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Sample Grant Application Introduction On the following pages you will find one of the Sample R01 Applications and Summary Statements indexed here: http://www.niaid.nih.gov/ncn/grants/app/default.htm Visit the Web site for the most recent information. We may add more in the future. We are truly indebted to the grantees who've allowed us to post their outstanding applications to help the next generation of investigators write applications. Copyright Please note that the application text is copyrighted. It may be used only for nonprofit educational purposes provided the document remains unchanged and the PI, the grantee organization, and NIAID are credited. Contact deaweb@niaid.nih.gov with any questions. Please note that the application text is copyrighted. It may be used only for nonprofit educational purposes provided the document remains unchanged and the PI, the grantee organization, and NIAID are credited. See more online: http://funding.niaid.nih.gov/researchfunding/grant/pages/appsamples.aspx PI: STRIEPEN, BORIS Title: Biology of the apicomplexan plastid Received: 03/05/2010 FOA: PA10-067 Competition ID: ADOBE-FORMS-B FOA Title: Research Project Grant (Parent R01) 2 R01 AI064671-06 Dual: IPF: 676602 Organization: UNIVERSITY OF GEORGIA (UGA) Former Number: Department: CTEGD IRG/SRG: PTHE AIDS: N Expedited: N Subtotal Direct Costs (excludes consortium F&A) Year 6: 225,000 Year 7: 225,000 Year 8: 225,000 Year 9: 225,000 Year 10: 225,000 Animals: N Humans: N Clinical Trial: N Current HS Code: 10 HESC: N New Investigator: N Early Stage Investigator: N Senior/Key Personnel: Organization: Role Category: Boris Striepen The University of Georgia Research Foundation, Inc. PD/PI Council: 10/2010 Accession Number: 3279005 APPLICATION FOR FEDERAL ASSISTANCE 3. DATE RECEIVED BY STATE SF 424 (R&R) 1. * TYPE OF SUBMISSION Pre-application AI064671 4. a. Federal Identifier Application Changed/Corrected Application OMB Number: 4040-0001 Expiration Date: 06/30/2011 State Application Identifier b. Agency Routing Identifier Applicant Identifier 2. DATE SUBMITTED 03/05/2010 * Organizational DUNS: 004315578 5. APPLICANT INFORMATION * Legal Name: The University of Georgia Research Foundation, Inc. Department: Division: * Street1: 617 Boyd GSRC Street2: D. W. Brooks Drive * City: Athens County / Parish: Clarke * State: Province: GA: Georgia * Country: * ZIP / Postal Code: 30602-7411 USA: UNITED STATES Person to be contacted on matters involving this application Prefix: Dr. * Last Name: Rachel * First Name: Gary Middle Name: Suffix: PhD * Phone Number: 706-542-5905 Email: gcrachel@uga.edu Fax Number: 706-542-5946 6. * EMPLOYER IDENTIFICATION (EIN) or (TIN): 7. * TYPE OF APPLICANT: xxxxxxx M: Nonprofit with 501C3 IRS Status (Other than Institution of Higher Education) Other (Specify): Women Owned Small Business Organization Type If Revision, mark appropriate box(es). 8. * TYPE OF APPLICATION: New A. Increase Award Resubmission Renewal Socially and Economically Disadvantaged Continuation C. Increase Duration E. Other (specify): Revision * Is this application being submitted to other agencies? B. Decrease Award Yes No 9. * NAME OF FEDERAL AGENCY: What other Agencies? 10. CATALOG OF FEDERAL DOMESTIC ASSISTANCE NUMBER: TITLE: National Institutes of Health 11. * DESCRIPTIVE TITLE OF APPLICANT'S PROJECT: Biology of the apicomplexan plastid * 13. CONGRESSIONAL DISTRICT OF APPLICANT 12. PROPOSED PROJECT: * Start Date * Ending Date 12/01/2010 11/30/2015 GA-010 14. PROJECT DIRECTOR/PRINCIPAL INVESTIGATOR CONTACT INFORMATION Prefix: Dr. * First Name: Boris Middle Name: * Last Name: Striepen Position/Title: Suffix: Associate Professor * Organization Name: The University of Georgia Research Foundation, Inc. Department: CTEGD Division: * Street1: 500 D.W. Brooks Drive Street2: * City: County / Parish: Clarke Athens * State: * Country: * Phone Number: 706-583-0588 * Email: striepen@cb.uga.edu GA: Georgia USA: UNITED STATES Fax Number: Province: * ZIP / Postal Code: 30602-7411 D. Decrease Duration SF 424 (R&R) Page 2 APPLICATION FOR FEDERAL ASSISTANCE 15. ESTIMATED PROJECT FUNDING 16. * IS APPLICATION SUBJECT TO REVIEW BY STATE EXECUTIVE ORDER 12372 PROCESS? a. Total Federal Funds Requested b. Total Non-Federal Funds 0.00 c. Total Federal & Non-Federal Funds 1,670,625.00 d. Estimated Program Income THIS PREAPPLICATION/APPLICATION WAS MADE AVAILABLE TO THE STATE EXECUTIVE ORDER 12372 PROCESS FOR REVIEW ON: a. YES 1,670,625.00 0.00 DATE: b. NO PROGRAM IS NOT COVERED BY E.O. 12372; OR PROGRAM HAS NOT BEEN SELECTED BY STATE FOR REVIEW 17. By signing this application, I certify (1) to the statements contained in the list of certifications* and (2) that the statements herein are true, complete and accurate to the best of my knowledge. I also provide the required assurances * and agree to comply with any resulting terms if I accept an award. I am aware that any false, fictitious. or fraudulent statements or claims may subject me to criminal, civil, or administrative penalities. (U.S. Code, Title 18, Section 1001) * I agree * The list of certifications and assurances, or an Internet site where you may obtain this list, is contained in the announcement or agency specific instructions. 18. SFLLL or other Explanatory Documentation Add Attachment Delete Attachment View Attachment 19. Authorized Representative * First Name: Gary Prefix: Dr. Middle Name: Suffix: PhD * Last Name: Rachel * Position/Title: Grants Officer * Organization: The University of Georgia Research Foundation, Inc. Department: Sponsored Programs Division: * Street1: U. of Georgia Research Foundation Street2: 622A Boyd * City: Athens Office of V. President for Res County / Parish: Clarke * State: Province: GA: Georgia * Country: USA: UNITED STATES * Phone Number: 706-542-5905 * ZIP / Postal Code: 30602-7411 Fax Number: 706-542-5946 * Email: gcrachel@uga.edu * Date Signed * Signature of Authorized Representative Gary 20. Pre-application 03/05/2010 Rachel Add Attachment Delete Attachment View Attachment Principal Investigator/Program Director (Last, first, middle): Striepen, Boris 424 R&R and PHS-398 Specific Table Of Contents Page Numbers SF 424 R&R Face Page------------------------------------------------------------------------------------------ 1 Table of Contents--------------------------------------------------------------------------------------------- 3 Performance Sites--------------------------------------------------------------------------------------------- 4 Research & Related Other Project Information------------------------------------------------------------------ 5 Project Summary/Abstract (Description)---------------------------------------- 6 Public Health Relevance Statement (Narrative attachment)---------------------------------------- 7 Facilities & Other Resources---------------------------------------- 8 Equipment---------------------------------------- 10 Research & Related Senior/Key Person-------------------------------------------------------------------------- 11 Biographical Sketches for each listed Senior/Key Person---------------------------------------- 13 Current and Pending Support for each listed Senior/Key Person---------------------------------------- 17 PHS 398 Specific Cover Page Supplement------------------------------------------------------------------------ 19 PHS 398 Specific Modular Budget------------------------------------------------------------------------------- 21 Personnel Justification---------------------------------------- 24 PHS 398 Specific Research Plan-------------------------------------------------------------------------------- 25 Specific Aims---------------------------------------- 26 Research Strategy---------------------------------------- 27 List of