Kate Bateman Mentors: Dr. Dennis Hruby …… Tove’ Bolken Department of Microbiology

-Gram+ bacterial pathogens: ANTIBIOTIC RESISTANCE - Staphylococcus aureus: HOSPITAL-ACQUIRED INFECTIONS 95% penicillin resistant 50% methicillin resistant Vancomycin resistance on the rise

-Anti-infective compounds -Block tissue attachment -Keep bacteria from hiding from the immune system -Work on antibiotic-resistant strains -Less likely to lead to resistance -Compounds don’t kill bacteria  Less selective pressure

S. aureus adhering to host tissue S. aureus infecting wound tissue

-Highly conserved in G+ bacteria -High Throughput Screening (HTS) assays -Test potential drug candidates

-Bacteria growth inhibition assays Incubate strains in the presence of the inhibiting compound, or a control Measure optical density of each treatment

S. aureus (G+) has the sortase enzyme. Growth slightly inhibited as concentration of compound increases

E. coli (G-) does not have the sortase enzyme Growth not inhibited at higher concentrations of compound

-Surface protein assays Western blot for protein A Protein A: Surface factor that inhibits phagocytes from engulfing the bacterium

-Grow S. aureus in presence or absence of sortase-inhibiting compounds -Harvest by centrifugation, retain supernatant -Resuspend cells in Tris-HCl and EDTA -Lyse cells with lysostaphin and add DNase -Dilute 10X and add gel loading dye with SDS to cell lysate and to supernatant, boil samples -Control (no compounds) Berberine chloride (known inhibitor of sortase)

-Run samples (and marker) on an SDS polyacrylamide gel Protein A: 50 kDa when cut at LPXTGX 56.7 kDa when uncut -Place gel in a transfer cassette with a PVDF membrane -Develop with antibodies

Antibody development

Every treatment looks the same in Intensity and Size

- Harvest cells during exponential phase of growth - Do an assay for another surface protein (fibrinogen clumping assay)

-Surface protein assays Fibrinogen assay Clumping factor/fibrinogen binding proteins anchored in cell wall: -Recognize and bind to fibrinogen in blood, forming clumps. -Promote attachment to blood clots and traumatized tissue

-Grow S. aureus in presence or absence of sortase-inhibiting compounds -Harvest by centrifugation, retain cells -Resuspend cells in solution containing fibrinogen -Measure optical density over time -Control (no compounds) Berberine chloride (known inhibitor of sortase) O.D. Decreases  Fibrinogen clumping  Proteins present  Sortase Active O.D. Constant  Fibrinogen not clumping  Proteins not present  Sortase Inactive

-Compounds do not inhibit growth of S. aureus -Assay for protein A needs to be more refined -Fibrinogen assay shows promise

-Compounds do not inhibit growth of S. aureus -Assay for protein A needs to be more refined -Fibrinogen assay shows promise *Several new compounds identified by HTS at Siga

- Howard Hughes Medical Institute - Dr. Hruby - Tove’ Bolken - Everyone in the Hruby lab - Kevin Ahern