persisters

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