genome/transcriptome/proteome databases. The rhoptry organelles contain effector molecules secreted by T. gondii into the host cell and several possess kinase activity that likely alter host cell function. The annotated genomes of S. neurona contain obvious ROP orthologues, and transcriptome analysis revealed transcripts for some of these ROP genes in the merozoite stage (unpublished data). To identify S. neurona proteins that are secreted into the host cell, a subset of ROP orthologues will be investigated. In silico prediction tools available in CLC Genomics Workbench and online will be used to better categorize the S. neurona-specific ROP genes based on predicted motifs, secretory leader peptides, subcellular location, glycosylation, etc. As we have done previously for a variety of S. neurona proteins, reagents will be generated to establish basic protein characteristics (immunolocalization, solubility, etc.) of the putative ROP proteins. As an alternative approach if antibody production proves unfeasible or uninformative, epitope tagged versions of the S. neurona proteins will be produced. To assess the role played by the S. neurona ROP proteins selected for investigation, the genes encoding these proteins will be targeted for disruption using the CRISPR/Cas9 gene editing system. Parameters such as host cell invasion, growth rate, and morphology of the resulting SNDgene clones will be compared to wild-type parasites to determine if disruption of the targeted ROP gene has resulted in phenotypic alteration.B) Clone and characterize the S. neurona PP2C-hn orthologue. Toxoplasma gondii expresses a PP2C that is secreted into the host cell cytoplasm and directed to the host nucleus, where it may play a role in altering host cell transcription. The S. neurona genome annotations predict one PP2C gene (ToxoDB; gene ID SN3_01700285) and at least 14 PP2C domain-containing proteins. As described for ROP genes in Objective 2A, the SnPP2C sequences will undergo in silico analyses to evaluate putative homology to TgPP2C-hn. Best candidates will be investigated using the experimental approaches outlined above in Objective 2A.C) Clone and characterize the two putative perforin-like protein 1 (PLP1) orthologues in S. neurona. A T. gondii perforin-like protein (TgPLP1) has been described as a microneme protein secreted by intracellular parasites to form pores in the PVM and host cell membrane, thus permitting egress by the parasite. Two genes predictions in the S. neurona genome have been annotated as PLP1 orthologues (ToxoDB; SN3_00200465, SN3_02300330), and might play a role in egress of S. neurona from the host cell. Moreover, it is conceivable that one or both of the putative SnPLP1 proteins could be involved in disrupting the PV soon after S. neurona invasion into the host cell (hypothesized in Objective 1C). As described in Objective 2A, the SnPLP1 sequences will undergo in silico analyses to evaluate putative homology to TgPLP1. Additionally, the putative SnPLP1 paralogues will be investigated using the experimental approaches outlined above in Objective 2A.D) Examine the kinetics of protein secretion by S. neurona during host cell invasion. In vitro, exocytosis of the dense granules and micronemes of T. gondii are temperature dependent, and discharge of the micronemes can be stimulated by elevating intracellular calcium. Secretion of ROP proteins is not apparent during incubation in vitro, so the trigger for rhoptry discharge remains uncertain. However, ROP proteins are found in the vesicles termed "evacuoles" (empty vacuoles) formed in the host cell cytoplasm when T. gondii tachyzoites are treated with CytD to prevent their invasion into the cell. We have previously shown that S. neurona secretion in vitro of a microneme protein SnMIC10 and a putative dense granule protein SnNTP1 is temperature dependent. The reagents produced in Objectives 2A, 2B, and 2C will be used in secretion assays to similarly assess the in vitro secretion kinetics of the SnROP, SnPP2C-hn, and SnPLP1 proteins. Based on what is known for T. gondii, it is expected that the SnROP proteins will not be present in the secreted fractions. Additionally, the potential role of calcium in stimulating exocytosis of the different S. neurona secretory proteins will be examined. Again, secretion of the SnROPs by calcium stimulation is not expected, although secretion of SnMIC10 should be enhanced. Finally, CytD-treated S. neurona merozoites will be used to generate evacuoles in host cells, and immunofluorescence will be performed to assess the protein content of the evacuoles. It is anticipated that some or all of the SnROP proteins will be localized to the evacuoles.