Publications---------------------------------------- 39 Bibliography & References Cited---------------------------------------- 41 Letters of Support---------------------------------------- 46 PHS 398 Checklist--------------------------------------------------------------------------------------------- Table of Contents 47 Page 3 Principal Investigator/Program Director (Last, first, middle): Striepen, Boris OMB Number: 4040-0010 Expiration Date: 08/31/2011 Project/Performance Site Location(s) Project/Performance Site Primary Location I am submitting an application as an individual, and not on behalf of a company, state, local or tribal government, academia, or other type of organization. Organization Name: The University of Georgia Research Foundation, Inc. DUNS Number: 0043155780000 * Street1: 617 Boyd GSRC Street2: D. W. Brooks Drive * City: Athens * State: GA: Georgia County: Clarke Province: * Country: USA: UNITED STATES * ZIP / Postal Code: 30602-7411 Project/Performance Site Location 1 * Project/ Performance Site Congressional District: GA-010 I am submitting an application as an individual, and not on behalf of a company, state, local or tribal government, academia, or other type of organization. Organization Name: DUNS Number: * Street1: Street2: * City: County: * State: Province: * Country: USA: UNITED STATES * ZIP / Postal Code: * Project/ Performance Site Congressional District: Add Attachment Additional Location(s) Performance Sites Delete Attachment Page 4 View Attachment Principal Investigator/Program Director (Last, first, middle): Striepen, Boris RESEARCH & RELATED Other Project Information 1. * Are Human Subjects Involved? 1.a No Yes If YES to Human Subjects Is the Project Exempt from Federal regulations? Yes If yes, check appropriate exemption number. If no, is the IRB review Pending? 1 Yes No 2 3 4 5 6 No IRB Approval Date: Human Subject Assurance Number: 2. * Are Vertebrate Animals Used? 2.a. Yes No If YES to Vertebrate Animals Is the IACUC review Pending? Yes No IACUC Approval Date: Animal Welfare Assurance Number 3. * Is proprietary/privileged information included in the application? Yes 4.a. * Does this project have an actual or potential impact on the environment? No Yes No 4.b. If yes, please explain: 4.c. If this project has an actual or potential impact on the environment, has an exemption been authorized or an environmental assessment (EA) or environmental impact statement (EIS) been performed? Yes No 4.d. If yes, please explain: 5. * Is the research performance site designated, or eligible to be designated, as a historic place? Yes No 6. * Does this project involve activities outside of the United States or partnerships with international collaborators? Yes 5.a. If yes, please explain: No 6.a. If yes, identify countries: 6.b. Optional Explanation: Add Attachment 7. * Project Summary/Abstract summary1004083417.pdf 8. * Project Narrative generalpublic1004083418.pdf Add Attachment Add Attachment 10. Facilities & Other Resources FACILITIES1004083317.pdf 12. Other Attachments Add Attachments Delete Attachments Other Information Delete Attachment Add Attachment 9. Bibliography & References Cited refs1004083316.pdf 11. Equipment Equipmenttarle1004083318.pdf Delete Attachment Add Attachment Delete Attachment View Attachment Delete Attachment Delete Attachment View Attachment View Attachments Page 5 View Attachment View Attachment View Attachment Principal Investigator/Program Director (Last, first, middle): Striepen, Boris Please note that the application text is copyrighted. It may be used only for nonprofit educational purposes provided the document remains unchanged and the PI, the grantee organization, and NIAID are credited. See more online: http://funding.niaid.nih.gov/researchfunding/grant/pages/appsamples.aspx Apicomplexa are responsible for a number of important human diseases including malaria, toxoplasmosis, cryptosporidiosis and cyclosporidiosis. Management of these diseases rests heavily on chemotherapy but anti-parasitic drug treatment faces multiple challenges. These include poor overall potency, restriction to certain life-cycle stages, unwanted side effects, and rapidly emerging multiple drug resistance. A constant stream of new drugs and potential drug targets is required to stay abreast of the threat posed by these pathogens. One of the most promising sources of such parasite specific targets is the apicomplexan plastid or apicoplast. The apicoplast is unique to the parasite and its function is essential to parasite survival. This organelle is a holdover from a free-living photosynthetic past. The structure and biology of the apicoplast is remarkably complex as it is derived from the endosymbiotic marriage of two eukaryotes: a red alga and an auxotrophic protist. The goal of this application is to unravel the complexity of this biology in mechanistic detail and to identify future targets for intervention. Using Toxoplasma as a model organism we will conduct genetic, cell biological and biochemical approaches to characterize the function of two pathways that unfold in the outer compartments of the organelle and that we hypothesize are essential to the organelle and the parasites. We will complement this focused approach with a broader effort to define a comprehensive set of plastid proteins to continue to feed a pipeline of hypothesis-driven mechanistic experiments with strong candidate genes. Project Description Page 6 Principal Investigator/Program Director (Last, first, middle): Striepen, Boris Toxoplasma gondii is an important human pathogen that causes disease in the unborn fetus, young children and patients with a weakened immune system. We are a studying a unique cellular structure of the parasite that is related to the chloroplast of plants. A detailed understanding of the biology of this structure will lead us to new parasite specific interventions to treat and prevent disease. Public Health Relevance Statement Page 7 Principal Investigator/Program Director (Last, first, middle): Striepen, Boris FACILITIES  &  OTHER  RESOURCES  –  The  Center  for  Tropical  and  Emerging  Global  Diseases,  The   Coverdell  Center,  University  of  Georgia       Environment  –  Contribution  to  Success:     The  PI’s  lab,  office  and  most  supporting  facilities  are  in  the  state-­‐of-­‐the-­‐art  Coverdell  Center  for   Biomedical  Research  on  the  UGA  campus.  The  Coverdell  Center  houses  9  of  the  19  faculty  in  the   UGA  Center  for  Tropical  and  Emerging  Global  Diseases  (CTEGD;  www.ctegd.uga.edu).    The   CTEGD  administers  the  main  UGA  Flow  Facility,  established  in  1999  and  also  housed  in  the   Coverdell  Center.    This  facility  maintains  multiple  analyzers  and  a  new  high-­‐speed  sorter  (see   detailed  list  in  the  “Equipment”  page).    The  Coverdell  Center  also  has  an  AAALAC  accredited   Rodent  Vivarium  (CRV)  in  its  lowest  floor.    In  this  facility  of  nearly  20,000  sq  ft,  rodents  are   housed  in  individually  ventilated  racks  with  automated  watering.    Other  facilities  within  the  CRV   include  a  dedicated  rodent  surgery  suite,  a  rodent  import  quarantine  suite,  a  necropsy  room,   and  a  whole  animal  imaging  room  (equipped  with  a  CRi  Maestro  II  and  an  Olympus  OV100   Intravital  Observation  System  (both  for  whole  animal  fluorescent  imaging)  and  a  soon  to  be   added  Xenogen  IVIS  Lumina  system  for  bioluminescent  imaging).  Other  core  facilities   maintained  by  the  CTEGD  include  two  Delta  Vision  microscope  suites  for  high  resolution  in  vivo   imaging  including  time-­‐lapse,  laser  bleaching  and  recovery  analyses,  a  BD  Pathways  high-­‐ content  microscope  to  screen  fluorescence  phenotypes  in  96  and  384  well  plates  and  numerous   plate  readers  for  fluorescence  absorbence  and  luminescence.       