Unknown,Transcriptomics,Genomics,Proteomics

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Identification of the teixobactin-induced CroRS regulon in Enterococcus faecalis using RNA-seq.

ABSTRACT: CroRS is a cell envelope stress response two-component system in the bacterial pathogen Enterococcus faecalis. Antimicrobial tolerance is the ability of an organism to survive, but not proliferate, upon antimicrobial challenge, and is a known precursor to the development of antimicrobial resistance. We have previously shown that CroRS is essential for antimicrobial tolerance in E. faecalis. Therefore, the aim of this experiment was to determine the teixobactin-induced CroRS regulon to identify key pathways of antimicrobial tolerance. To do this, E. faecalis wild-type and a croRS deletion mutant were grown to mid-exponential phase and challenged with and without teixobactin (0.5 ug/ml) for 1 hour. RNA was subsequently extracted, purified and RNA sequenced. RNA libraries were prepped using the Zymo-Seq RiboFree Total RNA-Seq Library Kit. Sequencing was completed using an Illumina MiSeq (v3) system generating 150 bp paired-end reads.

INSTRUMENT(S): Illumina MiSeq

ORGANISM(S): Enterococcus faecalis

SUBMITTER: Rachel Darnell

PROVIDER: E-MTAB-12526 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Emerging antibiotic resistance among clinically relevant bacteria, paired with their ability to form biofilms on medical and technical devices, represents a serious problem in terms of effective and long-term decontamination in health care environments and gives rise to an urgent need for new antimicrobial materials. Here we present the first study of the impact of AGXXÂ®, a novel broad-spectrum antimicrobial surface coating consisting of micro galvanic elements formed by silver and ruthenium, on the transcriptome of the nosocomial pathogen Enterococcus faecalis. E. faecalis was subjected to metal stress by growing it for different periods of time in the presence of AGXXÂ® or silver-coated steel meshes. Subsequently, total RNA was isolated and next-generation RNA sequencing was performed to analyze variations in gene expression levels in the presence of the antimicrobial materials with focus on known stress genes. Exposure to AGXXÂ® had a large impact on the transcriptome of E. faecalis. After 24 minutes almost 1/5 of the E. faecalis genome displayed differential expression. At each time-point the cop operon was strongly up-regulated, providing indirect evidence for the presence of free Ag+-ions. Moreover, exposure to AGXXÂ® induced a broad general stress response in E. faecalis. Genes coding for the chaperones GroEL and GroES as well as the Clp proteases ClpE and ClpB were among the top up-regulated heat shock genes. Furthermore, differential expression of genes coding for thioredoxin, superoxide dismutase and glutathione synthetase indicates a high level of oxidative stress. We postulate a mechanism of action where the combination of Ag+-ions and reactive oxygen species generated by AGXXÂ® results in a synergistic antimicrobial effect, which is superior to that of conventional silver coatings. Gene expression analysis of Enterococcus faecalis 12030 either subjected to metal stress by exposure to an antimicrobial AGXXÂ®- or Ag-coated V2A steel mesh or exposed to an uncoated V2A steel mesh or left untreated performing RNA Sequencing with an Ion ProtonTM Sequencer and subsequent data analysis with a T-REx RNA-Sequencing expression analysis pipeline.

Project description:Teixobactin is the first novel antimicrobial to be discovered in decades and represents a new class of antimicrobials. Teixobactin shows great promise with proven efficacy against multi-drug resistant organisms such as methicillin-resistant S. aureus (MRSA), vancomycin-resistant Enterococci (VRE), and Mycobacterium tuberculosis. VRE infections are notoriously difficult to treat with complex and adaptable cell wall stress response systems, which confer intrinsic resistance to a wide variety of antimicrobials. The aim of this study was to isolate the teixobactin-induced transcriptional response by challenging lab strain Enterococcus faecalis JH2-2 with sub-MIC levels of teixobactin using RNA sequencing. Two cultures of E. faecalis were grown to an OD600 of 0.2 and subsequently split into three to form a total of six cultures (two samples, with three technical replicates each), and grown to an OD600 of 0.5. One set of three cultures were treated with 0.5 ug/ml (0.25 x MIC), and all six cultures were grown for a further 1h. Cells were harvested by centrifugation and stored at -80 degrees C. RNA was extracted using TRIzol-chloroform and RNA samples were run through the RNAeasy Minikit (Qiagen). The Agilent RNA 6000 Nano kit and the Agilent 2100 Bioanalyzer (RIN >8), was used to verify RNA quality as per the manufacturer’s instructions, and RNA concentration was determined using a NanoDrop ND-100 spectrophotometer. Ribosomal RNA was removed from total RNA using Ribo-Zero and cDNA libraries were created using the Illumina TruSeq™ stranded total RNA library prep kit. Sequencing was completed using Illumina MiSeq_v3 generating 150 bp single end reads. Adapter sequences were removed from raw fastq files using Flexbar and reads shorter than 50bp were discarded. Sequence reads from each sample were mapped against the E. faecalis JH2-2 genome (NZ_KI518257.1) using Bowtie to produce a table of raw read counts for JH2-2 genes for each of the replicates. Statistical and principal component analysis were performed using the Bioconductor DESeq package.

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Identification of the teixobactin-induced CroRS regulon in Enterococcus faecalis using RNA-seq.

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