Progress 02/15/17 to 09/30/21

Outputs
Target Audience:This project serves research scientists, veterinary practitioners, and horse owners. Changes/Problems:Research activities were significantly curtailed during the Covid-19 pandemic. Additionally, a research scientist in the lab decided to retire during the pandemic, and her position was not re-filled due to the budget cut mandated by the university. Consequently, it was not possible for us to accomplish some of the objectives of the project that required extensive wet-bench research. During the time that laboratory research was restricted, we focused on using existing sequence information for S. neurona to conduct in silico analyses. This work has revealed interesting and important aspects of the parasite's biology, including unique aspects of its mRNA polyadenylation machinery. This foundational research has the prospect of leading to the identification of potential new drug targets for treatment of infections caused by Sarcocystis and related pathogens such as Toxoplasma gondii and Neospora spp. What opportunities for training and professional development has the project provided?During the current review period, the project has provided training for three graduate students (Parul Suri, Izabela Rocha, and Jamie Norris). How have the results been disseminated to communities of interest?The findings of our studies have been published in journal articles and presented at research conferences. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective #1, Examine motility, invasion into the host cell, and establishment of the initial intracellular niche by S. neurona merozoites. Sub-objective A) Confirm that S. neurona motility is actin-based. This sub-objective has been accomplished. Experiments examining S. neurona merozoites in the presence of the actin inhibitor cytochalasin D yielded non-motile parasites. These assays confirmed that S. neurona motility is actin based. Sub-objective B) Confirm that S. neurona microneme secretion and motility/invasion are dependent on intracellular calcium. To demonstrate that S. neurona organelle secretion and motility/invasion dependent on calcium signaling, we have used BAPTA-AM, a chelator of calcium that is able to permeate the parasite cellular membrane. Contradictory to our expectations, gliding assays of S. neurona merozoites treated with BAPTA-AM showed parasites that were still motile; no reduction in gliding was observed. These results suggest that organelle secretion is not required for S. neurona motility. Sub-objective C) Establish the events during and after invasion that lead to "naked" S. neurona in the host cell cytoplasm. These experiments have encountered significant complications involving cross-labeling of the parasites during the experiments, thus requiring amendment and repeat of the experiments with the modifications. After significant unsuccessful efforts to troubleshoot, it was decided that these experiments should be suspended. Objective #2, Investigate protein secretion by S. neurona during invasion and intracellular growth. Sub-objective A) Clone and characterize representative ROP protein orthologues that have been identified in the S. neurona genome/transcriptome/proteome databases. Due to the Covid-19 pandemic, research activities were significantly curtailed. Additionally, a research scientist in the lab decided to retire during the pandemic, and her position was not re-filled due to the budget cut mandated by the university. Consequently, it was not possible for us to accomplish this sub-objective. Sub-objective B) Clone and characterize the S. neurona PP2C-hn orthologue. Further in-depth in silico analyses of the predicted SnPP2C gene (ToxoDB; gene ID SN3_01700285) implied that this gene is not a homologue of the T. gondii TgPP2C-hn, which is targeted to the infected host cell nucleus. Consequently, this S. neurona gene will not be pursued further. Although there were significant restrictions on wet-lab research activities during the Covid-19 pandemic, we were able to focus on in silico studies that utilized existing sequence information that had been generated for S. neurona. These studies include an ongoing collaboration with Dr. Art Hunt in the UK Department of Plant and Soil Sciences examining the critical function of mRNA polyadenylation in S. neurona and related parasites and its potential as a target for chemotherapeutic intervention. This work has resulted in two journal articles. The results from these studies have also led to several grant applications to federal funding agencies (USDA NIFA and NIH). Additional bioinformatic studies that have been feasible over the past 8 months include examination of genomic variation across strains of S. neurona and within a single strain (SN3) after decades of in vitro propagation. These studies are expected to yield interesting and important information regarding parasite evolution in nature and in cell culture.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Ojo, K.K., S. Dangoudoubiyam, S.K. Verma, S. Scheele, A.E. DeRocher, M. Yeargan, R. Choi, T.R. Smith, K.L Rivas, M.A. Hulverson, L.K. Barrett, E. Fan, D.J. Maly, M. Parsons, J.P. Dubey, D.K. Howe, W.C. Van Voorhis. Sarcocystis neurona and antiprotozoal bumped kinase inhibitors. EPM Society 2nd EPM Workshop, Lake Tahoe, CA, 2017.
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Hunt, A.G., D.K. Howe, A. Brown, M. Yeargan. 2021. Transcriptional dynamics in the protozoan parasite Sarcocystis neurona and mammalian host cells after treatment with a specific inhibitor of apicomplexan mRNA polyadenylation. PLOS ONE 16(10): e0259109. doi.org/10.1371/journal.pone.0259109.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Gubbels, M.J., C.D. Keroack, S. Dangoudoubiyam*, H.L. Worliczek, A.S. Paul, C. Bauwens, B. Elsworth, K. Engleberg, D.K. Howe, I. Coppens, and M.T. Duraisingh. 2020. Fussing about fission: defining variety among mainstream and exotic apicomplexan cell division modes. Frontiers in Cellular and Infection Microbiology, 10:269, doi: 10.3389/fcimb.2020.00269.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Stevens, A.T., D.K. Howe, A.G. Hunt. 2018. Characterization of mRNA polyadenylation in the Apicomplexa. PLOS ONE 13(8):e0203317. doi: 10.1371/journal.pone.0203317.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Howe, D.K., M. Yeargan*, L. Simpson*, S. Dangoudoubiyam*. 2018. Molecular Genetic Manipulation of Sarcocystis neurona. Current Protocols in Microbiology 48, 20D.2.1-20D.2.14. doi: 10.1002/cpmc.48.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Dubey, R., B. Harrison, S. Dangoudoubiyam*, G. Bandini, K. Cheng, A. Kosber, C.A. Nersesian, D.K. Howe, J. Samuelson, D.J.P. Ferguson, and M.J. Gubbels. 2017. Differential roles for IMC proteins across Toxoplasma gondii and Sarcocystis neurona development. mSphere, 2(5): e00409-17. DOI: 10.1128/mSphere.00409-17.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Van Voorhis, W.C., J.S. Doggett, M. Parsons, M.A. Hulverson, R. Choi, S. Arnold, M.W. Riggs, A. Hemphill, D.K. Howe, R.H. Mealey, A.O.T. Lau, E.A. Merritt, D.J. Maly, E. Fan, K.K. Ojo. 2017. Extended-spectrum antiprotozoal bumped kinase inhibitors: A review. Experimental Parasitology, doi.org/10.1016/j.exppara.2017.01.001
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Rocha, I., M.A. Hulverson, R. Choi, L.K. Barrett, S.L. Arnold, W.C. Van Voorhis, S. Reed, D.K. Howe, A.E. Page. Pharmacokinetics of bumped kinase inhibitor 1748 in horses: investigational therapeutic for EPM. Conference of Research Workers in Animal Diseases, Chicago, IL 2021.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Norris, J*., S. Dangoudoubiyam*, D. Howe. Whole genome sequencing to identify genetic variations in isolates of Sarcocystis neurona. 5th International Meeting on Apicomplexan Parasites in Farm Animals (ApicoWplexa), Berlin, Germany 2019.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Norris, J*., S. Dangoudoubiyam*, D. Howe. The Effect of Long-Term Cell-Culture Passage on Sarcocystis neurona Genome Content and Predicting Genes Necessary for Completion of the Parasite Life Cycle. 27th Conference of the World Association for the Advancement of Veterinary Parasitology (WAAVP 2019), Madison, WI 2019.