The  intellectual  environment  at  UGA  and  particularly  in  the  CTEGD  is  extremely  rich.  UGA  has   long  recognized  the  importance  of  parasitology  and  in  December  1998  established  the  CTEGD   as  a  multi-­‐disciplinary  UGA-­‐wide  center.    CTEGD’s  research,  training  and  service  efforts  are   focused  on  global  health  challenges  that  involve  parasitic  diseases.    The  original  CTEGD  faculty   of  8  provided  a  strong  research  and  training  foundation  in  parasitology,  immunology,  cell   biology  and  molecular  biology.    CTEGD  has  now  grown  to  a  faculty  of  19,  who  individually  and   collectively  represent  broad  and  in-­‐depth  expertise  in  the  field.    Although  diverse  in  interest   and  focus,  this  is  highly  interactive  group  with  weekly  Research  in  Progress  and  Journal  Club   meetings,  5  domestic  and  international  training  grants,  and  an  annual  Symposium  that  attracts   more  that  125  attendees  from  throughout  the  Southeast.    In  2007,  UGA  made  another  major   commitment  to  CTEGD  by  assigning  the  entire  third  floor  and  part  of  the  first  floor  of  a  new   research  building  (the  Coverdell  Center)  to  CTEGD  for  its  offices,  its  core  facility  and  9  of  its   faculty.    This  consolidation  of  CTEGD  into  identifiable  space,  especially  in  the  showcase  building   for  UGA  biomedical  research,  has  greatly  strengthened  the  Center  and  provided  a  home-­‐base   for  our  trainees,  whether  they  are  in  laboratories  housed  in  the  Coverdell  Center  or  with  faculty   elsewhere  on  campus.  Research  funding  to  the  CTEGD  in  its  10  year  history  of  existence   exceeds  $50  million.               Facilities:       LABORATORY:   PI   has   lab   space   in   the   Paul   D.   Coverdell   Building   for   Biomedical   &   Health   Sciences,  a  105,000  sq  ft.  state-­‐of-­‐the-­‐art  building.    The  PI's  lab  is  located  on  the  3rd  floor  of  this   facility,   which   houses   other   members   of   the   Center   for   Tropical   &   Emerging   Global   Diseases.   Facilities Page 8 Principal Investigator/Program Director (Last, first, middle): Striepen, Boris PI's  assigned  space  in  the  open  lab  set-­‐up  is  approximately  1,500  sq  ft  and  includes  procedure   rooms  and  equipment  hallways.     ANIMAL:  The  Coverdell  Building  has  an  approximately  20,000  sq.  ft  vivarium  to  house  rodents.     The  PI's  lab  1  animal  rooms  capable  of  housing  >1000  rodent  cages  and  a  procedure  room  with   2  BSL2  cabinets,  centrifuges,  microsopes,  etc.  for  the  exclusive  use  of  his  lab.         COMPUTER:   The   PI   has   an   Apple   macbook   pro.   There   are   3   PCs   and   5   Macs   for   use   by   his   laboratory.  The  lab  maintains  a  web/database  server.     OFFICE:   The   PI   has   an   office   approximately   168   sq   ft.   which   is   adjacent   to   his   laboratory.   There   are  also  offices  for  Research  Scientists  and  Post-­‐docs.     Facilities Page 9 Principal Investigator/Program Director (Last, first, middle): Striepen, Boris Equipment Tarleton, R.L. In the Striepen Lab (within Coverdell Center): type II biosafety hoods (2), refrigerated centrifuges (2), low speed centrifuge, CO2 incubators (7), ultralow freezers (2), refrigerators (4), freezers (3), inverted phase microscope, nitrogen freezer for cell storage, thermocyclers for PCR (4), hybridization oven, incubator shaker, multiple gel apparati and power supplies for protein and nucleic acid analysis, electroporation apparatus, Leica motorized inverted fluorescence microscope with Hammamatsu camera and Openlab and Volocity software, BD Pathways fully automated high content fluorescence microscope. (additional microscopy facilities within the Coverdell building include two new Zeiss confocals and two Delta Vision in vivo imaging stations). Other CTEGD Core facilities (within Coverdell Center): The CTEGD Core Flow Cytometry laboratory, also located in the Coverdell Building, houses a DAKO-Cytomation 9-color CyAn analyzer, a new (2009), all digital Dako/Cytomation Mo-Flo sorter, a B-D. 4-color FACScalibur and a BioRad Luminex bead array reader. A successful NIH equipment proposal added an additional Cyan Analyzer to the facility in 2010. Other UGA facilities potentially relevant to the project: Molecular Genetics Instrumentation Facility. The Sequencing and Synthesis Facility provides sequencing and synthesis capabilities for protein and nucleic acids. The Proteomics Resource Facility provides protein purification and characterization facilities. The facility is equipped with two state-of-the-art mass spectrometers, robotics for mass spectrometry sample preparation, high-throughput two-dimensional gel electrophoresis units, and an automated chromatography station for multi-dimensional chromatography. The Functional Genomics Resources Facility is a full service microarray and genotyping laboratory. Services include gene expression analysis include custom microarray printing, RNA purification, probe labeling, hybridization, laser scanning and real time quantitative PCR. The facility also offers genotyping services, including microsatellite analysis, SNP validation, and AFLP fragment analysis. The Complex Carbohydrate Research Center: The CCRC offers custom synthesis and analysis of complex carbohydrates as a service to scientists in university, industrial, and government laboratories. The CCRC also offers assistance to those who need information on complex carbohydrates derived from animal, plant, and microbial sources. The facility is equipped with three high-field NMR spectrometers (300-, 500-, and 600-MHz), fully equipped for biomolecular studies of liquids and solids. In addition, the GRA- UGA 800-MHz NMR facility is a regional resource for high-field NMR studies of biological macromolecules. The CCRC has one magnetic sector (Jeol SX/SX102A), four electrospray (PE-Sciex API III, Micromass Q-TOF-2, Finnigan LCQ Advantage LC/MS/MS and Finnigan LCQ Deca XP Plus LC/MS/MS), and three laser-desorption (HP G2025A LD-TOF, Kratos Kompact SEQ and Applied Biosystems 4700 Proteomics Analyzer) mass spectrometers. The Center for Ultrastructural Research provides additional access to a series of high-end light and electron microscopes. These include a new Leica TCS SP2 two photon confocal, a Bio-Rad MRC600 conventional confocal microscope, a very powerful new 200 kV Technai 20 transmission EM as well as a Jeol 100 kV transmission EM and a state of the art Leo 982 scanning EM. The Glass Shop provides custom production and modification of flasks and laboratory glassware. The Instrument Shop offers the ability to machine, form and fabricate special laboratory devices, one of a kind research instrument or modifications to existing equipment. Equipment Page 10 Principalnote that the application text is copyrighted. ItStriepen, Boris only for nonprofit educational purposes Please Investigator/Program Director (Last, first, middle): may be used provided the document remains unchanged and the PI, the grantee organization, and NIAID are credited. See more online: http://funding.niaid.nih.gov/researchfunding/grant/pages/appsamples.aspx OMB Number: 4040-0001 Expiration Date: 06/30/2011 RESEARCH & RELATED Senior/Key Person Profile (Expanded) PROFILE - Project Director/Principal Investigator Prefix: Dr. * First Name: Boris Middle Name: * Last Name: Striepen Suffix: Position/Title: Associate Professor Department: CTEGD Organization Name: The University of Georgia Research Foundation, Inc. Division: * Street1: 500 D.W. Brooks