Source: PURDUE UNIVERSITY submitted to
CHARACTERIZATION OF THE SARCOCYSTIS NEURONA PROTEOME BASED ON CELL-CULTURED MEROZOITES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
ANIMAL HEALTH
Reporting Frequency
Annual
Accession No.
0202803
Grant No.
(N/A)
Project No.
IND020390AH
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2009
Project End Date
Sep 30, 2012
Grant Year
(N/A)
Project Director
Camp, J.
Recipient Organization
PURDUE UNIVERSITY
(N/A)
WEST LAFAYETTE,IN 47907
Performing Department
Veterinary Comparative Pathobiology
Non Technical Summary
Equine protozoal myeloencephalitis (EPM) is caused by a protozoan (one-celled) parasitic organism that infects horses when they ingest the infective stage as they eat contaminated grain, hay, or pasture grasses. The infective stage of the parasite is found in the feces of opossums which are especially common in the eastern U.S. Hence, horse facilities often have populations of opossums on-site or nearby and contamination is a very real threat. Horses are abnormal (aberrant) hosts and the parasites migrate to and then infect the CNS (brain and spinal cord) after ingestion. Infection can lead to many nervous system problems that may pass if the horse's immune system destroys the parasites. In some horses the nervous system problems get progressively worse and may lead to recumbency and death. Unfortunately, the clinical signs associated with EPM are also characteristic of other nervous system problems and it is very difficult for veterinarians to diagnose this disease. Even the methods that exist for diagnosis can be inaccurate due to contamination or the possibility of false positive reactions. The purpose of the proposed research is to identify proteins produced by the parasites that are found in the fluid that flows through the brain and spinal cord spaces. This fluid is called cerebrospinal fluid. At this time, these proteins have not been identified but once they are identified they will be used as biomarkers for the early diagnosis of the disease. These proteins should also provide a more accurate diagnosis without the problems associated with false positive tests. These proteins may also be used in research designed to create new drug treatments for the disease.
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
31338101110100%
Knowledge Area
313 - Internal Parasites in Animals;

Subject Of Investigation
3810 - Horses, ponies, and mules;

Field Of Science
1110 - Parasitology;
Goals / Objectives
The overall goal of this research project is to characterize key proteins in the Sarcocystis neurona proteome. The research hypothesis is that proteins unique to S. neurona will be identified. The specific objective of this research project is to identify S. neurona surface membrane and excretory-secretory proteins. Together, these proteins will provide a general proteome signature of S. neurona that will be added to appropriate international proteomics databases. These studies are considered to be discovery in nature as little information currently exists on the proteome of S. neurona. Upon completion of the aforementioned objective, this project has the potential to classify unique proteins that can be used to diagnose equine protozoal myeloencephalitis (EPM) and provide the opportunity to develop a rapid, specific diagnostic test for the disease. However, consideration of the diagnostic potential of these unique proteins will not be done in the current study but in separate, follow-up studies.
Project Methods
Objective 1. Cultures of S. neurona will be maintained in tissue culture flasks containing monolayers of bovine turbinate (BT) cells. Monolayers will be supplemented with 10% FBS in RPMI 1640 medium, pH 7.4 with 100 U/mL streptomycin, and 0.25 mg/mL fungizone. After cell lysis, S. neurona merozoites will be passed through 20 G, 23 G, and 25 G needles and filtered through a 3 micrometer filter to remove host cell debris. The desired inoculum will then be added to the cell culture flask containing fresh BT cells. S. neurona passage to new culture flasks and fresh BT host cells will occur every 5 to 7 days. Uninfected BT host cells will be fed once per week and split every 2-3 weeks to maintain a viable population. Merozoites from the S. neurona cultures will be used for the proteomic studies that utilize two-dimensional gel electrophoresis (2-DGE) and matrix-assisted laser desorption/ionization-time of flight tandem mass spectrometry (MALDI-TOF/MS). Peptide sequence interpretation and protein identification will be accomplished using computer search algorithms that correlate MS/MS data with predicted amino acid sequences contained in protein or genome sequence databases. S. neurona merozoites will be resuspended in PBS. S. neurona proteins will be concentrated using molecular weight cut off columns (MWCO). Individual proteins in each fraction will then be separated and analyzed using 2-DGE, followed by in-gel trypsin digestion. MALDI-TOF/MS/MS analysis of the resultant tryptic peptides from each protein spot will be used to determine the peptide masses and the molecular weights of the individual amino acid sequences. These peptide mass fingerprints will be used to identify the specific amino acid sequences by comparing them to peptide sequences from a cDNA library or EST (expressed sequence tag) database. The experimental, tryptic peptide masses will be matched with peptide masses derived in silico in database search engines such as the Swiss-Prot ExPASy Molecular Biology Server (http://www.expasy.ch/), the basic local alignment search tool (BLAST) available on the National Center for Biotechnology Information (NCBI) server, Protein Pilot, or the MASCOT databases. These database search engines compare the experimental precursor m/z ions from each MS/MS scan with hypothetical peptide m/z values from the database. Hypothetical peptide masses from the database that correspond with the experimental mass values are then assigned probability scores for protein identification. dbESTs for closely related parasites such as Toxoplasma gondii and Neospora caninum will also be searched to identify homologous sequences.