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:This project serves research scientists, veterinary practitioners, and horse owners. Changes/Problems:Many of the experiments associated with this project are fully dependent on cell culture of the parasites. These experiments were drastically curtailed over the past 8 months dur to Covid-19 pandemic-mandated restrictions on laboratory activities. Only recently have these experiments resumed. What opportunities for training and professional development has the project provided?During the current review period, the project has provided training for three graduate students (Parul Suri, Izabela Rocha, and Jamie Norris). How have the results been disseminated to communities of interest?Results from work associated with the project have been reported as scientific conferences (2 conferences) and published in scientific journals (3 articles). What do you plan to do during the next reporting period to accomplish the goals?As it becomes possible to lessen restrictions on laboratory activities, we will resume experiments that are heavily dependent on cell culture. Additional experiments addressing Objective 1B, confirm that S. neurona microneme secretion and motility/invasion are dependent on intracellular calcium, include host cell invasion assays using parasites that have been pre-treated with BAPTA-AM and secretion assays to assess whether microneme protein secretion by S. neurona is inhibited in the presence of BAPTA-AM. To address experimental issues with Objective 1C, establish the events during and after invasion that lead to "naked" S. neurona in the host cell cytoplasm, the experiments are being repeated using different host cells (3T3 murine fibroblasts and HeLa cells), a different lipid label that is less likely to transfer between cells, and different host cell surface molecules (Sca-1 and Melanotransferrin). These experiments will be conducted by graduate student Parul Suri as part of her dissertation research. For Objective 2A, our studies will start to focus on individual ROP proteins to determine their localization in extracellular merozoites, intracellular schizonts, and in infected host cells. Additionally, attempts will be made to disrupt these genes using CRISPR/Cas9 technology to assess the function of these ROP proteins. To aid characterization of the putative secretory proteins, we are currently working on generating a strain of S. neurona that has a disrupted KU80 gene. The KU80 enzyme is responsible for the non-homologous end-joining pathway of DNA repair. Toxoplasma gondii deficient in this pathway have been shown to preferentially integrate transgenes into homologous sites, thus providing much greater opportunity for endogenous gene tagging. The S. neurona KU80 knockout strain that we produce will be used to fuse epitope tags (e.g., HA tag) to the genes of interest (e.g., ROP or PLP1 homologues). These clones containing HA-tagged genes will then be used to characterize the proteins, particularly their cellular localization in the parasites and the infected host cells.