Drive Street2: * City: * State: County/ Parish: Clarke Athens Province: GA: Georgia * Country: USA: UNITED STATES * Phone Number: 706-583-0588 * Zip / Postal Code: 30602-7411 Fax Number: * E-Mail: striepen@cb.uga.edu Credential, e.g., agency login: xxxxxxx * Project Role: Other Project Role Category: PD/PI Degree Type: Degree Year: *Attach Biographical Sketch Attach Current & Pending Support Striepen_biosketch_New1004083 Add Attachment Delete Attachment View Attachment OtherSupport_Stiepen201010040 Add Attachment Delete Attachment View Attachment PROFILE - Senior/Key Person 1 Prefix: * First Name: Middle Name: * Last Name: Suffix: Position/Title: Department: Organization Name: Division: * Street1: Street2: * City: County/ Parish: * State: Province: * Country: USA: UNITED STATES * Zip / Postal Code: * Phone Number: Fax Number: * E-Mail: Credential, e.g., agency login: * Project Role: Other Project Role Category: Degree Type: Degree Year: *Attach Biographical Sketch Add Attachment Delete Attachment View Attachment Attach Current & Pending Support Add Attachment Delete Attachment View Attachment To ensure proper performance of this form; after adding 20 additional Senior/ Key Persons; please save your application, close the Adobe Reader, and reopen it. Key Personnel Page 11 Principal Investigator/Program Director (Last, first, middle): Striepen, Boris ADDITIONAL SENIOR/KEY PERSON PROFILE(S) Add Attachment Delete Attachment View Attachment Additional Biographical Sketch(es) (Senior/Key Person) Add Attachment Delete Attachment View Attachment Additional Current and Pending Support(s) Add Attachment Delete Attachment View Attachment Key Personnel Page 12 Please note that the application text is copyrighted. It may be used only for nonprofit educational purposes Principal Investigator/Program Director (Last, first, middle): Striepen, Boris provided the document remains unchanged and the PI, the grantee organization, and NIAID are credited. See more online: http://funding.niaid.nih.gov/researchfunding/grant/pages/appsamples.aspx Principal Investigator/Program Director (Last, First, Middle): Striepen, Boris BIOGRAPHICAL SKETCH Provide the following information for the key personnel and other significant contributors in the order listed on Form Page 2. Follow this format for each person. DO NOT EXCEED FOUR PAGES. NAME POSITION TITLE Boris Striepen Associate Professor eRA COMMONS USER NAME EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and include postdoctoral training.) INSTITUTION AND LOCATION DEGREE (if applicable) YEAR(s) FIELD OF STUDY Rheinische Friedrich Wilhelms Universität, Bonn, Germany Philipps-Universität Marburg, Marburg, Germany Vordipl./B.S. 1987 Biology Diplom/M.S. 1991 Biology Philipps-Universität Marburg, Marburg, Germany University of Pennsylvania, Philadelphia, PA Dr. rer. nat Post-Doc 1995 1999 Biochemistry Cell/Molecular Biology A. Personal Statement: The goal of the current application is to dissect the biology of a unique chloroplast-like organelle found in apicomplexan parasites. We have considerable expertise in the specific area of apicoplast biology and the laboratory is well suited and equipped to conduct the studies outlined in this application. Conceptually and technologically the project straddles the line between parasitology, cell biology and genetics. I have been a student of parasite biology for 25 years. I have studied zoology, botany and cell biology in Bonn, was trained in biochemistry as a graduate student of Ralph Schwarz in Marburg, and learned cell biology and genetics from David Roos at the Univ. of Pennsylvania, before starting my own group in a new center for parasite research at the Univ. of Georgia in 2000. In the past ten years I have pursued studies to understand the cell biology and biochemistry of apicomplexan parasites (Toxoplasma, Cryptospordiium and Sarcocystis), most of these studies were rooted in genetic approaches. I feel that my group has contributed to the knowledge of unique parasite organelles and their biogenesis, the mechanism of parasite cell division and replication, and to our understanding of parasite metabolism and its value as a drug target (in particular for lipid and nucleotide metabolism). Many members of my laboratory including myself have enjoyed honing the experimental technology for Toxoplasma research. We have developed several useful tools including GFP expressing parasites, a number of organelle markers, phenotypic complementation of mutant parasites, genetic screening protocols, and we have continuously worked to generate mutant parasites faster and with less effort. We have a solid reputation for sharing tools that we develop with the community. I also have an interest in teaching and training. I teach parasitology to undergraduate and graduate students, I serve as chair of graduate admissions for my cell biology department, I co-direct an NIH T32 training program in parasitology at UGA, and I have a long and enjoyable association with the Biology of Parasitism course at the MBL, for which I currently serve as one of the directors. B. Positions and Honors: Positions: 201020102005-2010 200420001995 - 1999 1991 - 1995 1991 1987 - 1988 Professor of Cellular Biology (8/1/2010) Co-Director Biology of Parasitism, MBL, Woods Hole, MA Associate Professor with tenure, University of Georgia Adjunct Professor, Department of Microbiology, University of Georgia Assistant Professor, Department of Cellular Biology & CTEGD, University of Georgia Postdoctoral fellow, University of Pennsylvania. Molecular and cell biology of the protozoan parasite Toxoplasma gondii, with Dr. David S. Roos. Doctoral student, Philipps-Universität. Worked on the structure and biosynthesis of glycolipid membrane anchors in the laboratory of Dr. Ralph T. Schwarz. Parasitological field work at the Centre de Recherche sur les Trypanosomoses animales, Bobo Dioulasso, Burkina Faso, with Dr. Peter Clausen. Undergraduate research on parasitic flatworms, with Dr. Hans Komnick, Universität Bonn. PHS 398/2590 (Rev. 09/04) Page Biosketches Biographical Sketch Format Page Page 13 Principal Investigator/Program Director (Last, first, middle): Striepen, Boris Principal Investigator/Program Director (Last, First, Middle): Striepen, Boris Honors and Awards: University of Georgia Creative Research Medal, 2007; Postdoctoral training grant, Deutsche Forschungsgemeinschaft, 1996-98; Dissertation ‘summa cum laude’ Philipps-Universität Marburg 1995; Predoctoral fellowship, Friedrich-Ebert-Foundation, 1991-94; Undergraduate fellowship, Friedrich-EbertFoundation, 1988-90. Other Professional Activities: Member NIH study section AOIC, AIDS associated Opportunistic Infections and Cancer (2005-09) Ad hoc reviewer NIH study sections AAR4 Opportunistic Infections and Malignancies associated with AIDS (7/01, 4/02,11/02, 8/03, 11/03) and TMP Tropical Medicine and Parasitology (2/03); MBL course Biology of Parasitism, Woods Hole, MA, Lecturer (2001-2010), module head (2006-08) director (2010- ); Editorial Board: Molecular Microbiology (1/05-9), International Journal of Parasitology (1/07-), PLoS Pathogens (1/08); Eukaryotic Cell (1/09). Chair, Coccidiosis Conference, Mobile, AL (7/05); Organizer 9th International Congress on Toxoplasmosis, Chico Hotsprings, MT (6/07), Organizer Symposium Global Health through Research, Athens, GA (9/08), Vice chair, Gordon Research Conf. Host-Parasite Interactions, New Port, RI (6/10) C. Selected peer-reviewed publications (out of 64): Recent publications most relevant to the current application: Brooks, C.F., Johnsen, H., van Dooren, G.G., Muthalagi, Liu, S.S., M, Bohne, W., Fischer, K.*, and Striepen, B.