1. NIH AREA/R15 Grant Proposals Mohamed Elasri Department of Biological Sciences

2. The Details NIH Academic Research Enhancement Award (AREA) Program (R15) – Goals of the Program – Support meritorious research – Expose students to research – Strengthen the research environment of the institution

3. Key Features – Project period is limited to 3 years – Direct costs are limited to $300,000 over the entire project period – Grants are renewable – Preliminary data are not required

4. R15 is a great grant mechanism for USM – Eligibility: The institution may not receive more than $6 million per year in NIH support in each of 4 of the last 7 years.

5. My Experience with the R15 Pending Research Support: – 1R21AI117160-01Elasri (PI)04/01/2015-03/31/2017 NIH/NIAID Riboregulation of Biofilm Development in Staphylococcus aureus Ongoing Research Support: – 1R15AI099922-01A1Elasri (PI)12/01/2012- 11/30/2015 NIH/NIAID Biofilm Dissemination in Staphylococcus aureus Selected Completed Research Support: – 1R15AI062727-01A1 Elasri (PI) 8/1/2005-5/31/2009 NIH/NIAID Regulation of Virulence by LuxS in Staphylococcus aureus. – 1 R15 EB003399-01Lowe (PI) Elasri (Co-PI)4/1/2004-3/31/2007 NIH/NIBIB New Polymeric Betaines as Antiadherent Materials

6. Framing Mechanisms Three framing mechanisms for your NIH proposal: – fill the gap – cure the disease – save the world

7. What makes for a good R15 proposal? Good idea Good proposal And some luck…

8. What you need to know… Grantsmanship is a skill that can be cultivated through practice. Grantsmanship has nothing to do with scientific ability.

9. Anatomy of my latest R15 Specific Aims (most important part of your proposal) Research Strategy – Preliminary Data (absolutely essential) – Have a good plan (A, B, and C) – Collaborators (even if you don’t think you need them)

10. Specific Aims This section should include who, what, where, when, how, and why Should be very clear This is where reviewers make up their mind about your proposal

11. Specific Aims Staphylococcus aureus is a versatile pathogen that causes a wide variety of infections. In addition to its large arsenal of virulence factors, S. aureus can grow in biofilm communities that are recalcitrant to antibiotics treatment and host defenses. It is estimated that 80% of S. aureus hospital infections are associated with biofilm. Biofilm development can generally be divided into four stages: adherence, accumulation, maturation and dispersion. Mature staphylococcal biofilms are characterized as bacterial communities that are encased in complex structures comprised of extrapolymeric substances that include extracellular DNA (eDNA) released from active death of cells. Biofilm communities contain a variety of niches that are metabolically heterogeneous. Cells are actively released from biofilm leading to dissemination of infection within the host. Previously, we have characterized a new gene, msa that is involved in regulation of virulence and biofilm development. In this application, we will test the hypothesis that msa plays a key role in biofilm maturation and/or dispersion by regulating structuring and dispersion factors both in vitro and in vivo. We also hypothesize that the structural defects in biofilm caused by mutation of msa will lead to reduced pathogenic impact in biofilm-associated infection and increased clearance by the host. Finally, since the ultimate goal of this study is to exploit msa as a therapeutic target to enhance conventional therapy, we will examine the impact of msa on biofilm susceptibility to antibiotic treatment in vivo. We have developed two specific aims to test this overall hypothesis:

