Differentially expressed genes in rough and smooth colony isolates of Clostridioides difficile
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA664385 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR12671773_1.fastq.gz | Fastqsanger.gz | |||
| SRR12671773_2.fastq.gz | Fastqsanger.gz | |||
| SRR12671774_1.fastq.gz | Fastqsanger.gz | |||
| SRR12671774_2.fastq.gz | Fastqsanger.gz | |||
| SRR12671775_1.fastq.gz | Fastqsanger.gz |
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Project description:Purpose: to determine the differentially expressed genes in the phase-variable rough and smooth colony isolates of C. difficile Methods: C. difficile R20291 was grown on BHIS agar to obtain distinct colonies. Individual rough and smooth colonies were chosen for propagation on BHIS-agar for 24 hours as described in Garrett, et al., PLoS Biology, 2019. Growth was collected from n = 2 biological replicates. RNA was purified using TriSure and chloroform, beadbeating, and isopropanol/ethanol precipitation. Quality was verified with Bioanalyzer Assay. Samples were submitted to Genewiz for depletion of rRNA using the TruSeq RiboZero Gold Kit, library preparation, and single-end sequencing on the Illumina HiSeq 2500 platform. RNA sequencing analysis was done using CLC Genomic Workbench v20. Reads were mapped to C. difficile R20291 genome using the software's default menalties for mismatch, deletion, and insertion differences from the reference genome. Transcript reads were normalized as RPKM. Results: 88 genes were differentially expressed between bacteria from rough versus smooth colonies, with equal to or greater than a 2-fold change and p < 0.05 with FDR correction.
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Project description:Salmonella enterica serovar Typhimurium (S. Typhimurium) is a major cause of salmonellosis, with various multidrug-resistant pathovariants emerging. Here, we investigated a distinct rough colony variant with potentially enhanced environmental adaptation in China. Whole-genome sequencing of 2,212 Chinese isolates and 1,739 publicly available isolates revealed the population structure and evolutionary history of the rough colony variants. These variants showed macro, red, dry, and rough (mrdar) colonies with better biofilm-forming abilities at 28 °C and 37 °C compared to typical rdar colonies. The mrdar variants displayed expansive multidrug resistance, mainly attributed to an IncHI2 plasmid carrying 19 antimicrobial resistance genes. Phylogenomic analysis divided global collections into six lineages, with the majority of mrdar variants in sublineage L6.5, which originated from Chinese smooth colony strains and possibly emerged circa 1977. Upregulation of the csgDEFG operons was observed among mrdar variants, probably due to a distinct point mutation (-44G>T) in the csgD gene promoter. Pangenome and genome-wide association analyses identified 87 specific accessory genes and 72 distinct single nucleotide polymorphisms associated with the mrdar morphotype. This study presents the first large-scale, genome-based study of a distinct rdar colony morphology variant of S. Typhimurium in China, highlighting its global epidemic potential. Ongoing surveillance and genomic epidemiological studies are required to control epidemics, monitor bacterial population changes, and assess associated risks to global public health.
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Project description:The protein inventory of the anaerobic pathogen Clostridioides difficile grown in colony or aggregate biofilms was investigated in a comprehensive LC-MS/MS approach. C. difficile has a versatile metabolism and is perfectly equipped to survive and persist inside the mammalian intestine. However, knowledge on how C. difficile exactly resides inside the intestine is still scare. Aggregate or biofilms attached to the epithelium are discussed as possible growth forms and have become a focus in research. However, recent data suggest that the choice of the model strain, timepoint of analysis and the biofilm model significantly impact the outcome of biofilm experiments leading to contradictory results. To address some of these issues, a comprehensive proteomics approach was applied to investigate the metabolism of C. difficile strain 630erm grown in colony or aggregate biofilms. Besides verification of recent findings, a comprehensive overview on the differential expression profile of C. difficile’s cell surface proteins as well as its energy and stress metabolism during the two different forms of biofilm growth could be provided. Additionally, regulatory circuits that were activated in the two different biofilm models were analysed whereby RpoN was determined to be an important regulator of aggregate biofilm formation in C. difficile.
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