Impacts
What was accomplished under these goals? Objective #1, Examine motility, invasion into the host cell, and establishment of the initial intracellular niche by S. neurona merozoites. Sub-objective A) Confirm that S. neurona motility is actin-based. This sub-objective has been accomplished. Experiments examining S. neurona merozoites in the presence of the actin inhibitor cytochalasin D yielded non-motile parasites. These assays confirmed that S. neurona motility is actin based. Sub-objective B) Confirm that S. neurona microneme secretion and motility/invasion are dependent on intracellular calcium. To demonstrate that S. neurona organelle secretion and motility/invasion dependent on calcium signaling, we have used BAPTA-AM, a chelator of calcium that is able to permeate the parasite cellular membrane. Contradictory to our expectations, gliding assays of S. neurona merozoites treated with BAPTA-AM showed parasites that were still motile; no reduction in gliding was observed. These results suggest that organelle secretion is not required for S. neurona motility. Sub-objective C) Establish the events during and after invasion that lead to "naked" S. neurona in the host cell cytoplasm. These experiments have encountered significant complications involving cross-labeling of the parasites during the experiments, thus requiring amendment and repeat of the experiments with the modifications. These studies are fully dependent on cell culture of the parasites, which has been drastically curtailed over the past 8 months. Only recently have these experiments resumed. Objective #2, Investigate protein secretion by S. neurona during invasion and intracellular growth. Sub-objective A) Clone and characterize representative ROP protein orthologues that have been identified in the S. neurona genome/transcriptome/proteome databases. The rhoptry organelles of apicomplexans contain effector molecules (ROP proteins) that are secreted into the host cell and likely possess activities that alter host cell function. Our prior studies analyzing transcriptome data revealed transcripts for multiple ROP and rhoptry neck (RON) proteins. These significant findings provide novel information about the biology of this ostensibly rhoptry-less stage of S. neurona. As mentioned, cell culture of the parasites has been curtailed over the past 8 months. Consequently, we have not yet had opportunities to progress further with the characterization of the ROP and RON proteins. Sub-objective B) Clone and characterize the S. neurona PP2C-hn orthologue. Further in-depth in silico analyses of the predicted SnPP2C gene (ToxoDB; gene ID SN3_01700285) implied that this gene is not a homologue of the T. gondii TgPP2C-hn, which is targeted to the infected host cell nucleus. Consequently, this S. neurona gene will not be pursued further. During the time it has not been possible to proceed with cell culture-dependent experiments, we have focused on in silico studies that utilize existing sequence information for S. neurona. These studies include an ongoing collaboration with Dr. Art Hunt in the UK Department of Plant and Soil Sciences examining the critical function mRNA polyadenylation in S. neurona and related parasites. This work has generated one prior journal article, with a second manuscript in preparation. The results from these studies have also led to several grant applications to federal funding agencies (USDA NIFA and NIH). Additional bioinformatic studies that have been feasible over the past 8 months include examination of genomic variation across strains of S. neurona and within a single strain (SN3) after decades of in vitro propagation. These studies are expected to yield interesting and important information regarding parasite evolution in nature and in cell culture.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Stevens, A.T., D.K. Howe, A.G. Hunt. 2018. Characterization of mRNA polyadenylation in the Apicomplexa. PLOS ONE 13(8):e0203317. doi: 10.1371/journal.pone.0203317.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Gubbels, M.J., C.D. Keroack, S. Dangoudoubiyam, H.L. Worliczek, A.S. Paul, C. Bauwens, B. Elsworth, K. Engleberg, D.K. Howe, I. Coppens, and M.T. Duraisingh. 2020. Fussing about fission: defining variety among mainstream and exotic apicomplexan cell division modes. Frontiers in Cellular and Infection Microbiology, 10:269, doi: 10.3389/fcimb.2020.00269.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Dubey, R., B. Harrison, S. Dangoudoubiyam, G. Bandini, K. Cheng, A. Kosber, C.A. Nersesian, D.K. Howe, J. Samuelson, D.J.P. Ferguson, and M.J. Gubbels. 2017. Differential roles for IMC proteins across Toxoplasma gondii and Sarcocystis neurona development. mSphere, 2(5): e00409-17. DOI: 10.1128/mSphere.00409-17.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Norris, J., S. Dangoudoubiyam, D. Howe. The Effect of Long-Term Cell-Culture Passage on Sarcocystis neurona Genome Content and Predicting Genes Necessary for Completion of the Parasite Life Cycle. 27th Conference of the World Association for the Advancement of Veterinary Parasitology (WAAVP 2019), Madison, WI 2019.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Norris, J., S. Dangoudoubiyam, D. Howe. Whole genome sequencing to identify genetic variations in isolates of Sarcocystis neurona. 5th International Meeting on Apicomplexan Parasites in Farm Animals (ApicoWplexa), Berlin, Germany 2019.