Progress 10/01/09 to 09/30/12

Outputs
OUTPUTS: There were no outputs this year as my new graduate student left the program in June of 2012 and she has not been replaced. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Veterinarians PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Although the graduate student left the program, Dr. Camp recently submitted a Sarcosystis neurona sample to the Bindley Proteomics Facility for an analysis of the proteins. The analysis identified six unique Sarcocystis neurona proteins. The following specific proteins were identified in this preliminary proteomics studies: surface antigen 1 (snSAG1), surface antigen 2 (snSAG2), surface antigen 3 (snSAG3), enolase-2, microneme 10 antigen (snMIC10), and nucleoside triphosphate hydrolase. Numerous other proteins were found when databases for Toxoplasma gondii and Neospora caninum were searched in addition to the S. neurona database. The fact that most of these proteins were not specifically identified as S. neurona proteins highlights the lack of proteome information in the current databases and emphasizes the need for such research. Our work is designed to meet this need. There are no other impacts or outcomes at this time.

Publications

  • No publications reported this period


Progress 10/01/10 to 09/30/11

Outputs
OUTPUTS: There were no outputs this year as my previous graduate student left the program in December of 2010 and I did not get a replacement for that student until June of 2011. The new graduate student is trying to come up to speed at this time. PARTICIPANTS: Kavita Sharma joined the laboratory and will begin working on the project in 2012. She is a Ph.D. student who replaced Lisa Keefe. Ms. Keefe left the laboratory to join another graduate program on campus at Purdue University. TARGET AUDIENCES: Research Veterinarians PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Changes in knowledge revolve around the identification of the proteins as described here. The following specific proteins were identified in the preliminary proteomics studies: heat shock protein 70 (Toxoplasma gondii), microneme 10 antigen (snMIC10, S. neurona), β-tubulin (T. gondii), actin (T. gondii), enolase-2 (T. gondii), and merozoite surface antigen (snSAG1, S. neurona). Several other proteins were found but could only be identified as hypothetical T. gondii proteins. The fact that most of these proteins were not specifically identified as S. neurona proteins highlights the lack of proteome information in the current databases and emphasizes the need for such research. Our work is designed to meet this need. There are no other impacts or outcomes at this time.

Publications

  • No publications reported this period


Progress 10/01/09 to 09/30/10

Outputs
OUTPUTS: All information is the same as the report from last year including communities of interest. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Veterinarians PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Changes in knowledge revolve around the identification of the proteins as described here. One of the S. neurona proteins identified was actin based on its peptide homology with actin from Toxoplasma gondii. Other proteins identified were a membrane skeletal protein (related to Toxoplasma gondii), enolase 2 (related to Toxoplasma gondii), heat shock protein 70 (related to Toxoplasma gondii), tubulin beta chain protein, microneme antigen 10 (Sarcocystis neurona), myosin A (related to Sarcocystis muris), and merozoite surface antigen SAG1 (Sarcocystis neurona. Characterization of these proteins indicates a biochemical similarity of S. neurona proteins with other apicomplexan parasites including the tissue-cyst forming T. gondii, which also causes encephalitis. The results from the western blot suggest that there are immunogenic proteins present in S. neurona. These proteins might serve as biomarkers for diagnosis of EPM or for development of a vaccine. However, these proteins must be identified first and the identification process is ongoing. There are no other impacts or outcomes at this time.

Publications

  • No publications reported this period


Progress 10/01/08 to 09/30/09

Outputs
OUTPUTS: The only additional output to report this year is a presentation of the data from last year at the Annual Midwest Conference of Parasitologists held at Ohio Wesleyan University in June 2009. All other information is the same as the report from last year including communities of interest. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Changes in knowledge revolve around the identification of the proteins as described here. One of the S. neurona proteins identified was actin based on its peptide homology with actin from Toxoplasma gondii. Other proteins identified were a membrane skeletal protein (related to Toxoplasma gondii), enolase 2 (related to Toxoplasma gondii), heat shock protein 70 (related to Toxoplasma gondii), tubulin beta chain protein, microneme antigen 10 (Sarcocystis neurona), myosin A (related to Sarcocystis muris), and merozoite surface antigen SAG1 (Sarcocystis neurona. Characterization of these proteins indicates a biochemical similarity of S. neurona proteins with other apicomplexan parasites including the tissue-cyst forming T. gondii, which also causes encephalitis. The results from the western blot suggest that there are immunogenic proteins present in S. neurona. These proteins might serve as biomarkers for diagnosis of EPM or for development of a vaccine. However, these proteins must be identified first and the identification process is ongoing. There are no other impacts or outcomes at this time.

