ABSTRACT: Bacterial antibiotic resistance is as a serious health problem. Antibiotic resistance appears either because of mutations or as a result of a bacteria dormant state without heritable genetic change. This non-growing state allows bacteria to survive antibiotic treatment. The mechanisms of entrance to the bacterial dormant state are unknown. It has been suggested that toxin-antitoxin systems (TASs) are possible controlling factors for cell dormancy. In Staphylococcus aureus, the role of TASs genome-wide and their link to the dormancy induction mechanisms has not been investigated in detail. In this study, we analyzed the role of MazF toxin on transcriptome, translatome and proteome of S. aureus using RNA-Seq, Ribo-Seq and quantitative proteomics. We characterized the correlation between transcription, translation, and protein levels, and demonstrated that the MazF endonuclease decreases translation directly by cleaving mRNA, and indirectly, by decreasing translation factors and by promoting ribosome hibernation. Thus, MazF represses transcription and translation of many genes rather than a particular set of genes. Nevertheless, several specific pathways affected by MazF were identified: we demonstrated that cell wall thickness is increased and cell division is decreased upon MazF induction. MazF cleaves mRNA in vivo, creating stop-less transcripts and stalled ribosomes. These stalled ribosomes are rescued by SsrA-system, which is activated upon MazF induction. Finally, we described the overall impact of MazF on S. aureus metabolism, and propose one of the mechanisms by which MazF may induce bacterial dormancy.
Samples in dupicates RNA-Seq and Ribo-Seq of WT with pRAB11 vector, DELTAmazEF with pRAB11, and DELTAmazEF with pRAB11 vector expressing inducible MazF. All samples submited to RNA-Seq and Ribo-Seq.
ATc induction was done for 10 min. For total protein extraction 25 ml of cultures with OD600 of 0.7 were collected and washed 3 times with 1 ml of Phosphate-buffered saline (PBS) buffer. Cells were lysed in the presence of 400 microl of lysis buffer (LB) (PBS, 200 microg/ml lysostaphin (AMBI, LLC), 200 microg/ml DNAse I, protease inhibitors (Roche)) for 20 min at 37 C, chilled on ice, and sonicated 10 times with 30-sec cycles using Cell Disrupter B-30 (Branson). Extracts were clarified by centrifugation for 10 min at 14000 g 4 C. Total protein concentration was measured in supernatants by the Bradford protein assay. This method permitted to obtain of about 1 mg of total protein with a concentration of about 2.5-3.0 mg/ml. Samples were mixed with Laemmli Sample Buffer (SB) and analyzed by SDS polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie Blue staining or western blot.
Tandem Mass Tags Mass Spectrometry (TMT-MS2) was done at the Proteome Sciences R&D GmbH&Co KG. Samples were digested with trypsin, TMT-labelled, and subjected to Strong Cation Exchange (SCX) chromatography. Each fraction was analyzed by nano LC-MS/MS and MS2 (Thermo LTQ-Orbitrap Velos mass spectrometer). Data were analyzed with the MASCOT and SEQUEST databases. Filtering, normalization and quantification was achieved using Proteome Discoverer (Thermo Scientific).
In the raw data sample with WT with pRAB11 vector expressing inducible MazF is present, which is not used for the analysis.
Olesya Panasenko 
