Project description:The enterococci comprise a genus of 49 low-GC content Gram-positive commensal species within the Firmicutes phylum that are known to occupy diverse habitats, notably the gastrointestinal core microbiota of nearly every phylum, including human. Of particular clinical relevance are two rogue species of enterococci, Enterococcus faecalis and the distantly related Enterococcus faecium, standing among the nefarious multi-drug resistant and hospital-acquired pathogens. Despite increasing evidence for RNA-based regulation in the enterococci, including regulation of virulence factors, their transcriptome structure and arsenal of regulatory small sRNAs (sRNAs) are not thoroughly understood. Using dRNA-seq, we have mapped at single-nucleotide resolution the primary transcriptomes of E. faecalis V583 and E. faecium AUS0004. We identified 2517 and 2771 transcription start sites (TSS) in E. faecalis and E. faecium, respectively. Based on the identified TSS, we created a global map of s70 promoter motifs. We also revealed features of 5’ and 3’UTRs across the genomes. The transcriptome maps also predicted 150 and 128 sRNA candidates in E. faecalis and E. faecium, respectively, some of which have been identified in previous studies and many of which are new. Finally, we validated several of the predicted sRNAs by Northern Blot in biologically relevant conditions. Comprehensive TSS mapping of two representative strains will provide a valuable resource for the continued development of RNA biology in the Enterococci.
Project description:The transcriptome of Enterococcus faecium E1162 growing in Brain heart Infusion Broth was compared in the mid-exponential growth phase (A660 = 0.3) at 25 C and 37 C.
Project description:This study aimed to investigate the transcriptional response of human cervical cancer HeLa cells to metabolites derived from Enterococcus faecium. HeLa cells were treated with filter-sterilized E. faecium supernatant (equivalent to 1×10⁸ CFU/mL) for 24 hours, with three biological replicates each for the control (HC) and treatment (HT) groups. Total RNA was extracted and subjected to transcriptome sequencing. Bioinformatic analysis identified a set of differentially expressed genes involved in cellular metabolism, stress response, and key signaling pathways. The results demonstrate that E. faecium metabolites significantly alter the transcriptional profile of HeLa cells, providing new insights into the molecular mechanisms underlying host–bacterial metabolic interactions.
Project description:Parkinson's disease (PD) is defined by neurodegeneration, muscle atrophy, and bone deterioration, largely due to dopamine depletion. This study evaluates the therapeutic potential of heat-killed Enterococcus faecium FBL1 (HEF) in mitigating PD-related dysfunction through osteoblastogenesis and neurogenesis pathways. In the rotarod test, MPTP-treated mice exhibited a significant reduction in walking time, 4.1 times lower than that of the normal group. However, treatment with HDF notably improved the retention time, with a dose-dependent increase, compared to MPTP group. Wire-hanging test showed enhanced muscle strength, as HEF-treated mice demonstrated a 2.1- and 3.3-fold increase in the latency to fall at low and high doses, respectively, when compared to MPTP group. Grip strength and forced swim test, further supported the findings of neuromuscular recovery and reduced immobility in the HEF treated mice. The alpha-synuclein aggregation in the brain and muscle induced by MPTP were attenuated by HEF. Volcano plot analysis of muscle tissue revealed that MPTP treatment caused significant dysregulation, with 142 upregulated and 163 downregulated genes, including the downregulation of Wnt signaling-related genes Astn1 and Frat2, which are involved in neurogenesis and muscle regeneration. Conversely, the osteogenesis-related gene Pbx1 was upregulated by HEF, compared to MPTP treated group. Treatment with HEF also restored gene expression, notably increasing Tnxb, essential for tissue integrity, and Gsn, involved in various biological processes, compared with MPTP. Key markers of skeletal muscle differentiation (Myf5, MyoG, Myh1), osteoblastogenesis (Bmp2, Bmp4, SMAD1/5/8, RUNX2), and neurogenesis (Wnt3a, Beta-catenin, TCF1, LEF1) were downregulated in MPTP-induced PD but restored by HEF. Inflammatory markers (TNF-alpha, iNOS, and NF-kappaB) were significantly elevated in the MPTP group. However, these levels were reduced by HEF in a dose-dependently. This study highlights the pivotal role of osteoblastogenic (BMP/SMAD signaling) and neurogenic (Wnt signaling) pathways in maintaining muscle and bone homeostasis in PD. HEF offers a novel therapeutic approach targeting the BMP/SMAD and Wnt signaling pathways to mitigate muscle and bone degeneration in patients with PD.
Project description:A set of small RNAs was identified in Vancomycin-resistant Enterococcus faecium, a leading cause of MDR infections. We described here the function of srn_2050, acting as a T-box riboswitch to regulate expression of downstream genes encoding the HisRS and AspRS aminoacyl-tRNA synthetases. Comparative RNAseq between Aus0004 and isogenic srn_2050 mutant identified the genes whose expression is impacted by the RNA. srn_2050 structure in its ‘off state’ was deciphered by in-line probing, containing T-box consensus sequences, a pseudoknot, a specifier loop and a terminator. Transcription binding assays between the riboswitch and either tRNAAsp or tRNAHis indicate that each deacylated tRNA interacts with the T-box. Their anticodons bind to a GACAC sequence within the specifier loop (GAC and CAC are Asp and His codons, respectively), whereas tRNATyr (UA/C-U) does not. A pioneering evaluation of E. faecium amino acid auxotrophy, with emphasis on E. faecium strain Aus0004, revealed auxotrophy for Histidine but not for Aspartic acid. Based on comparative growths and RNAseq between Aus004 and Aus004-srn2050, the riboswitch is shown essential for growth under aspartate starvation. This is the first example of a functional riboswitch in E. faecium with two overlapping codons allowing a dual tRNA-dependent regulation at transcriptional level.