12. Specific Aims (continued) Specific Aim 1: Define the role of msa in biofilm development. We will use the wild type strain, the msa deletion mutant, and the complemented msa mutant of three clinical strains from three dominant human lineages of S. aureus: HA-MRSA N315 (CC5), CA-MRSA USA300 LAC (CC8), and MSSA UAMS1 (CC30). – To examine the maturation stage of the msa mutant, we will grow biofilms in flow cells and assess maturity using confocal microscopy and image analysis to spatially map and define the composition of the biofilm. We will use fluorescent stains to visualize and quantify viable cells, dead cells, eDNA, and poly-N-acetylglucosamine (PNAG) in biofilm. – We will use a transcriptional fusion (msa–egfp) and confocal microscopy to monitor expression of msa in various niches of biofilm and correlate the temporal and spatial expression pattern of msa with its regulatory function during biofilm development. – To examine the dispersion stage of the msa mutant, we will determine the rate of detachment of the msa mutant by measuring detached biomass in the effluent of biofilms grown under low- and high-shear conditions. We will use a proteomics approach on the effluent from flow cells to define proteins that are differentially secreted/shed by the msa mutant biofilm. This sub-aim (1C) will be done in collaboration with Dr. Lindsey Shaw (USF), who has extensive experience in proteomics. Specific Aim 2: Define the role of msa in biofilm-associated infection, host responses and susceptibility to antibiotics in vivo. We have established that msa is essential to pathogenesis in three animal models: C. elegans, septic arthritis murine model, and a sepsis murine model. We hypothesize that the role of msa in biofilm development is key to its pathogenesis in biofilm-associated infections. We will use a murine model to evaluate the role of msa during in vivo biofilm development, and to correlate it with pathogenesis, and host responses. In addition, we will evaluate the impact of msa on susceptibility to daptomycin. In this study, we will use a biofilm-development murine model in which we surgically implant catheters subcutaneously and then inoculate them with the test strain (i.e. CA-MRSA USA300 LAC). – To assess the ability of the msa mutant to develop biofilm in vivo, we will enumerate the adherent bacteria present on the surface of the explanted catheters and quantify biofilm by confocal microscopy at three time points post-infection (2, 4, and 6 days). This sub-aim will be done in collaboration with Dr. Mark Smeltzer (UAMS), who has extensive experience in this animal model. – To define the role of msa in biofilm-associated infection, we will evaluate the ability of the mutant to form subcutaneous abscesses by microbiological, histological and inflammation quantification analyses of tissue removed from the implant sites. This sub-aim will be done in collaboration with Dr. Kenneth Butler (UMMC), who has extensive experience in analysis of host immune responses. – To evaluate the impact of msa on susceptibility of biofilm to antibiotics, we will treat one group of mice with daptomycin daily and quantify biofilm clearance from the explanted catheters by confocal microscopy and enumeration of bacteria at 2, 4, and 6 days post-infection.

13. Advice about your research strategy… Be clear and concise (get people to read it for you) Grants are NOT a creative writing contest! Show evidence that you can do the techniques you are proposing to use – If not possible, get a collaborator who is an expert in the technique

14. Use figures to tell stories

16. Gotta have collaborators… Collaborators help beef up your feasibility factor Collaborations are also great opportunity to develop interdisciplinary projects

17. Preliminary Data show feasibility Summary: – “Good preliminary results, well-addressed responses to most previous critiques” Reviewer 2: – Strengths “The studies are well justified by preliminary results.” Reviewer 3: – Strengths “Preliminary data were added that increase the probability of success, particularly regarding the animal models. The C. elegans story was interesting.”

18. Framing Mechanisms Three framing mechanisms for my current R15: – fill the gap (lack of understanding of biofilm development in staph infections) – cure the disease (my gene is a great target for future therapy) – save the world (antibiotic resistant staph will take over the world if I don’t do something about it)

19. The R15 Education bit… Reviewer: “Dr. Elasri is committed to both undergraduate and graduate education in biomedical research. His discussion of the potential impact of an AREA award on his program specifically and the university as a whole is a strength of this application.”

20. Other resources: – Profile of Students at the University of Southern Mississippi (USM) and the Biological Sciences Department. – AREA award would strengthen the PI’s effort to provide training in biomedical research for undergraduate and graduate students. – AREA award would strengthen the research environment at USM. The R15 Education bit…

21. “Internal” reviews of proposal are essential Put together an informal study section for your proposal Internal reviewers do not have to be in your area of research Very difficult to accomplish

22. NIH Reporter Use NIH Reporter to research what topics your study section is funding. Great tool to see how your idea stacks up against funded research