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  1. Susan A. McDowell, Ph.D. Department of Biology Biotechnology Program Ball State University Muncie, IN samcdowell@bsu.edu Outsmarting the Superbug: developing alternatives to antibiotics

  2. Staphylococcus aureus (staph, MRSA) • Morbidity endocarditis osteomyelitis • Mortality most common infectious agent leading to sepsis 1 million sepsis-associated deaths (1999-2005)

  3. Risk factors for virulent S. aureus infections Hospitalization most common cause of nosocomial cases of pneumonia and surgical wound infection Invasive procedures hip or knee replacement, catheterization Immunosuppression age, HIV Community exposure contact sports

  4. Treatment of infection

  5. Mechanisms of antibiotic resistance Antibiotic Antibiotic Enzymes that alter antibiotics (chloramphenicol aceyltransferase) Enzymes that degrade antibiotics (penicillinase) Plasmid with resistance genes (ampicillin) Antibiotic Chromosomal changes – alter amino acid sequence/binding site of antibiotic (MRSA – new transpeptidase) Pumps that transport antibiotics out of the cell Figure adapted from Lisa Melton: http://www.wellcome.ac.uk/doc_WTX026110.html

  6. Challenge: Increase in antibiotic resistance Adapted from Clinical Infectious Diseases, 2006, 42: 389-91

  7. Hypothesis • Blocking infection at the level of the host is protective.

  8. How we study host cell invasion Human umbilical vein endothelial cell (HUVEC)

  9. Host cell Cell Membrane

  10. Host cell Cell Membrane Nucleus

  11. Host cell Cytoplasm Cell Membrane Nucleus

  12. Host cell Cytoplasm Cell Membrane Nucleus Vimentin

  13. Host cell Cytoplasm Cell Membrane S. aureus Nucleus Fluorescent secondary antibody binds to protein A of S. aureus Fc region of IgG Protein A S. aureus ATCC#29213

  14. Primary mechanism of host invasion Fibronectin S. aureus Fibronectin binding protein a5b1 Integrin

  15. S. aureus Fibronectin binding protein Fibronectin a5b1 Integrin

  16. S. aureus a5b1 Integrin Fibronectin binding protein Fibronectin

  17. Our focus – investigate host cell responses that facilitate invasion and could be blocked pharmacologically

  18. The first host cell response we noticed: Actin stress fibers disassemble during invasion by S. aureus – WHY? Uninfected Infected (1 hr)

  19. The second host cell response we noticed: Filopodia formation (a different actin structure) is stimulated during S. aureus invasion Is this assembly of filopodia related to the disassembly of actin? Stankiewicz, et al., BBRC 391: 2010

  20. What we found: When CDC42, a known regulator of actin, is mutated, host cell invasion is inhibited 120 100 80 Internalized bacteria (% Control +/- SEM) 60 40 * 20 HEK CDC42 C507V/V5 Horn, et al., JPET 326: 2008 *less than control; p < 0.05 Student’s t-test

  21. Could a compound that blocks CDC42 activity inhibit invasion? ML 141 (recently identified inhibitor of CDC41) Could we improve upon that compound? Derivatives of ML 141 **

  22. More derivatives of ML 141 ML 141

  23. Is the inhibition of CDC42 limiting stress fiber disassembly?

  24. Do the derivative compounds work? RSM 06

  25. Potential Outcomes • We will identify a compound with greater efficacy in inhibition of invasion. • Alternatively, several compounds of similar efficacy as that of ML 141 will be identified in vitro that may demonstrate greater bioavailability in vivo.

  26. Significance • Addressing questions of basic science • how S. aureus is internalized • how infection spreads • Evaluation of the therapeutic benefit of inhibitors • during systemic infection • as a prophylactic for invasive procedures

  27. Indiana Academy of Science Acknowledgements The National Institutes of Health George Tegos Mark Haynes Larry Sklar Tom Kirchhausen Rob Sammelson Sponsored Programs Office Laura Michael Chris Vlahos College of Sciences and Humanities Honors College John McKillip Derron Bishop Lilly V Heather Bruns Jim Mitchell Sharm Knecht Xiaoling Liu Parker Siddall Ashley Zahrt Fran Rushing Nickie May Mary Horn Lindy Caffo Kelsey Haaning Julie Clark (Birdsong) Robin DeWalt Kelly Gammon Janelle Owens Amy Pierce (Brown) Charron Johnson Katie Reed Julie Pratt Alys Jordan Terri Abraham Tiera Liby Catherine Volk Sarita Tony Dan Horan Jacy Willis Amber Hampton Diana Santana Daniel Petrovich Ray Kenney Amanda Reese Kristin Abel Traci Stankiewicz Shana Ellis Jacob Henry