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:This project serves research scientists, veterinary practitioners, and horse owners. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?During the current review period, the project has provided training for two graduate students (Parul Suri and Jamie Norris). How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?Additional experiments addressing Objective 1B, confirm that S. neurona microneme secretion and motility/invasion are dependent on intracellular calcium, include host cell invasion assays using parasites that have been pre-treated with BAPTA-AM and secretion assays to assess whether microneme protein secretion by S. neurona is inhibited in the presence of BAPTA-AM. To address experimental issues with Objective 1C, establish the events during and after invasion that lead to "naked" S. neurona in the host cell cytoplasm, the experiments are being repeated using different host cells (3T3 murine fibroblasts and HeLa cells), a different lipid label that is less likely to transfer between cells, and different host cell surface molecules (Sca-1 and Melanotransferrin). These experiments will be conducted by graduate student Parul Suri as part of her dissertation research. For Objective 2A, our studies will start to focus on individual ROP proteins to determine their localization in extracellular merozoites, intracellular schizonts, and in infected host cells. Additionally, attempts will be made to disrupt these genes using CRISPR/Cas9 technology to assess the function of these ROP proteins. To aid characterization of the putative secretory proteins, we are currently working on generating a strain of S. neurona that has a disrupted KU80 gene. The KU80 enzyme is responsible for the non-homologous end-joining pathway of DNA repair. Toxoplasma gondii deficient in this pathway have been shown to preferentially integrate transgenes into homologous sites, thus providing much greater opportunity for endogenous gene tagging. The S. neurona KU80 knockout strain that we produce will be used to fuse epitope tags (e.g., HA tag) to the genes of interest (e.g., ROP or PLP1 homologues). These clones containing HA-tagged genes will then be used to characterize the proteins, particularly their cellular localization in the parasites and the infected host cells.