* (2010) The phosphate translocator is the source of carbon and energy for the Toxoplasma apicoplast and essential for parasite survival. Cell Host & Microbe 7: 63-73 (*joint senior authors). Agrawal, S., van Dooren, G.G., Beatty, W., and Striepen, B. (2009) An endosymbiont-derived ERAD system is required for apicoplast protein import. J. Biol Chem 284: 33683-33691. van Dooren, G.G., Reiff, S, Tomova, C., Meissner, M., S., Humbel, B., M., and Striepen, B. (2009) A novel Dynamin-related protein has been recruited for apicoplast fission in Toxoplasma gondii. Current Biology. 19:267-276. Breinich, M., Ferguson, D.P., van Dooren, G.G., Bradley, P.J., Striepen, B., Carruthers, V.B., and Meissner, M. The dynamin-related protein B, DrpB, is required for intracellular trafficking to specialised secretory organelles in apicomplexan parasites. Current Biology 19: 277-286. van Dooren, G.G., Tomova, C., Agrawal, S., Humbel, B. , M., and Striepen, B. (2008) Toxoplasma gondii Tic20 is essential for apicoplast protein import. Proc. Natl. Acad. Sci. U.S.A. 105:13574-13579 Gubbels, M.J., Lehmann, M., Muthalagi, M., Maria E. Jerom, Brooks, C., Szatanek, T. Flynn, J., Parrot, B., Radke, J., Striepen, B. * and White, M.W. * (2008) Forward genetic analysis of the apicomplexan cell division cycle in Toxoplasma gondii, PLoS Pathogens 4: e36. (*joint senior authors). Mazumdar, J., Wilson, E., Masek, K., Hunter, C and Striepen, B (2006) Apicoplast fatty acid synthesis is essential for organelle biogenesis and survival in Toxoplasma gondii. Proc. Natl. Acad. Sci. 103: 13192– 13197. Gubbels, M.J., Vaishnava, S., Boot, N., Dubremetz, J.F. and Striepen, B., (2006) A MORN-repeat protein is a dynamic component of the Toxoplasma gondii cell division apparatus. J. Cell Sci. 119, 2236-2245. Vaishnava, S. and Striepen, B. (2006) The cell biology of endosymbiosis – How parasites build, divide and segregate the apicoplast. Mol. Microbiol. 61: 1380–1387. Vaishnava, S., Morrison, D., Gaji, R.Y., Entzeroth, R.K., Howe, D.L., and Striepen, B. (2005) Development and segregation of the plastid in the apicomplexan parasite Sarcocystis neurona. J. Cell Sci.,118: 3397-407. Additional recent publications important to the field: Chtanova, T., Han, S.J., Schaeffer, M., van Dooren, G.G., Herzmark, P., Striepen, B., and Robey, E.A. (2009) Dynamics of T cell, antigen presenting cell, and pathogen interactions during recall responses in the lymph node, Immunity 31: 342-355. Schaeffer, M., Han, S.J., Chtanova, T., van Dooren, G.G., Herzmark, P., Striepen, B.*, and Robey, E.A.* (2009) Dynamic imaging of T cell – parasite interactions in the brains of mice chronically infected with Toxoplasma gondii. J. Immunol., 182: 6379-6393. (*joint senior authors). PHS 398/2590 (Rev. 09/04) Page Biosketches Continuation Format Page Page 14 Principal Investigator/Program Director (Last, First, Middle): Striepen, Boris Chtanova T, Schaeffer M, Han SJ, van Dooren GG, Nollmann M, Herzmark P, Chan SW, Satija H, Camfield K, Aaron H, Striepen B, Robey EA. (2008) Dynamics of Neutrophil Migration in Lymph Nodes during Infection. Immunity 29: 1-10 Umejiego, N.N., Gollapalli, D., Sharling, L., Volftsun, A., Lu, J., Benjamin, N., Stroupe, A.H. Striepen, B. and Hedstrom, L. (2008) Targeting a prokaryotic protein in a eukaryotic pathogen: identification of lead compounds against Cryptosporidiosis. Chemistry & Biology 15:70-77. Striepen, B., Jordan, C.N., Reiff, S., and van Dooren, G. (2007) Building the perfect parasite: Apicomplexan cell division. PLoS Pathogens 3: e78. Patents: European Patent T/95161 ALG, Toxoplasma gondii glycoconjugates U.S. Provisional Patent Application No.: 60/810,276. Compounds and Methods for Treating Mammalian Gastrointestinal Parasitic Infections Research Projects Ongoing or Completed During Last 3 Years: Biosketches PHS 398/2590 (Rev. 09/04) Page Page 15 Continuation Format Page Principal Investigator/Program Director (Last, first, middle): Striepen, Boris PHS 398 Cover Page Supplement OMB Number: 0925-0001 Please note that the application text is copyrighted. It may be used only for nonprofit educational purposes provided the document remains unchanged and the PI, the grantee organization, and NIAID are credited. See more online: http://funding.niaid.nih.gov/researchfunding/grant/pages/appsamples.aspx 1. Project Director / Principal Investigator (PD/PI) Prefix: * First Name: Boris Dr. Middle Name: * Last Name: Striepen Suffix: 2. Human Subjects Clinical Trial? No Yes * Agency-Defined Phase III Clinical Trial? No Yes 3. Applicant Organization Contact Person to be contacted on matters involving this application Prefix: * First Name: Dr. Gary Middle Name: * Last Name: Suffix: Rachel PhD * Phone Number: 706-542-5905 Fax Number: 706-542-5946 Email: gcrachel@uga.edu * Title: Grants Officer * Street1: Street2: * City: 617 Boyd GSRC D. W. Brooks Drive Athens County/Parish: Clarke * State: GA: Georgia Province: * Country: USA: UNITED STATES Clinical Trial & HESC * Zip / Postal Code: 30602-7411 Page 19 Principal Investigator/Program Director (Last, first, middle): Striepen, Boris PHS 398 Cover Page Supplement 4. Human Embryonic Stem Cells * Does the proposed project involve human embryonic stem cells? No Yes If the proposed project involves human embryonic stem cells, list below the registration number of the specific cell line(s) from the following list: http://stemcells.nih.gov/research/registry/. Or, if a specific stem cell line cannot be referenced at this time, please check the box indicating that one from the registry will be used: Cell Line(s): Specific stem cell line cannot be referenced at this time. One from the registry will be used. Clinical Trial & HESC Page 20 Principal Investigator/Program Director (Last, first, middle): Striepen, Boris PHS 398 Modular Budget, Periods 1 and 2 Please note that the application text is copyrighted. It may be used only for nonprofit educational purposes provided the document remains unchanged and the PI, the grantee organization, and NIAID are credited. See more online: http://funding.niaid.nih.gov/researchfunding/grant/pages/appsamples.aspx OMB Number: 0925-0001 Budget Period: 1 Start Date: 12/01/2010 End Date: 11/30/2011 * Funds Requested ($) A. Direct Costs * Direct Cost less Consortium F&A 225,000.00 Consortium F&A * Total Direct Costs B. Indirect Costs Indirect Cost Base ($) Indirect Cost Rate (%) Indirect Cost Type 1. Research On Campus 48.5 225,000.00 * Funds Requested ($) 109,125.00 225,000.00 2. 3. 4. Cognizant Agency (Agency Name, POC Name and Phone Number) DHHS, Division of Cost Allocation, 202-401-2808 Total Indirect Costs 109,125.00 Funds Requested ($) 334,125.00 Indirect Cost Rate Agreement Date 12/23/2008 C. Total Direct and Indirect Costs (A + B) Budget Period: 2 Start Date: 12/01/2011 End Date: 11/30/2012 * Funds Requested ($) A. Direct Costs * Direct Cost less Consortium F&A 225,000.00 Consortium F&A * Total Direct Costs B. Indirect Costs Indirect Cost Type 1. Research On Campus Indirect Cost Rate (%) 48.5 Indirect Cost Base ($) 225,000.00 * Funds Requested ($) 225,000.00 109,125.00 2. 3. 4. Cognizant Agency (Agency Name, POC Name and Phone Number) DHHS, Division of Cost Allocation, 202-401-2808 Indirect Cost Rate Agreement Date 12/23/2008 Total Indirect Costs 109,125.00 C. Total Direct and Indirect Costs (A + B) Funds Requested ($) 334,125.00 Modular Budget Page 21 Principal Investigator/Program Director (Last, first, middle): Striepen, Boris PHS 398 Modular Budget, Periods 3 and 4 Budget Period: 3 Start Date: 12/01/2012 End Date: 11/30/2013 * Funds Requested ($) A. Direct Costs * Direct Cost less Consortium F&A 225,000.00 Consortium F&A * Total Direct Costs B. Indirect Costs Indirect Cost Type 1. Research On Campus Indirect Cost Base ($) Indirect Cost Rate (%) 48.5 225,000.00 * Funds Requested ($) 225,000.00 109,125.00 2. 3. 