Unknown,Transcriptomics,Genomics,Proteomics

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RNA-seq of differential gene expression in response to teixobactin in Enterococcus faecalis

ABSTRACT: 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.

INSTRUMENT(S): Illumina MiSeq

ORGANISM(S): Enterococcus faecalis

SUBMITTER: Rachel Darnell

PROVIDER: E-MTAB-6484 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:ClpP is important for bacterial growth and plays an indispensable role in cellular protein quality control systems by refolding or degrading damaged proteins, but the role of ClpP on Enterococcus faecalis is still obscure. A clpP deletion mutant (△clpP) was constructed in E. faecalis strain OG1RF in order to elucidate a more comprehensive picture of the effect of ClpP on E. faecalis. Then the global RNA level of genes was detected by RNA sequencing and the global abundance of proteins was determined by mass spectrometer with Tandem Mass Tags (TMT) labeling. We found the ΔclpP mutant strain showed impaired growth at 20℃ or 45℃, at 5% NaCl or 2 mM H2O2. The surviving bacteria of the ΔclpP mutant strain was decreased after exposure to the high concentration of linezolid or minocycline (100 x MIC) for 96 h compared with that of the parent strain. The ΔclpP mutant strain also demonstrated decreased biofilm formation, but increased virulence in a Galleria mellonella model. The abundances of heat shock proteins, regulatory protein Spx (spxA), heat-inducible transcription repressor HrcA, transcriptional regulator CtsR, ATPase/chaperone ClpC and chaperonin GroEL increased in the ΔclpP mutant strain. The FsrA response regulator and its regulating protein gelatinase GelE, acetyl esterase/lipase, adapter protein MecA and fructose-1,6-bisphosphatase (fbp) also up regulated in the ΔclpP mutant strain. However, the abundances of ribosomal protein L4/L1 family protein (rplD) , ribosomal protein L7/L12 (rplL2), 50S ribosomal protein L13 (rplM), L18 (rplR), L20 (rplT), 30S ribosomal protein S14 (rpsN2) and S18 (rpsR) all reduced in the ΔclpP mutant strain. The dihydroorotase (pyrC), orotate phosphoribosyltransferase (pyrE) and orotidine-5'-phosphate decarboxylase (pyrF) also down regulated in the ΔclpP mutant strain. In conclusion, these results suggest that ClpP participates in stress tolerance, biofilm formation, antimicrobials tolerance, and virulence of E. faecalis.

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RNA-seq of differential gene expression in response to teixobactin in Enterococcus faecalis

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