Impacts
What was accomplished under these goals? Objective #1, Examine motility, invasion into the host cell, and establishment of the initial intracellular niche by S. neurona merozoites. Sub-objective A) Confirm that S. neurona motility is actin-based. This sub-objective has been accomplished. Experiments examining S. neurona merozoites in the presence of the actin inhibitor cytochalasin D yielded non-motile parasites. These assays confirmed that S. neurona motility is actin based. Sub-objective B) Confirm that S. neurona microneme secretion and motility/invasion are dependent on intracellular calcium. To demonstrate that S. neurona organelle secretion and motility/invasion dependent on calcium signaling, we have used BAPTA-AM, a chelator of calcium that is able to permeate the parasite cellular membrane. Contradictory to our expectations, gliding assays of S. neurona merozoites treated with BAPTA-AM showed parasites that were still motile; no reduction in gliding was observed. These results suggest that organelle secretion is not required for S. neurona motility. Sub-objective C) Establish the events during and after invasion that lead to "naked" S. neurona in the host cell cytoplasm. In contrast to the related apicomplexan Toxoplasma gondii, intracellular S. neurona merozoites and developing schizonts do not have a parasitophorous vacuole (PV) surrounding them and instead are free in the host cell cytoplasm. To assess whether S. neurona merozoites invade directly into the host cell cytoplasm or into a host-derived vacuole that is subsequently lysed, pulse invasions have been conducted using the lipid label DiIC16 or antibodies against host surface proteins to reveal host surface membrane. Our expectation is that the newly-invaded parasites will be surrounded by the DiIC16 and surface protein label at early time points (e.g., within 5 minutes post invasion), indicating that the parasite invades initially into a PV derived from the host cell. We then expect that the host membrane labels surrounding the parasite will disappear at later time points, indicative of PV dissolution. These experiments are being conducted by a graduate student, Parul Suri. Thus far, Ms. Suri has encountered significant complications involving cross-labeling of the parasites during the experiments, which prevents our ability to interpret the results. Currently, Ms. Suri is re-doing the experiments using different host cells (3T3 murine fibroblasts and HeLa cells), a different lipid label that is less likely to transfer between cells, and different host cell surface molecules (Sca-1 and Melanotransferrin). Objective #2, Investigate protein secretion by S. neurona during invasion and intracellular growth. Sub-objective A) Clone and characterize representative ROP protein orthologues that have been identified in the S. neurona genome/transcriptome/proteome databases. The rhoptry organelles of apicomplexans contain effector molecules (ROP proteins) that are secreted into the host cell and likely possess activities that alter host cell function. Our prior studies analyzing transcriptome data revealed transcripts for multiple ROP and rhoptry neck (RON) proteins. These significant findings provide novel information about the biology of this ostensibly rhoptry-less stage of S. neurona. Sub-objective B) Clone and characterize the S. neurona PP2C-hn orthologue. Further in-depth in silico analyses of the predicted SnPP2C gene (ToxoDB; gene ID SN3_01700285) implied that this gene is not a homologue of the T. gondii TgPP2C-hn, which is targeted to the infected host cell nucleus. Consequently, this S. neurona gene will not be pursued further.

Publications

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:This project serves research scientists, veterinary practitioners, and horse owners. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?During the current review period, the project has provided training for two graduate students (Parul Suri and Jamie Norris). How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?The key reagents are in hand to accomplish experiments addressing Objective 1B, confirm that S. neurona microneme secretion and motility/invasion are dependent on intracellular calcium. These experiments will be conducted by graduate student Parul Suri as part of her dissertation research. For Objective 2A, studies will focus on individual ROP proteins to determine their localization in, extracellular merozoites, intracellular schizonts, and in infected host cells. Additionally, attempts will be made to disrupt these genes using CRISPR/Cas9 technology to assess the function of these ROP proteins.