Publications

  • No publications reported this period


Progress 10/01/07 to 09/30/08

Outputs
OUTPUTS: The western blot that was run in 2007 was compared with the latest 2-D gel to help choose spots to be excised from the gel for identification. The western blot used antiserum from an EPM-positive horse to probe for immunogenic proteins of interest. Several proteins on the immunoblot paper were immunogenic and these spots lined up with spots on the 2-D gel. The comparable spots on the 2-D gel were excised and characterized. We used two-dimensional electrophoresis and matrix-assisted laser desorption ionization-time of flight (MALDI-ToF) ToF mass spectrometry to identify several proteins from S. neurona based on comparative peptide homology with other apicomplexan proteins. A poster presentation of the current research progress will be presented in the summer of 2009 at an appropriate parasitology society meeting. PARTICIPANTS: The Purdue University Bindley Proteomics Facility was used to process the S. neurona samples for proteomics analysis. Mr. Kevin Kowalski is the lead technician who processed the samples. Other unnamed technicians may have been involved. Funding for the proteomics analysis and parasite culture was provided by a grant from the Purdue University School of Veterinary Medicine Equine Research Advisory Board. Dr. Camp is the principal investigator. TARGET AUDIENCES: At this time, the target audience consists of basic scientists interested in the proteomics of Sarcocystis neurona. If biomarkers for diagnosis or vaccine development can be identified, the target audience will change to those individuals who can develop diagnostic kits and/or vaccines. If diagnostic kits and/or vaccines can be developed, the target audience will become practitioners and others interested in EPM caused by S. neurona. At this time the only effort that qualifies by the definition is the brief mention I make of my research to the second-year veterinary students in the parasitology class I teach. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Changes in knowledge revolve around the identification of the proteins as described here. One of the S. neurona proteins identified was actin based on its peptide homology with actin from Toxoplasma gondii. Other proteins identified were a membrane skeletal protein (related to Toxoplasma gondii), enolase 2 (related to Toxoplasma gondii), heat shock protein 70 (related to Toxoplasma gondii), tubulin beta chain protein, microneme antigen 10 (Sarcocystis neurona), myosin A (related to Sarcocystis muris), and merozoite surface antigen SAG1 (Sarcocystis neurona. Characterization of these proteins indicates a biochemical similarity of S. neurona proteins with other apicomplexan parasites including the tissue-cyst forming T. gondii, which also causes encephalitis. The results from the western blot suggest that there are immunogenic proteins present in S. neurona. These proteins might serve as biomarkers for diagnosis of EPM or for development of a vaccine. However, these proteins must be identified first and the identification process is ongoing. There are no other impacts or outcomes at this time.

Publications

  • No publications reported this period


Progress 10/01/06 to 09/30/07

Outputs
OUTPUTS: We used two-dimensional electrophoresis and matrix-assisted laser desorption ionization-time of flight (MALDI-ToF) ToF mass spectrometry to identify several proteins from S. neurona based on comparative peptide homology with other apicomplexan proteins. One of the S. neurona proteins identified was actin based on its peptide homology with actin from Toxoplasma gondii. Other proteins identified were a hypothetical Plasmodium sp. protein, apical membrane antigen 1 (Plasmodium yoelii yoelii), and immunoglobulin heavy chain binding protein (Eimeria tenella). Characterization of these proteins indicates a biochemical similarity of S. neurona proteins with other apicomplexan parasites including the tissue-cyst forming T. gondii, which also causes encephalitis. The proteins from the two dimensional gel were transferred to immunoblot paper. A western blot was run using antiserum from an EPM-positive horse to probe for immunogenic proteins of interest. Several proteins on the immunoblot paper were immunogenic and these spots will be compared with the 2-D gel to identify proteins in the gel that will be excised and characterized. In this way, we will identify the proteins that elicited an immune response to S. neurona. A poster presentation of the current research progress was presented in June 2007 at the First North American Conference of Parasitology. PARTICIPANTS: The Purdue University Bindley Proteomics Facility was used to process the S. neurona samples for proteomics analysis. Mr. Kevin Kowalski is the lead technician who processed the samples. Other unnamed technicians may have been involved. Dr. Dan Howe at the University of Kentucky allowed the use of his laboratory and the necessary antibodies and reagents to perform the western blot analysis. There was no cost associated with the work done in Dr. Howe's laboratory. Funding for the proteomics analysis and parasite culture was provided by a grant from the School of Veterinary Medicine Equine Research Advisory Board. Dr. Camp is the principal (and only) investigator. TARGET AUDIENCES: At this time, the target audience consists of basic scientists interested in the proteomics of Sarcocystis neurona. If biomarkers for diagnosis or vaccine development can be identified, the target audience will change to those individuals who can develop diagnostic kits and/or vaccines. If diagnostic kits and/or vaccines can be developed, the target audience will become practitioners and others interested in EPM caused by S. neurona. At this time the only effort that qualifies by the definition is the brief mention I make of my research to the second-year veterinary students in the parasitology class I teach. PROJECT MODIFICATIONS: The project was modified to include Western Blot analysis of proteins that are potentially immunogenic to S. neurona.