4. Cognizant Agency (Agency Name, POC Name and Phone Number) DHHS, Division of Cost Allocation, 202-401-2808 Total Indirect Costs 109,125.00 Funds Requested ($) 334,125.00 Indirect Cost Rate Agreement Date 12/23/2008 C. Total Direct and Indirect Costs (A + B) Budget Period: 4 Start Date: 12/01/2013 End Date: 11/30/2014 * Funds Requested ($) A. Direct Costs * Direct Cost less Consortium F&A 225,000.00 Consortium F&A * Total Direct Costs B. Indirect Costs Indirect Cost Type 1. Research On Campus Indirect Cost Rate (%) 48.5 Indirect Cost Base ($) 225,000.00 * Funds Requested ($) 225,000.00 109,125.00 2. 3. 4. Cognizant Agency (Agency Name, POC Name and Phone Number) DHHS, Division of Cost Allocation, 202-401-2808 Indirect Cost Rate Agreement Date 12/23/2008 Total Indirect Costs 109,125.00 C. Total Direct and Indirect Costs (A + B) Funds Requested ($) 334,125.00 Modular Budget Page 22 Principal Investigator/Program Director (Last, first, middle): Striepen, Boris PHS 398 Modular Budget, Periods 5 and Cumulative Budget Period: 5 Start Date: 12/01/2014 End Date: 11/30/2015 * Funds Requested ($) A. Direct Costs * Direct Cost less Consortium F&A 225,000.00 Consortium F&A * Total Direct Costs B. Indirect Costs Indirect Cost Type Indirect Cost Rate (%) 1. Research On Campus 48.5 Indirect Cost Base ($) 225,000.00 * Funds Requested ($) 225,000.00 109,125.00 2. 3. 4. Cognizant Agency (Agency Name, POC Name and Phone Number) DHHS, Division of Cost Allocation, 202-401-2808 Total Indirect Costs 109,125.00 Funds Requested ($) 334,125.00 Indirect Cost Rate Agreement Date 12/23/2008 C. Total Direct and Indirect Costs (A + B) Cumulative Budget Information 1. Total Costs, Entire Project Period *Section A, Total Direct Cost less Consortium F&A for Entire Project Period $ Section A, Total Consortium F&A for Entire Project Period $ *Section A, Total Direct Costs for Entire Project Period $ 1,125,000.00 *Section B, Total Indirect Costs for Entire Project Period $ 545,625.00 *Section C, Total Direct and Indirect Costs (A+B) for Entire Project Period $ 1,670,625.00 1,125,000.00 2. Budget Justifications Add Attachment Delete Attachment View Attachment Consortium Justification Add Attachment Delete Attachment View Attachment Additional Narrative Justification Add Attachment Delete Attachment View Attachment Personnel Justification Personaljustification100408341 Modular Budget Page 23 Principal Investigator/Program Director (Last, first, middle): Striepen, Boris . Personal Justification: Boris Striepen, PhD. Dr. Striepen will direct and oversee all aspects of the project, analyze data, and write publications and reports to the NIH. Dr. Striepen has extensive experience in parasite genetics, cell biology and biochemistry. Dr. Striepen will devote 2 months per year to this project and two months of summer salary are requested. Maria Francia M.S. Ms. Francia is a new graduate student in the laboratory. She will devote 12 months per year to this project. Sarah Reiff. Sarah Reif is a graduate student in the laboratory. She will devote 12 months per year to the genetic and cell biological analysis of apicoplast proteins. Lilach Sheiner. Postdoctoral fellow. Dr. Sheiner is a molecular biologist with outstanding graduate training from the laboratory of Prof. Dominique Soldati at the University of Geneva, Switzerland. She has published several strong papers on the biology of rhomboid proteases in parasite biology. She will devote 12 months per year to this project. Dr. Sheiner has taken an lead on studies described in specific Aim 3 and she has developed the Ku80 TATi parasite model described in the application. Carrie Brooks B.S., Research coordinator. Ms. Brooks is a highly skilled molecular biologist with more than 15 years of laboratory experience, she has been a lead contributor in the development of the cosmid-mediated knockout system. Ms. Brooks will provide assistance to the team by culturing cells and parasites and by constructing modified cosmid clones for gene targeting experiments. She will devote six months of her annual time to this project. (Note: Swati Agrwawal, M.S., a senior graduate student who has lead our efforts to understand the apicoplast ERAD system will work on this project as well. As Swati recently received a graduate student training fellowship from the American Heart Association that covers her stipend no funds are requested for her under this proposal.) Personnel Justification Page 24 Principal Investigator/Program Director (Last, first, middle): Striepen, Boris Please note that the application text is copyrighted. It may be used only for nonprofit educational purposes provided the document remains unchanged and the PI, the grantee organization, and NIAID are credited. See more online: http://funding.niaid.nih.gov/researchfunding/grant/pages/appsamples.aspx OMB Number: 0925-0001 PHS 398 Research Plan 1. Application Type: From SF 424 (R&R) Cover Page. The response provided on that page, regarding the type of application being submitted, is repeated for your reference, as you attach the appropriate sections of the Research Plan. *Type of Application: New Resubmission Renewal Continuation Revision 2. Research Plan Attachments: Please attach applicable sections of the research plan, below. 1. Introduction to Application Add Attachment Delete Attachment View Attachment (for RESUBMISSION or REVISION only) 2. Specific Aims ApicoplastGrantRenewalSPAIM Add Attachment Delete Attachment View Attachment 3. *Research Strategy researchstrategy1004083310.p Add Attachment Delete Attachment View Attachment Add Attachment Delete Attachment View Attachment Add Attachment Delete Attachment View Attachment 6. Protection of Human Subjects Add Attachment Delete Attachment View Attachment 7. Inclusion of Women and Minorities Add Attachment Delete Attachment View Attachment 8. Targeted/Planned Enrollment Table Add Attachment Delete Attachment View Attachment 9. Inclusion of Children Add Attachment Delete Attachment View Attachment 10. Vertebrate Animals Add Attachment Delete Attachment View Attachment 11. Select Agent Research Add Attachment Delete Attachment View Attachment 12. Multiple PD/PI Leadership Plan Add Attachment Delete Attachment View Attachment 13. Consortium/Contractual Arrangements Add Attachment Delete Attachment View Attachment Boris_Letter_2_28_1010040832 Add Attachment Delete Attachment View Attachment Add Attachment Delete Attachment View Attachment 4. Inclusion Enrollment Report 5. Progress Report Publication List pubsfromproject1004083311.p Human Subjects Sections Other Research Plan Sections 14. Letters of Support 15. Resource Sharing Plan(s) 16. Appendix Add Attachments Remove Attachments List of Research Plan Attachments View Attachments Page 25 Principal Investigator/Program Director (Last, first, middle): Striepen, Boris Specific Aims: Apicomplexa are responsible for a number of important human diseases including malaria, toxoplasmosis, cryptosporidiosis and cyclosporidiosis. Management of these diseases rests heavily on chemotherapy but antiparasitic drug treatment faces multiple challenges. These include poor overall potency, restriction to certain lifecycle stages, unwanted side effects, and rapidly emerging multiple drug resistance. A constant stream of new drugs and potential drug targets is required to stay abreast of the threat posed by these pathogens. One of the most promising sources of such parasite specific targets is the apicomplexan plastid or apicoplast. The apicoplast is unique to the parasite and its function is essential to parasite survival. This organelle is a holdover from a free-living photosynthetic past. The structure and biology of the apicoplast is remarkably complex as it is derived from the endosymbiotic marriage of two eukaryotes: a red alga and an auxotrophic protist. The goal of this application is to unravel the complexity of this biology in mechanistic detail. We