Impacts
What was accomplished under these goals? Objective #1, Examine motility, invasion into the host cell, and establishment of the initial intracellular niche by S. neurona merozoites. Sub-objective C) Establish the events during and after invasion that lead to "naked" S. neurona in the host cell cytoplasm. In contrast to the related apicomplexan Toxoplasma gondii, intracellular S. neurona merozoites and developing schizonts do not have a parasitophorous vacuole (PV) surrounding them and instead are free in the host cell cytoplasm. To assess whether S. neurona merozoites invade directly into the host cell cytoplasm or into a host-derived vacuole that is subsequently lysed, pulse invasions are being conducted using the lipid label DiIC16 or antibodies against the host surface protein CD55 to reveal host surface membrane. Our expectation is that the newly-invaded parasites will be surrounded by the DiIC16 and CD55 label at early time points (e.g., within 5 minutes post invasion), indicating that the parasite invades initially into a PV derived from the host cell. We then expect that the host membrane labels surrounding the parasite will disappear at later time points, indicative of PV dissolution. These experiments are being conducted by a new graduate student, Parul Suri. Much of Ms. Suri's efforts thus far have focused on optimization of the pulse-invasion assay and the IFA conditions. Once completed, these studies will provide important information about the intracellular biology of this important agricultural pathogen. Objective #2, Investigate protein secretion by S. neurona during invasion and intracellular growth. Sub-objective A) Clone and characterize representative ROP protein orthologues that have been identified in the S. neurona genome/transcriptome/proteome databases. The rhoptry organelles of apicomplexans contain effector molecules (ROP proteins) that are secreted into the host cell and likely possess activities that alter host cell function. To aid characterization of ROP proteins and their functions in S. neurona, we have generated transcriptome data that reveal differential expression of ROP proteins during the merozoite and schizont stages. Transcripts for the rhoptry protein homologues ROP9, 14, 19, 20, 27, 24, 26, 39, and two homologues to ROP40 were found to be abundant in the merozoite stage. Evidence for ROP9, 19A, 21, 26, 27, 39, and 40 was also found in the merozoite proteome, thus confirming expression of these homologues. Additionally, homologues to three rhoptry neck (RON) proteins, RON6, RON8 and RON9, showed distinct transcript upregulation in merozoites. Both RONs and ROPs identified in the merozite transcriptome appeared to be distinctly upregulated in merozoite stage, with the FPKM values of these transcripts in the schizont stages being low across the entirety of schizont development (all time points). Interestingly, transcripts of ROP21 and ROP30 and toxolysin TLN1 were found to be abundant in the schizont stages. Based on FPKM values, ROP21 appears to be abundant only during the early schizont stage (24h time point) while ROP30 and TLN1 appear to be abundant during both early (24h) and mid schizont (48h) stages and maintain similar transcript levels. Collectively, these findings are significant since they provide novel information about the biology of this ostensibly rhoptry-less stage of S. neurona.

Publications

Progress 02/15/17 to 09/30/17

Outputs
Target Audience:This project serves research scientists, veterinary practitioners, and horse owners. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?During the current review period, the project has provided training for two first-year graduate students (Rachel Womack and Jamie Norris). How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?Gliding assays conducted with CytD thus far have generated qualitative results (i.e. presence/absence of surface antigen trails). Additional gliding assays in the presence of CytD will be performed to generate quantitative data. Although not yet fully defined, it is probable that these data will be obtained using ImageJ software to measure trail length. The key reagents have been obtained to perform experiments to address Objective 1B, confirm that S. neurona microneme secretion and motility/invasion are dependent on intracellular calcium, and Objective 1C, establish the events during and after invasion that lead to "naked" S. neurona in the host cell cytoplasm. These experiments will be initiated in early 2018.

Impacts
What was accomplished under these goals? Objective #1, Examine motility, invasion into the host cell, and establishment of the initial intracellular niche by S. neurona merozoites. Sub-objective A) Confirm that S. neurona motility is actin-based. As shown initially for T. gondii, motility and host cell invasion by multiple apicomplexans is known to be dependent on the actin-myosin motor of the parasite. To confirm that S. neurona motility is actin-based, initial gliding assays have been performed in the presence or absence of cytochalasin D (CytD), which inhibits actin filament polymerization and paralyzes the parasite. Trails of surface antigen deposited by gliding S. neurona merozoites were labeled with polyclonal antibody against the major S. neurona surface antigen SnSAG1. These experiments demonstrated that trail deposition was inhibited by CytD in a dose dependent fashion, supporting the hypothesis that S. neurona motility is dependent on the fidelity of parasite actin polymerization.

Publications