Impacts
The results from the western blot suggest that there are immunogenic proteins present in S. neurona. These proteins might serve as biomarkers for diagnosis of EPM or for development of a vaccine. However, these proteins must be identified first and the identification process is ongoing. There are no other impacts or outcomes at this time.

Publications

  • No publications reported this period


Progress 10/01/05 to 09/30/06

Outputs
In this study, proteomics techniques were used to characterize and identify several proteins from Sarcocystis neurona based on the match between proteins and protein subunits of S. neurona with proteins and protein subunits from related protozoan parasites. Two forms of actin were identified in S. neurona; one related to actin from Toxoplasma gondii and the second related to actin from Pyrocystis lunula. Actin is a very common protein molecule in protozoa as well as in multi-celled organisms and is associated with motility (movement) and with the cytoskeleton. Given the functional importance of actin, it was not surprising to find it in S. neurona. T. gondii is another protozoan parasite that can cause neurologic problems in its hosts including encephalitis. In T. gondii, actin undergoes rapid turnover and assembly as part of the host-cell invasion process known as gliding. Perhaps the actin in S. neurona exhibits similar properties and functions. Three other proteins were identified, beta-tubulin similar to that found in Babesia microti and two types of heat shock protein 70 (hsp70). The hsp70 proteins in S. neurona were similar to forms found in T. gondii and Paramecium tetraurelia. Beta-tubulin is a common structural protein and component of microtubules. As in the case of actin, it is not surprising to find this protein in S. neurona. The hsp70 molecules are of interest because they serve as molecular chaperones in the cytosol, mitochondria, and endoplasmic reticulum of many organisms and are important in translation, protein translocation, proteolysis, protein folding, suppressing activation and reactivating denatured proteins. Hsp70 has been found in T. gondii, but it has not as yet been localized to a particular part of the organism nor has a specific function been assigned. Characterization of these proteins indicates a biochemical similarity of S. neurona proteins with other protozoan parasites including the closely related T. gondii. These results verify that the techniques that were used to prepare and analyze the samples of S. neurona worked well. Although these particular proteins may not prove to be useful as biomarkers or possible targets for new drugs, these early results are very encouraging because they show that S. neurona proteins can be identified with standard proteomics techniques. We are now in the process of analyzing additional proteins in S. neurona to try and identify those that are immunogenic and/or might serve as useful biomarkers.

Impacts
We still expect to find some proteins in the CSF of horses infected with Sarcocystis neurona that will serve as potential biomarkers for diagnostic purposes and that are immunogenic. The global proteome profile of the parasite will provide basic information that will be added to the existing databases for proteomes of parasites.

Publications

  • No publications reported this period


Progress 10/01/04 to 09/30/05

Outputs
At this time we have succeeded in getting Sarcocystis neurona to survive in culture in our laboratory. We have also been able to harvest the parasites from the culture and spin them down into pellets that are then frozen for use in future analyses. We have encountered the common problem of fungal contamination of the cultures and have lost several culture flasks of bovine turbinate (BT) host cells and infected BT cell flasks. We are taking great care in processing, but the ubiquitous nature of fungal spores makes some contamination inevitable. In spite of this problem, the cultures are still in place in the incubator. We are also in discussion with several laboratories to determine the best techniques for proteomic analyses of the horse CSF as well as the parasites themselves. We anticipate reaching agreement with one or more facilities in the next two months with the prospect of having preliminary results by the end of the year. Once we have results we will prepare a manuscript of the preliminary results. We will also prepare one or more additional grant proposals to seek additional funds for our research. In addition to searching for proteins that are in common between the EPM parasite and the CSF from parasite-infected horses, we hope to produce a global profile of the proteome of Sarcocystis neurona. To date, there is no published information on the global profile for this parasite.

Impacts
We still expect to find some proteins in the CSF of horses infected with Sarcocystis neurona that will serve as potential biomarkers for diagnostic purposes. The global proteome profile of the parasite will provide basic information that will be added to the existing databases for proteomes of parasites.

Publications

  • No publications reported this period