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Enterococcus Grad-seq and KhpB RIP-seq

ABSTRACT: We investigated the RNA-protein interactome of Enterococcus faecalis V583 and Enterococcus faecium Aus0004 by native gradient fractionation of complexes coupled to RNA-sequencing. Whole bacterial cell lysates were analysed by size and density in a glycerol gradient. At native conditions, RNA-protein complexes stay intact and sediment as a whole. Sedimentation profiles of individual RNAs appear correlated in case of interaction in a complex. The profile of KhpB caught our attention and we determined its RNA interactome by immunoprecipitation that suggests a role at the post-transcriptional level, binding notably several tRNAs, sRNAs, and 3’UTRs.

ORGANISM(S): Enterococcus faecalis V583 Enterococcus faecium Aus0004

PROVIDER: GSE196534 | GEO | 2022/11/03

REPOSITORIES: GEO

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Project description:Regulatory RNAs (sRNAs) are now considered as major players in many physiological and adaptive responses in pathogenic bacteria. sRNAs have been extensively studied in Gram-negative bacteria, but less information is available in Gram-positive pathogens. There is a spread of multidrug-resistant (MDR) opportunistic organisms, grouped as “ESKAPE” pathogens, which comprise enterococci, a leading cause of hospital-acquired infections and outbreaks with emergence of MDR isolates, especially vancomycin-resistant Enterococcus faecium (VREF). Note that no information about sRNA expression is known in this major opportunistic pathogen. By transcriptomic and genomic analyses using E. faecium Aus0004 reference strain, 249 transcribed IGRs, including sRNA candidates, were detected and, using a series of cut-offs, this set was lowered down to 54 sRNAs while 7 that were predicted based on comparative sequence analysis. RNA-seq was performed with and without subinhibitory concentrations (SIC) of daptomycin, a cyclic lipopeptide antibiotic used for VREF infections. Under daptomycin SIC exposure, 260 genes (9.1% of the genome) had a significant alteration of expression including 80 upregulated genes and 180 downregulated genes. Among the repressed genes, a large proportion (55%) coded for proteins involved in carbohydrate and transport metabolism. Also, we focused on the 9 sRNAs exhibiting the highest expression, and all of them were confirmed as expressed along bacterial growth by Northern blots and qPCR. Out of these 9 sRNAs, four had significantly lower or higher expression in the presence of daptomycin SIC, and therefore responded to antibiotic exposure. Finally, we also tested the expression of these 9 sRNAs in a collection of isogenic Aus0004 mutants with increasing levels of daptomycin resistance, and we observed by qPCR that some sRNAs had a significantly modified expression in daptomycin resistance mutants. It highlights the significant implication of some of the E. faecium sRNAs in the early steps of the development of daptomycin resistance. This is the first experimental genome-wide sRNA identification in Gram-positive E. faecium, a leading cause of hospital acquired infections.

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Enterococcus Grad-seq and KhpB RIP-seq

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