Isolation and Proteomic Characterization of Escherichia Coli Persisters

Abstract

Bacteria are known to adapt in various unfavorable situations. The term ‘persister’ has emerged to signify their ability to survive under physiologically adverse environments. Persisters can even tolerate multiple lethal antibiotic treatments. They are of major clinical importance due to their involvement in many fatal chronic bacterial infections such as tuberculosis, lung infection associated with cystic fibrosis, urinary tract infection, and many more. Physiological dormancy is the main basis of their survival in antibiotic stress or host induced environmental stress. Persisters serve as a ‘biological memory’ and are capable of resuming their growth to repopulate when the favourable conditions are restored. Only little do we know about the diverse cascade of events taking place in persisters. Eradication of chronic infection requires elimination of persisters using a suitable anti-persister drug. It is imperative to analyze their physiology thoroughly for a better insight and to explore effective drug-targets. Naturally occurring persisters are present at a very low frequency in any bacterial population and also they lack specific biomarker to selectively fish them out. To isolate native persisters from a heterogeneous population, we first needed to develop a highly specific and quantitative method that selectively identifies and separates them. Using E. coli as a model, we developed an innovative and sophisticated flow cytometry method to isolate native persisters from planktonic liquid cultures. Taking advantage of their non-dividing nature, we exploited their inability to dilute fluorescence and thereby based on the pattern of fluorescent intensity they were quantitatively detected and sorted through flow cytometry. We also performed microscopic analysis to address the ‘fluorescent dilution’ ability of normal dividing cells. Whole proteomic analysis using Mass spectrometry of the sorted persisters revealed an involvement of tryptophanase enzyme as a key regulator of persister physiology. Further we performed microscopic and flow cytometric analysis to demonstrate the crucial role of tryptophanase in bacterial persistence. Thereby tryptophanase can be proposed as an attractive drug-target to develop effective anti-persister drug. Overall, our successful attempt to isolate and analyze persisters in a modern and more authentic manner paid off by reflecting a unique aspect of their physiology.