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

Overview Persisters Method to follow cell division My results

Bacterial “lifecycle” in lab

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

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

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

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

Persisters - dormant cells Balaban, et al 2004

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

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

Method to follow cell division Roostalu, et al 2008

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

Age of the culture and persistence

Growth resumption dynamics

Length of antibiotic treatment

Persister dynamics in mutants

Growth resumption of sucB mutant

Other TCA cycle mutants

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

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

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

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