Dataset Information

Endogenous antisense walR RNA modulates biofilm organization and pathogenicity of Enterococcus faecalis.

ABSTRACT: Enterococcus faecalis (E. faecalis) is regarded as the major pathogen for persistent periapical periodontitis. The aim of the present study was to investigate the role of antisense walR RNA in the regulation of adjacent downstream genes. Reverse transcription-PCR assays were performed to validate walR. Adjacent downstream genes walK, EF1195, EF1196, and EF1197 were co-transcribed and detect antisense walR RNA. Northern blotting and 5'-rapid amplification of cDNA ends (5'-RACE) assays were conducted to detect and confirm a novel walR antisense (ASwalR) RNA. ASwalR overexpression mutants were constructed, and the biofilm biomass was determined using a crystal violet microtiter assay. The present study detected and confirmed a 550-bp noncoding antisense RNA with the potential to attenuate the activities of the essential response regulator WalR. The levels of antisense walR RNA transcripts were inversely associated with the production of WalR protein. It was showed that overexpression of ASwalR leads to reduced biofilm formation and exopolysaccharide synthesis. Furthermore, the pathogenicity of E. faecalis was markedly decreased by ASwalR overexpression in an in vivo periapical periodontitis model. In summary, the present study detected a novel antisense walR RNA that leads to a reduction in biofilm formation and the pathogenicity of E. faecalis. Collectively, the data suggest a role for ASwalR as a post-transcriptional modulator of the WalR regulator in E. faecalis.

SUBMITTER: Wu S

PROVIDER: S-EPMC7716642 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Endogenous antisense walR RNA modulates biofilm organization and pathogenicity of Enterococcus faecalis.

Wu Shizhou S Liu Yunjie Y Lei Lei L Zhang Hui H

Experimental and therapeutic medicine 20201123 1

Enterococcus faecalis (E. faecalis) is regarded as the major pathogen for persistent periapical periodontitis. The aim of the present study was to investigate the role of antisense walR RNA in the regulation of adjacent downstream genes. Reverse transcription-PCR assays were performed to validate walR. Adjacent downstream genes walK, EF1195, EF1196, and EF1197 were co-transcribed and detect antisense walR RNA. Northern blotting and 5'-rapid amplification ...[more]

PMID: 33365069

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Project description:Enterococcus faecalis is a common commensal organism and a prolific nosocomial pathogen that causes biofilm-associated infections. Numerous E. faecalis OG1RF genes required for biofilm formation have been identified, but few studies have compared genetic determinants of biofilm formation and biofilm morphology across multiple conditions. Here, we cultured transposon (Tn) libraries in CDC biofilm reactors in two different media and used Tn sequencing (TnSeq) to identify core and accessory biofilm determinants, including many genes that are poorly characterized or annotated as hypothetical. Multiple secondary assays (96-well plates, submerged Aclar discs, and MultiRep biofilm reactors) were used to validate phenotypes of new biofilm determinants. We quantified biofilm cells and used fluorescence microscopy to visualize biofilms formed by six Tn mutants identified using TnSeq and found that disrupting these genes (OG1RF_10350, prsA, tig, OG1RF_10576, OG1RF_11288, and OG1RF_11456) leads to significant time- and medium-dependent changes in biofilm architecture. Structural predictions revealed potential roles in cell wall homeostasis for OG1RF_10350 and OG1RF_11288 and signaling for OG1RF_11456. Additionally, we identified growth medium-specific hallmarks of OG1RF biofilm morphology. This study demonstrates how E. faecalis biofilm architecture is modulated by growth medium and experimental conditions and identifies multiple new genetic determinants of biofilm formation. IMPORTANCE E. faecalis is an opportunistic pathogen and a leading cause of hospital-acquired infections, in part due to its ability to form biofilms. A complete understanding of the genes required for E. faecalis biofilm formation as well as specific features of biofilm morphology related to nutrient availability and growth conditions is crucial for understanding how E. faecalis biofilm-associated infections develop and resist treatment in patients. We employed a comprehensive approach to analysis of biofilm determinants by combining TnSeq primary screens with secondary phenotypic validation using diverse biofilm assays. This enabled identification of numerous core (important under many conditions) and accessory (important under specific conditions) biofilm determinants in E. faecalis OG1RF. We found multiple genes whose disruption results in drastic changes to OG1RF biofilm morphology. These results expand our understanding of the genetic requirements for biofilm formation in E. faecalis that affect the time course of biofilm development as well as the response to specific nutritional conditions.

Project description:Enterococci are the third leading cause of hospital associated infections and have gained increased importance due to their fast adaptation to the clinical environment by acquisition of antibiotic resistance and pathogenicity traits. Enterococcus faecalis harbours a pathogenicity island (PAI) of 153 kb containing several virulence factors including the enterococcal surface protein (esp). Until now only internal fragments of the PAI or larger chromosomal regions containing it have been transferred. Here we demonstrate precise excision, circularization and horizontal transfer of the entire PAI element from the chromosome of E. faecalis strain UW3114. This PAI (ca. 200 kb) contained some deletions and insertions as compared to the PAI of the reference strain MMH594, transferred precisely and integrated site-specifically into the chromosome of E. faecalis (intergenic region) and Enterococcus faecium (tRNAlys). The internal PAI structure was maintained after transfer. We assessed phenotypic changes accompanying acquisition of the PAI and expression of some of its determinants. The esp gene is expressed on the surface of donor and both transconjugants. Biofilm formation and cytolytic activity were enhanced in E. faecalis transconjugants after acquisition of the PAI. No differences in pathogenicity of E. faecalis were detected using a mouse bacteraemia and a mouse peritonitis models (tail vein and intraperitoneal injection). A 66 kb conjugative pheromone-responsive plasmid encoding erm(B) (pLG2) that was transferred in parallel with the PAI was sequenced. pLG2 is a pheromone responsive plasmid that probably promotes the PAI horizontal transfer, encodes antibiotic resistance features and contains complete replication and conjugation modules of enterococcal origin in a mosaic-like composition. The E. faecalis PAI can undergo precise intra- and interspecies transfer probably with the help of conjugative elements like conjugative resistance plasmids, supporting the role of horizontal gene transfer and antibiotic selective pressure in the successful establishment of certain enterococci as nosocomial pathogens.

Dataset Information

Endogenous antisense walR RNA modulates biofilm organization and pathogenicity of Enterococcus faecalis.

Publications

Endogenous antisense <i>walR</i> RNA modulates biofilm organization and pathogenicity of <i>Enterococcus faecalis</i>.

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