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

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.


E. faecalis RNase J1 and J2 are active exonucleases.(A) Diagram depicting the metallo-β-lactamase (red), β-CASP (orange) and dimerization domains. Residues that may be involved in zinc coordination were highlighted. Purified N-His6 recombinant E. faecalis RNase J1, J2 and three point mutations (H69A; H71A; D73A) of J2 were analyzed by SDS-PAGE. (B) and (C) Exonuclease activities of RNase J1, J2 and three J2 mutants were determined by RT-FeDEx analysis. Recombinant EbpC protein served as a negative control for the assay. (D) Binding of E. faecalis RNase J2 and two mutants (H69A and H71A) (all represented at 100 nM) to immobilized E. faecalis RNase J1 on a CM5 sensor surface is shown by the SPR sensorgrams.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5383250&req=5

pone.0175212.g002: E. faecalis RNase J1 and J2 are active exonucleases.(A) Diagram depicting the metallo-β-lactamase (red), β-CASP (orange) and dimerization domains. Residues that may be involved in zinc coordination were highlighted. Purified N-His6 recombinant E. faecalis RNase J1, J2 and three point mutations (H69A; H71A; D73A) of J2 were analyzed by SDS-PAGE. (B) and (C) Exonuclease activities of RNase J1, J2 and three J2 mutants were determined by RT-FeDEx analysis. Recombinant EbpC protein served as a negative control for the assay. (D) Binding of E. faecalis RNase J2 and two mutants (H69A and H71A) (all represented at 100 nM) to immobilized E. faecalis RNase J1 on a CM5 sensor surface is shown by the SPR sensorgrams.

Mentions: Sequence analysis revealed that the proteins coded by E. faecalis rnjA (ef2924) and rnjB (ef1185) are orthologs of the B. subtilis RNase J1 and J2, respectively. Both B. subtilis enzymes display endoribonuclease activity, while the exo-ribonuclease activity of J2 is much weaker than that of J1 [10]. In an effort to study E. faecalis RNase Js’ enzymatic activity, we cloned both E. faecalis rnjA and rnjB genes and expressed recombinant N-terminal His-tagged RNase J1 (M.W. 62.6 KDa) and RNase J2 (M.W. 64.2 KDa) in E. coli (Fig 2A). The exo-ribonuclease activities of E. faecalis RNase J1 and J2 were determined by RT-FeDex assay. The kinetic properties of E. faecalis RNase J1 are comparable to that of B. subtilis RNase J1 (KM of 0.48 μM and Kcat of 0.167 s-1, compared to KM of 0.47 μM and Kcat of 0.58 s-1 for the B. subtilis RNase J1). Due to the high concentration of enzyme required for this assay, we could not accurately measure Kcat and KM for RNase J2. However, RT-FeDex assay clearly demonstrated that RNase J2 is an active enzyme with much lower exonuclease activity as compared to J1(Fig 2B). For example, at a substrate DNA/RNAI concentration of 250 nM, the rate of the E. faecalis RNase J2 cleavage reaction is about half that of the E. faecalis J1 reaction, even though the J2 enzyme concentration is 40 times that of J1 (200 nM vs. 5 nM).


Functional studies of E . faecalis RNase J2 and its role in virulence and fitness
E. faecalis RNase J1 and J2 are active exonucleases.(A) Diagram depicting the metallo-β-lactamase (red), β-CASP (orange) and dimerization domains. Residues that may be involved in zinc coordination were highlighted. Purified N-His6 recombinant E. faecalis RNase J1, J2 and three point mutations (H69A; H71A; D73A) of J2 were analyzed by SDS-PAGE. (B) and (C) Exonuclease activities of RNase J1, J2 and three J2 mutants were determined by RT-FeDEx analysis. Recombinant EbpC protein served as a negative control for the assay. (D) Binding of E. faecalis RNase J2 and two mutants (H69A and H71A) (all represented at 100 nM) to immobilized E. faecalis RNase J1 on a CM5 sensor surface is shown by the SPR sensorgrams.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0175212.g002: E. faecalis RNase J1 and J2 are active exonucleases.(A) Diagram depicting the metallo-β-lactamase (red), β-CASP (orange) and dimerization domains. Residues that may be involved in zinc coordination were highlighted. Purified N-His6 recombinant E. faecalis RNase J1, J2 and three point mutations (H69A; H71A; D73A) of J2 were analyzed by SDS-PAGE. (B) and (C) Exonuclease activities of RNase J1, J2 and three J2 mutants were determined by RT-FeDEx analysis. Recombinant EbpC protein served as a negative control for the assay. (D) Binding of E. faecalis RNase J2 and two mutants (H69A and H71A) (all represented at 100 nM) to immobilized E. faecalis RNase J1 on a CM5 sensor surface is shown by the SPR sensorgrams.
Mentions: Sequence analysis revealed that the proteins coded by E. faecalis rnjA (ef2924) and rnjB (ef1185) are orthologs of the B. subtilis RNase J1 and J2, respectively. Both B. subtilis enzymes display endoribonuclease activity, while the exo-ribonuclease activity of J2 is much weaker than that of J1 [10]. In an effort to study E. faecalis RNase Js’ enzymatic activity, we cloned both E. faecalis rnjA and rnjB genes and expressed recombinant N-terminal His-tagged RNase J1 (M.W. 62.6 KDa) and RNase J2 (M.W. 64.2 KDa) in E. coli (Fig 2A). The exo-ribonuclease activities of E. faecalis RNase J1 and J2 were determined by RT-FeDex assay. The kinetic properties of E. faecalis RNase J1 are comparable to that of B. subtilis RNase J1 (KM of 0.48 μM and Kcat of 0.167 s-1, compared to KM of 0.47 μM and Kcat of 0.58 s-1 for the B. subtilis RNase J1). Due to the high concentration of enzyme required for this assay, we could not accurately measure Kcat and KM for RNase J2. However, RT-FeDex assay clearly demonstrated that RNase J2 is an active enzyme with much lower exonuclease activity as compared to J1(Fig 2B). For example, at a substrate DNA/RNAI concentration of 250 nM, the rate of the E. faecalis RNase J2 cleavage reaction is about half that of the E. faecalis J1 reaction, even though the J2 enzyme concentration is 40 times that of J1 (200 nM vs. 5 nM).

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.