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MazF toxin causes alterations in Staphylococcus aureus transcriptome, translatome and proteome that underlie bacterial dormancy

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.

ORGANISM(S): Staphylococcus aureus

PROVIDER: GSE155036 | GEO | 2020/12/28

REPOSITORIES: GEO

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Project description: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.

Project description:Bacterial toxin-antitoxin systems help bacteria to reduce their metabolism in various stressful conditions and also play a part in generating antibiotic tolerant bacterial subpopulation called persisters. Many toxins of the bacterial toxin-antitoxin systems are ribonucleases. Such toxins have been mostly viewed as degraders of mRNA, however recently it was demonstrated that they are also capable of cleaving non-coding RNA. Current libraries are a part of a project which aims to identify the major RNA cleavage sites (in mRNA, rRNA and regulatory non-coding RNA) of MazF and MqsR toxins in E. coli. We were also interested in the activity of promoters during toxin overexpression. We used a targeted RNA-sequencing approach that allowed us to map the distinct 5- and 3-ends of toxin-cleaved RNA and also the beginnings of transcripts. We extracted total RNA from cultures where the expression of MazF or MqsR was induced; RNA from log phase culture was used as the control. In addition, RNA extracted from stationary phase culture was used to test for the possible toxin cleavage in natural stress conditions. The cleaved RNA is rapidly recycled making it possible that we miss important cleavage sites due to RNA degradation during the prolonged stationary phase. Therefore, in addition to wt E. coli we also used stationary phase RNA from exoribonuclease deficient strain where RNA cleavage products accumulate. Identification of the 5 ends is based on ligation of RNA adapters to the cellular RNA molecules, which requires 5 -monophosphates. Mapping of the 3 -ends is based on poly(A) tailing of RNA, which requires 3 -OH groups. Transcription initiation, ordinary cellular RNases, and toxin endonucleases all produce different types of RNA ends. These can be enzymatically converted to 5 -phosphates and 3 -OHs, which allows one to separately quantify the RNA ends produced by each of these processes from a single biological sample. Briefly, (i) primary transcripts have 5 -triphosphate ends that can be converted to monophosphate by Tobacco Acid Pyrophosphatase (TAP), and 3 -hydroxyl ends that can be directly polyadenylated by poly(A) polymerase. (ii) Most cellular RNases (excluding RNase I and the toxins) produce 5 -monophosphates and 3 -hydroxyls, which are directly usable for ligation/polyadenylation. (iii) RNase I, MazF, and several other toxins produce 5 -hydroxyl ends that need to be phosphorylated by T4 Polynucleotide Kinase (PNK) prior to ligation, and 2,3 -cyclic phosphates that need to be dephosphorylated and converted to 3 -hydroxyls, also by T4 PNK. In short, for each of our RNA samples three 5 end and two 3 end libraries were made. The PNK treated libraries contain reads mapping specifically to 5- or 3-ends left by toxin (or RNaseI) cleavage and TAP treated libraries have extra reads mapping to beginnings of the transcripts.

Project description:Tan2012 - Antibiotic Treatment, Inoculum Effect The efficacy of many antibiotics decreases with increasing bacterial density, a phenomenon called the ‘inoculum effect’ (IE). This study reveals that, for ribosome-targeting antibiotics, IE is due to bistable inhibition of bacterial growth, which reduces the efficacy of certain treatment frequencies. This model is described in the article: The inoculum effect and band-pass bacterial response to periodic antibiotic treatment. Tan C, Phillip Smith R, Srimani JK, Riccione KA, Prasada S, Kuehn M, You L. Mol Syst Biol. 2012 Oct 9; 8:617 Abstract: The inoculum effect (IE) refers to the decreasing efficacy of an antibiotic with increasing bacterial density. It represents a unique strategy of antibiotic tolerance and it can complicate design of effective antibiotic treatment of bacterial infections. To gain insight into this phenomenon, we have analyzed responses of a lab strain of Escherichia coli to antibiotics that target the ribosome. We show that the IE can be explained by bistable inhibition of bacterial growth. A critical requirement for this bistability is sufficiently fast degradation of ribosomes, which can result from antibiotic-induced heat-shock response. Furthermore, antibiotics that elicit the IE can lead to 'band-pass' response of bacterial growth to periodic antibiotic treatment: the treatment efficacy drastically diminishes at intermediate frequencies of treatment. Our proposed mechanism for the IE may be generally applicable to other bacterial species treated with antibiotics targeting the ribosomes. This model is hosted on BioModels Database and identified by: MODEL1208300000 . To cite BioModels Database, please use: BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models . To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information.

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MazF toxin causes alterations in Staphylococcus aureus transcriptome, translatome and proteome that underlie bacterial dormancy

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