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Functional studies of E . faecalis RNase J2 and its role in virulence and fitness

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ABSTRACT

Post-transcriptional control provides bacterial pathogens a method by which they can rapidly adapt to environmental change. Dual exo- and endonucleolytic activities of RNase J enzymes contribute to Gram-positive RNA processing and decay. First discovered in Bacillus subtilis, RNase J1 plays a key role in mRNA maturation and degradation, while the function of the paralogue RNase J2 is largely unknown. Previously, we discovered that deletion of the Enterococcus faecalis rnjB gene significantly attenuates expression of a major virulence factor involved in enterococcal pathogenesis, the Ebp pili. In this work, we demonstrate that E. faecalis rnjB encodes an active RNase J2, and that the ribonuclease activity of RNase J2 is required for regulation of Ebp pili. To further investigate how rnjB affects E. faecalis gene expression on a global scale, we compared transcriptomes of the E. faecalis strain OG1RF with its isogenic rnjB deletion mutant (ΔrnjB). In addition to Ebp pili regulation, previously demonstrated to have a profound effect on the ability of E. faecalis to form biofilm or establish infection, we identified that rnjB regulates the expression of several other genes involved in bacterial virulence and fitness, including gls24 (a virulence factor important in stress response). We further demonstrated that the E. faecalis RNase J2 deletion mutant is more sensitive to bile salt and greatly attenuated in in vivo organ infection as determined by an IV-sublethal challenge infection mouse model, indicating that E. faecalis RNase J2 plays an important role in E. faecalis virulence.

No MeSH data available.


Bile-salt resistance assay.The percent survival of three mutant strains (ΔebpABC, Δgls24 and ΔrnjB) in BHI medium containing 0.3% bile salt relative to that of wild-type OG1RF was calculated as described in the Materials and Methods section. Each experiment was performed in triplicate and the experiments were repeated 3 times. Error bar represent the standard error of these repeats.
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pone.0175212.g006: Bile-salt resistance assay.The percent survival of three mutant strains (ΔebpABC, Δgls24 and ΔrnjB) in BHI medium containing 0.3% bile salt relative to that of wild-type OG1RF was calculated as described in the Materials and Methods section. Each experiment was performed in triplicate and the experiments were repeated 3 times. Error bar represent the standard error of these repeats.

Mentions: The transcriptome analysis revealed that the rnjB gene is required for the expression of multiple virulence-related gene loci, including ebpABC, gls24, and ef1097, a putative antimicrobial peptide [33]. We have previously shown that ΔrnjB is attenuated in biofilm formation analyzed by a microplate-binding adherence assay, which correlates with decreased Ebp pili surface display. Gls24, on the other hand, has been reported to be important for E. faecalis bile salts resistance and virulence [21]. To test whether deletion of rnjB has an effect on E. faecalis stress resistance, a bile-salt resistance assay was performed on ΔrnjB, Δgls24, and wild-type cells. As demonstrated in Fig 6, the gls24 deletion strain showed significant reduction in bile-salt resistance. In contrast, the rnjB deletion showed modestly reduced resistance to the bile salts compared to wild type (45%) with a P score of 0.03. This result is consistent with the results of transcriptome analysis and Western blot demonstrating that Gls24 expression is decreased but not eliminated in ΔrnjB.


Functional studies of E . faecalis RNase J2 and its role in virulence and fitness
Bile-salt resistance assay.The percent survival of three mutant strains (ΔebpABC, Δgls24 and ΔrnjB) in BHI medium containing 0.3% bile salt relative to that of wild-type OG1RF was calculated as described in the Materials and Methods section. Each experiment was performed in triplicate and the experiments were repeated 3 times. Error bar represent the standard error of these repeats.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5383250&req=5

pone.0175212.g006: Bile-salt resistance assay.The percent survival of three mutant strains (ΔebpABC, Δgls24 and ΔrnjB) in BHI medium containing 0.3% bile salt relative to that of wild-type OG1RF was calculated as described in the Materials and Methods section. Each experiment was performed in triplicate and the experiments were repeated 3 times. Error bar represent the standard error of these repeats.
Mentions: The transcriptome analysis revealed that the rnjB gene is required for the expression of multiple virulence-related gene loci, including ebpABC, gls24, and ef1097, a putative antimicrobial peptide [33]. We have previously shown that ΔrnjB is attenuated in biofilm formation analyzed by a microplate-binding adherence assay, which correlates with decreased Ebp pili surface display. Gls24, on the other hand, has been reported to be important for E. faecalis bile salts resistance and virulence [21]. To test whether deletion of rnjB has an effect on E. faecalis stress resistance, a bile-salt resistance assay was performed on ΔrnjB, Δgls24, and wild-type cells. As demonstrated in Fig 6, the gls24 deletion strain showed significant reduction in bile-salt resistance. In contrast, the rnjB deletion showed modestly reduced resistance to the bile salts compared to wild type (45%) with a P score of 0.03. This result is consistent with the results of transcriptome analysis and Western blot demonstrating that Gls24 expression is decreased but not eliminated in ΔrnjB.

View Article: PubMed Central - PubMed

ABSTRACT

Post-transcriptional control provides bacterial pathogens a method by which they can rapidly adapt to environmental change. Dual exo- and endonucleolytic activities of RNase J enzymes contribute to Gram-positive RNA processing and decay. First discovered in Bacillus subtilis, RNase J1 plays a key role in mRNA maturation and degradation, while the function of the paralogue RNase J2 is largely unknown. Previously, we discovered that deletion of the Enterococcus faecalis rnjB gene significantly attenuates expression of a major virulence factor involved in enterococcal pathogenesis, the Ebp pili. In this work, we demonstrate that E. faecalis rnjB encodes an active RNase J2, and that the ribonuclease activity of RNase J2 is required for regulation of Ebp pili. To further investigate how rnjB affects E. faecalis gene expression on a global scale, we compared transcriptomes of the E. faecalis strain OG1RF with its isogenic rnjB deletion mutant (ΔrnjB). In addition to Ebp pili regulation, previously demonstrated to have a profound effect on the ability of E. faecalis to form biofilm or establish infection, we identified that rnjB regulates the expression of several other genes involved in bacterial virulence and fitness, including gls24 (a virulence factor important in stress response). We further demonstrated that the E. faecalis RNase J2 deletion mutant is more sensitive to bile salt and greatly attenuated in in vivo organ infection as determined by an IV-sublethal challenge infection mouse model, indicating that E. faecalis RNase J2 plays an important role in E. faecalis virulence.

No MeSH data available.