2. Pseudomonas aeruginosa Human opportunistic pathogen – exploits some break in hosts’ defenses in which to initiate infection

3. Pseudomonas Antibiotic Resistance

5. Immuno- compromised individuals Cystic fibrosis infection less than 1yr – 21%, greater than 19yrs – 80% Responsible for death of majority 2 nd most common cause of ventilator-associated pneumonia (HAI) 4 th most common cause of catheter-associated urinary tract infection (HAI) Grows sometimes on skin, can infect through injury Nosocomial infections - originate or occur in a hospital or hospital-like setting [also called hospital- acquired infections (HAI)]

6. Quorum Sensing

7.  Signal diffuses  Cells and signal accumulate  Signal binds and activate LasR, a transcriptional regulator  Virulence gene expression LasI LasR LasI LasR Signal synthase Signal receptor

8. Discovery of anti-activator

9. LasI LasR LasI LasR Signal synthase Signal receptor  Signal produced  QteE inhibits the binding of the signal and LasR  Signal accumulates  Signal binds with and activates LasR, the transcriptional regulator  Virulence gene expression QteE LasR

12. To differentiate strains, integrate mCherry fluorescent protein into chromosome Insert the GFP plasmid into strains for QS determination Co-culture strains together and use microscopy to identify fluorescence

13. Integrating mCherry  Confirm integration:  Tecan – measure fluorescent excitation with a machine  Microscopy – check cellular fluorescence with a fluorescent microscope att site

14. Microscopy Image

15. GFP The green fluorescent protein is controlled by a QS promoter The cells will fluoresce green when LasR binds the signal and induces transcription of QS genes plasmid

16. Strain 1 Name (red) Strain 1 Function Strain 2 Name Strain 2 FunctionPredicted Results Initial Predicted Results Final WTProduces all proteins, QS proceeds as normal Δ lasI Doesn’t produce LasI signal synthase, QS can only respond to WT-produced signal Neither strain green Both turn green at the same time qteE- Doesn’t produce QteE, can “short- circuit” lasIqteE- Double mutant, requires signal from qteE-, depends on diffusion for signal and QS qteE- is green, lasIqteE- is not Both green LasI QteE Control Test