Novel strategies involved in bacterial stress adaptation

Bacteria frequently encounter changes in environmental conditions, e.g. shifts in temperature or pH, oxidative and osmotic stress or nutrient deprivation. In particular, pathogenic bacteria are subjected to rapidly changing environments when they enter their host. To survive these adverse conditions bacteria have developed several strategies to alter gene expression and protein activity, which allow a fast adaptation. In addition, some pathogenic bacteria are able to form persister cells, which are characterized by a dormant phenotype, which enables the bacteria to survive antibiotic treatment as well as the host immune response. Recent studies implicated toxin- antitoxin modules in establishment of persistent infections. The activation of these modules blocks several important processes in the cell, like translation and replication. However, the underlying molecular mechanism how these modules stimulate persistence remains to be established. Recently, we have shown that the aminoglycoside antibiotic Kasugamycin, a general translation initiation inhibitor, induces the formation of protein-depleted ribosomal particles in Escherichia coli in vivo. These particles are proficient in translation of leaderless mRNAs but fail to translate canonical mRNAs. Surprisingly, the prolonged presence of the antibiotic results in restored expression of several stress genes. Further studies of this phenomenon revealed that the respective mRNAs become leaderless upon antibiotic treatment, allowing their selective translation in the presence of Kasugamycin by protein-deficient ribosomes. Moreover, primer extension analyses upon over-expression of the toxin MazF implicated toxin-antitoxin systems in the appearance of conditional leaderless mRNAs. Taken together, these observations suggest a hitherto uncharacterized strategy of bacterial pathogens to cope with stress, e.g. upon infection of the host. The formation of conditionally leaderless mRNAs, which can be translated by aberrant ribosomes present under stress conditions, could lead to selective expression of genes, which might stimulate the formation of persister cells. Therefore, the objective of the study is the characterization of the molecular mechanisms underlying the protein-depletion of ribosomes and the formation of conditionally leaderless mRNAs. These studies are anticipated to shed light on key players and factors that are required for the adaptation to stress conditions and moreover, for the formation of persistent bacteria. Some of these factors might have the potential to serve as targets for the design of novel antimicrobial compounds, which could prevent the establishment of chronic infections or aid in eradicating persister cells of important human pathogens.

Bacteria frequently encounter changes in environmental conditions, e.g. shifts in temperature or pH, oxidative and osmotic stress or nutrient deprivation. In particular, pathogenic bacteria are subjected to rapidly changing environments when they enter their host. To survive bacteria have developed several strategies to adapt gene expression and protein activity to the given conditions. Besides, some bacteria are able to form persister cells, which are characterized by a dormant phenotype, enabling the bacteria to sustain antibiotic treatment as well as the host immune response. Recent studies implicated toxin-antitoxin (TA) modules in establishment of persistent infections. The activation of these modules blocks several important processes in the cell, like translation and replication. However, the underlying molecular mechanism how these modules stimulate persistence remained to be established. In the framework of this project we focused specifically on a post-transcriptional stress response pathway that is triggered by the stress-responsive activation of the TA-system mazEF. Our results show that besides degrading the majority of bulk mRNA the active endoribonuclease MazF cleaves at ACA sites at or closely upstream of the AUG start codon of some specific mRNAs. In addition, MazF also targets the ribosomal RNA within the translational machine thereby removing a functionally important region. Consequently, a subpopulation of ribosomes is formed that selectively translate the mRNAs processed by MazF. Together, this fast and energy-efficient reprogramming of protein synthesis by the stress-responsive activation of MazF represents a conceptually novel post-transcriptional regulation mechanism in bacteria that might contribute to heterogeneity within a bacterial population thereby stimulating persister cell formation. To further scrutinize this pathway, we determined the MazF-regulon, i.e. the entity of transcripts that are processed by MazF and selectively translated by the stress-ribosomes. The results reveal that the corresponding protein products are involved in all cellular processes, underpinning the general importance of the MazF-dependent response. In addition, the data collected within this project allowed us to define novel scientific questions, which are the basis for follow-up projects that address hitherto undescribed mechanisms in bacteria, like e.g. the reversibility of ribosome heterogeneity and the potential of the MazF-mediated stress response to enhance the biodiversity of proteins encoded by the bacterial genome, a mechanism comparable to alternative splicing in eukaryotes.