hypothesize that the photosynthetic past of Apicomplexa and the continued presence of a plastid has profound and lasting implications for their current metabolism and cell biology. Further we believe that discovering and characterizing this biology in its molecular detail will lead us to important insights into the biology of Apicomplexa, the evolution of the eukaryotic cell, and ultimately to novel targets for anti-parasitic interference. In our current funding period we have conducted genetic studies on proteins involved in apicoplast replication, protein import, and metabolism that were identifiable as plastid proteins in part based on their similarity to plant chloroplast proteins. We did so in a gene-by-gene fashion characterizing a limited number of proteins in considerable depth using a genetic approach. This has been an excellent strategy that served us well, we will continue to use this approach to dig deeper into mechanism in the current application. However, we also feel that we might have harvested the lower hanging fruit of candidates with a lot of function left unassigned. We therefore will complement this approach with a broader effort to define a comprehensive set of plastid proteins to continue to feed our pipeline of hypothesis-driven mechanistic experiments with strong candidate genes. Specific Aim1: Dissect the mechanism of apicoplast protein import. The bulk of the ~500 apicoplast proteins is nuclear encoded and post-translationally imported across four membranes. We (and others) have described three mechanistically distinct candidate protein translocons that reside in the three inner membranes of complex plastids. In the current funding period we will focus on a newly discovered mechanism that was derived from the ER-associated degradation system (ERAD) of the algal endosymbiont. We will use conditional gene disruptions and complementation assays to establish the importance of individual components and to define the energy source of the translocation process. Specific Aim2: Understand the function of the apicoplast ubiquitination pathway. The ER-localized ERAD pathway goes hand in hand with the ubiquitination and subsequent proteasomal degradation of translocated proteins. Our preliminary data indicates that aspects of this protein modification pathway are still present in the apicoplast. What is the enzymatic machinery involved in this process? What are its substrates? And most importantly, what is the molecular function of apicoplast ubiquitination? A combination of genetic and biochemical approaches will be used to answer these important questions. Specific Aim 3: Discover a comprehensive set of apicoplast proteins and characterize their function. Mining comparative and functional genomic information we have assembled an extensive list of proteins for which we hypothesize a role in apicoplast biology. We will establish the localization of their protein products for a comprehensive set of these candidate genes by epitope tagging. In the previous funding period we have found conditional null mutants to be highly informative to study apicoplast protein function and we have developed phenotypic assays to detect defects in apicoplast protein import, apicoplast division, and apicoplast metabolism. We will apply this genetic approach to a prioritized list of validated candidates. To increase the throughput of our analyses we will develop and test a new mutagenesis approach based on promoter replacement. Specific Aims Page 26 Principal Investigator/Program Director (Last, first, middle): Striepen, Boris Research Strategy (A) Significance Apicomplexa are important human pathogens responsible for numerous severe diseases around the World. These include the various forms of malaria (1-3) as well as opportunistic infections associated with AIDS (4, 5). Several of these organisms have been included on the NIH/CDC appendix B list of pathogens considered potential bioterrorism threats (Cryptosporidium, Toxoplasma, Cyclospora). This is due to the marked resistance of infectious oocysts to conventional water treatment, which has led to large water borne outbreaks in the past (6-11). No effective vaccines are available for use in humans and significant challenges remain in the antimicrobial drug treatment for diseases caused by Apicomplexa. These challenges include widespread multiple drug resistance (malaria (12-14), coccidosis (15-17)), lack of efficacy of current treatment against chronic stages (toxoplasmosis, with particular clinical importance in the case of ocular infection (18-21)), or absence of fully effective treatment (cryptosporidiosis (21, 22)). New treatment options with independent modes of action are urgently needed to overcome some of these challenges. One of the most promising sources for novel targets is the apicoplast. The apicoplast is a unique chloroplast-like organelle and essential for parasite growth and pathogenesis (23-25). As humans lack chloroplasts targeting plastid function has great potential to yield interventions that specifically inhibit the parasite but not the host (26, 27). Driven by the availability of full genome sequence we have made solid progress in our understanding of the potential metabolic functions of the organelle, however, its true raison d’être remains to be defined (28, 29). The apicoplast has a fascinating evolutionary history. As schematically depicted in Fig. 1 this organelle is the product of two subsequent endosymbiotic events. Most remarkably the second step reflects the union of two eukaryotes (a red alga and a pre-alveolate) and has let to a complex sub cellular structure that is delineated by four membranes (30). Figure 1: Schematic outline of apicoplast evolution. A cyanobacterium was endocytosed by a eukaryotic cell (primary endosymbiosis). Genes were transferred from the bacterial genome to the nucleus. Chloroplasts are bounded by two membranes. In a secondary endosymbiosis event a eukaryotic alga was phagocytosed by an ancestor of Apicomplexa. Gene transfer occurred from the endosymbiont to the host nuclear genome. The apicoplast is surrounded by four membranes. The goal of this research project, which was first funded in December 2005, is to produce mechanistic insights into the biology of the apicomplexan plastid using Toxoplasma gondii as a genetic model. We anticipate that a detailed understanding of the function and cell biology of this organelle will help to prioritize the list of currently considered plastid drug targets and furthermore will lead to the discovery of new targets beyond a narrow focus on anabolic metabolism (e.g. interference with enzymes involved in apicoplast biogenesis rather than interference with the fatty acid synthesis pathway). We also expect this work to contribute in meaningful ways to our general understanding of the evolution of the eukaryotic cell and the biogenesis of organelles. Lastly, to achieve our specific experimental goals we have developed novel genetic approaches and reagents and will continue to do so in the future. We expect these advances to spur on molecular research on T. gondii and other Apicomplexa beyond our own research focus. We feel that our first four years of work on this project have delivered on some of this promise. We have discovered genes and proteins with important roles in apicoplast division (31-34), apicoplast metabolism (24, 35), and apicoplast protein import (36, 37) and the detailed characterization of mutants in these genes has let us to strong mechanistic models for key aspects of apicoplast and parasite biology (29, 31, 38, 39). We have developed an approach to forward genetic analysis through chemical mutagenesis and complementation cloning and a highly efficient strategy to engineer conditional gene deletions (35, 40-42). We have shared these