Single-cell analysis of the MazF-mediated stress response

Bacteria adapt to adverse environmental conditions by altering gene expression patterns. Recently, a novel stress adaptation mechanism has been described that allows Escherichia coli to alter gene expression at the post-transcriptional level. The endoribonuclease MazF (the toxin component of the toxin-antitoxin module MazEF) is one of the key players responsible for the stress-induced production of leaderless mRNAs, which lack the ribosome binding site. MazF concomitantly modifies the ribosomes, making them selective for the translation of leaderless mRNAs. Intriguingly, the activation of MazF and other toxin- antitoxin systems has been implicated in the persistence phenotype in E. coli. Persistence is a prime example of phenotypic heterogeneity that arises in populations of genetically identical cells independently of genetic or environmental differences. Persister cells represent only a minor fraction of a clonal population that grows much slower than the majority of cells and can endure antibiotic treatments. However, the function of MazF in bacterial cells that persist antibiotic treatment or nutrient starvation still remains elusive. Thus, my main interest is to study the MazF-mediated post-transcriptional stress response from the single-cell perspective. Since only a small fraction of a bacterial population is able to express the persistence phenotype, I will address the question whether all cells or only a fraction of cells within a clonal population induce MazF under antibiotic stress or nutrient starvation. I hypothesize that the potential heterogeneous production and translation of leaderless mRNAs provide benefits for bacterial populations. Consequently, the cells with active MazF would selectively survive stress and thereby represent the subpopulation that will resume growth after the stress is removed. To tackle these questions, I will use flow cytometry combined with biochemical techniques as well as fluorescence time- lapse microscopy and microfluidics. This research project is anticipated to assess the physiological importance of the newly discovered MazF-dependent stress adaptation mechanism in single bacterial cells with respect to survival of the population. Hence, the expected results will provide novel insights into the molecular basis of phenotypic heterogeneity in the bacterial stress response and will contribute to a better understanding how bacterial populations endure antibiotic treatments and starvation upon host infection. Collectively, this is an important step towards development of strategies for the eradication of persister cells, which is currently one of the most challenging problems in medical microbiology.

Bacteria experience various stressful conditions in nature, from nutrient depletion and starvation, to exposure to antibiotics. To adapt fast and efficiently to environmental fluctuations, bacteria have evolved various protection programs called the bacterial stress response. Here we investigated one mechanism that helps bacteria to cope with stress. It is based on the activation of toxin-antitoxin systems that are one of the hallmarks of bacterial tolerance to antibiotics, and we were particularly interested in one toxin, MazF. MazF is called a "toxin" because its activity can be toxic for bacteria: MazF degrades cellular RNA and therefore its activation slows down the bacterial growth. However, MazF can also process mRNA and therefore contribute to the modulation of gene expression programs. Intriguingly, the activation of MazF and other toxin-antitoxin systems has been implicated in the persistence phenotype in the model bacterium Escherichia coli and, more importantly, in different species of pathogenic bacteria. Only a small percentage of bacterial cells within clonal populations can tolerate antibiotic treatments, and these cells are called persisters cells. In this project we aimed to research if genetically identical bacterial cells differ in the production and activation of the MazF toxin during stressful conditions, as well as how much of this variation is maintained in the affected cellular processes, i.e. growth and gene expression. Our results indicate a high level of variation in the MazF activity; in other words, some cells produce and activate more MazF than others. This variation is further exhibited at two levels. First, bacterial cells differ in their growth rates during mazF induction, so even though bacterial growth is on average reduced, some cells still grow faster than others. We also show that this growth heterogeneity is a consequence of the complex mechanisms that regulate the level of the MazF toxin. The complex regulation of the mazF expression enables cells to enter MazF-mediated stress quickly, as well as allows cells to exit the stress as fast as possible and resume the growth once the stressor is removed. Second, our results suggest that populations may exhibit a substantial level of variation in the gene expression during stressful conditions that trigger MazF and during resumption of growth upon stress relief. The results obtained in this project provide novel insights into the molecular basis of phenotypic variation in the bacterial stress response and contribute to a better understanding how bacterial populations endure antibiotic treatments and starvation upon host infection. This is an important step towards development of techniques for the eradication of persister cells, which is currently one of the most challenging problems in medical microbiology. Collectively, the knowledge about physiological importance of different toxin-antitoxin systems and their role in cellular processes could enable development of novel antimicrobial strategies.