1. Genetic and environmental conditions influencing persistence Hannes Luidalepp Institute of Technology University of Tartu August 2011

2. Overview Persisters Method to follow cell division My results

3. Bacterial “lifecycle” in lab

4. Resistance Including whole population Genetically determined Pumping out antibiotics Degradation of antibiotics Altering the target etc

5. Persistence Part of the population is insensitive – persisters Medically important – chronical and persistent infections Genetically identical to the mother cells Phenotypical property

6. Persisters frequency usually 10 -4 -10 -6 mainly originate from stationary phase common to all bacterial species investigated (also to fungi and cancer cells) Keren, et al 2004

7. High persistence (Hip) mutants persister frequency usually at least 100x higher (also some clinical isolates of C. candida, P. aeruginosa) most common mutant is hipA7 (point mutations in hipA gene) hipA is a toxin from hipBA antitoxin-toxin pair

8. Persisters - dormant cells Balaban, et al 2004

9. Persisters Type I persister: originate from stationary phase Type II persisters: originate from growth phase Balaban, et al 2004

10. Physiology of persisters Gene expression arrays different toxin/antitoxin genes, other unrelated genes Mutant collection screens different unrelated genes Over-expression studies all toxic stuff will increase persistence

11. Method to follow cell division Roostalu, et al 2008

12. Cell division LB medium 24h stationary phase culture diluted into fresh media Two population dividing non-dividing

13. Age of the culture and persistence

14. Growth resumption dynamics

15. Length of antibiotic treatment

16. Persister dynamics in mutants

17. Growth resumption of sucB mutant

18. Other TCA cycle mutants

20. Summary Growth resumption depends on: –media –age of the culture Level of persisters depends on growth resumption

23. Mutants and persistence –Mutants show different persister formation dynamics –Difference from wild type might be observed only in certain conditions

24. Medical applications –To make cell to wake up faster Allison et al., 2011 Kim et al., 2011 –To make cell permanently dormant

25. Acknowledgments People from Tanel Tenson’s lab Specially Arvi Jõers and Niilo Kaldalu