new reagents with the community and numerous researchers have put our fluorescent organelle markers, parasite strains, cosmid clones and libraries and KO cassettes to great use (see e.g. our collaboration with the Robey lab (43-45)). Lastly, we note that there are a number of studies that we have conducted that are still in submission or preparation. These include a fully characterized knock out in the MORN1 protein demonstrating that this protein is essential for apicoplast fission Research Strategy Page 27 Principal Investigator/Program Director (Last, first, middle): Striepen, Boris and segregation, a mutant in Tic22 showing that this protein is required for apicoplast import across the innermost membrane, and a mutant in the HU protein demonstrating that this protein is required for the maintenance of the apicoplast genome. We also have identified the genes for a suite of proteins with a likely role in apicoplast genome replication and we have tagged and successfully localized their protein products (PolA/Helicase/Primase, DNA-Helicase, Gyrase A and B). The space limitations of the new format preclude us from showing this extensive (yet still unpublished) data. (B) Innovation We would like to argue that our project has been highly innovative and we expect it to continue to be innovative. Innovation in this project is evident in the topic of the research, the concepts and hypotheses to be tested, and the approaches to be used. The apicoplast as a research topic has produced a truly new way to think about Apicomplexa that now permeates our view of their metabolism, development and cell biology. Studying the apicoplast has brought together biologist focused on different organisms that previously had little contact. This cross-fertilization has let parasitologists to consider pathways initially studied in plants and algae to explain parasite metabolism, drug sensitivity, gene expression control, and signaling and hormone action. Over the last year I have been invited to present our research on the apicoplast at Gordon Research Conferences in three different fields (parasite biology, chloroplast biology, and protein transport) and we view that as a testament to the fact that our specific questions and hypotheses have been innovative and are at the cutting edge of multiple fields. I am particularly excited about the potential role of ubiquitination in the apicoplast. This is a new concept that will lead us to discover new biology and might have strong potential for drug development. Lastly, as a research group and within this project we have invested considerable effort into the development of new experimental tools and approaches. This is particularly true for parasite genetics and cell biological analysis and the current proposal represents an extension of these efforts. We feel that overall this investment has paid off (at times in unexpected ways) and that taking the risk to develop new approaches in the future will keep our experiments fresh and will allow us to ask deeper and deeper mechanistic questions. (C) Approach Specific Aim1: Dissect the mechanism of apicoplast protein import. The apicoplast maintains its own genome, however the bulk of the estimated 400-500 apicoplast proteins are nuclear encoded and imported into the organelle (46). Nuclear encoded apicoplast proteins are synthesized with a bipartite leader and initially routed through the secretory pathway (47, 48). Vesicle fusion with the outermost membrane is believed to deliver proteins to the organelle. How they then cross the remaining three membranes to reach the lumen has been the topic of many spirited discussions and countless review articles, yet until very recently little experimental evidence was available to test these various hypotheses. This has changed dramatically and a model is emerging that proposes three consecutive protein translocons that enable transport over consecutive membranes. Work performed under this proposal has made significant contributions to this progress. We have identified, cloned and localized several members of two candidate translocons in T. gondii ((29, 36, 37), van Dooren & Striepen unpubl., and this proposal). This work described a translocon related to the Tic (translocon of the inner chloroplast membrane) in the innermost apicoplast membrane and a translocon of the second or periplastid membrane derived from an endoplasmic reticulum associated mechanism of the algal endosymbiont. We have adapted a split GFP assay to define the various subcompartments of the apicoplast (37) that has subsequently also been used in other complex plastid systems (49, 50). We also have developed three biochemical assays to quantify apicoplast protein import and most importantly we have used these assays in combination with knock out studies to provide rigorous genetic support for a direct role of two translocons in protein import (36, 37). A recent publication from an algal model organism suggests that a Toc (tranlocon of the outer chloroplast membrane) derived mechanism might be responsible for the transport across the middle membrane (50). The apicoplast ERAD system The initial focus of our mechanistic studies will be the system that is now believed to be responsible for protein import across the second outermost apicoplast membrane. This membrane is of particular interest as it is thought to be homologous to the plasma membrane of the algal endosymbiont. Key to the discovery of the Research Strategy Page 28 Principal Investigator/Program Director (Last, first, middle): Striepen, Boris mechanism was the sequencing of the nucleomorph genome of the cryptophyte alga Guillardia theta (the nucleomorph is the “fossil” remnant of the algal nucleus). Sommer and coworkers noted that this highly reduced genome encodes core elements of the endoplasmatic reticulum associated degradation (ERAD) system (51). ERAD usually acts in ER homeostasis by retrieving misfolded secretory proteins from the ER and funneling them for degradation to the proteasome in the cytosol. The core components of the ERAD transport machinery are Der-1, the ATPase Cdc48 and its co-factor Ufd-1. Der-1 is a favored candidate for the proteinaceous pore in the ER membrane and has been shown to be essential for retro translocation of misfolded luminal proteins (52). Protein substrates destined to be degraded are polyubiquitinated and subsequently extracted from the pore by the Cdc48-Ufd-1-Npl4 complex (53). Sommer and colleagues formulated the hypothesis that an ERAD translocon had been retooled to import proteins into complex plastids (51). The ERAD hypothesis has accumulated considerable support from a recent flurry of publications reporting the identification and plastid localization of ERAD components in cryptomonads, diatoms, and Apicomplexa (51, 54-56). In our own work supported by this grant we have demonstrated that the T. gondii genome encodes multiple homologs of Der1, Cdc48 and Ufd-1. Immunofluorescence analysis of parasite cell lines expressing epitope tagged forms of these proteins reveal that while one complete set of components is associated with the ER and likely performs their classical role in ERAD, at least one homolog of each of these components localizes to the outer membranes of the apicoplast. Furthermore phylogenetic analysis of the two T. gondii Cdc48 proteins demonstrates that they are of divergent evolutionary origins. The apicoplast localized Cdc48 forms a well-supported clade with its red algal lineage counterparts (including the protein encoded on the G. theta nucleomorph) while the cytoplasmic protein branches with proteins that reflect the current view of vertical evolution for Apicomplexa (56). Genetic ablation of Der1Ap in T. gondii results in swift and complete ablation of apicoplast protein import as measured u