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The production of extracellular proteins is regulated by ribonuclease III via two different pathways in Staphylococcus aureus.

Liu Y, Dong J, Wu N, Gao Y, Zhang X, Mu C, Shao N, Fan M, Yang G - PLoS ONE (2011)

Bottom Line: It was found that the extracellular proteins of Δrnc were decreased.We found during the lag phase of the bacterial growth cycle RNase III could influence the extracellular protein secretion via regulating the expression of secY2, one component of accessory secretory (sec) pathway.Our results suggest that RNase III could regulate the pathogenicity of S. aureus by influencing the level of extracellular proteins via two different ways respectively at different growth phases.

View Article: PubMed Central - PubMed

Affiliation: Beijing Institute of Basic Medical Sciences, Beijing, People's Republic of China.

ABSTRACT
Staphylococcus aureus ribonuclease III belongs to the enzyme family known to degrade double-stranded RNAs. It has previously been reported that RNase III cannot influence cell growth but regulates virulence gene expression in S. aureus. Here we constructed an RNase III inactivation mutant (Δrnc) from S. aureus 8325-4. It was found that the extracellular proteins of Δrnc were decreased. Furthermore, we explored how RNase III regulated the production of the extracellular proteins in S. aureus. We found during the lag phase of the bacterial growth cycle RNase III could influence the extracellular protein secretion via regulating the expression of secY2, one component of accessory secretory (sec) pathway. After S. aureus cells grew to exponential phase, RNase III can regulate the expression of extracellular proteins by affecting the level of RNAIII. Further investigation showed that the mRNA stability of secY2 and RNAIII was affected by RNase III. Our results suggest that RNase III could regulate the pathogenicity of S. aureus by influencing the level of extracellular proteins via two different ways respectively at different growth phases.

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RNAIII regulates the levels of extracellular proteins at 6 h and 12 h.A: The expression level of RNAIII was analyzed by Northern blot. The level of RNAIII in different strains at 6 h and 12 h was detected by Northern blot. 16s rRNA was used as the internal control. WT: wild type, S. aureus 8325-4; Δrnc: an RNase III inactivation mutant from 8325-4; rncR: the restoration of RNase III activity in Δrnc. B: Detection of the extracellular proteins of WT and ΔRNAIII at the different time points. The extracellular proteins from the equal number of cells were extracted at the indicated time points. The results of SDS-PAGE showed that the extracellular proteins of ΔRNAIII were decreased in comparing with WT at 6 h and 12 h. 1,4,7: wild type; 2,5,8: ΔRNAIII (RNAIII deletion mutant); 3,6,9: ΔRNAIIIR (the restoration of RNAIII in ΔRNAIII). C: Detection of the extracellular proteins from different strains. The pOS1-RNAIII plasmid was constructed to recover the level of RNAIII in Δrnc. At the same time, the double mutant Δrnc/RNAIII was constructed. Then the extracellular proteins were extracted. The results showed that the extracellular proteins were increased at 6 h and 12 h after the level of RNAIII was recovered in Δrnc. The level of RNAIII was measured by RT-PCR. 16s rRNA was used as the internal control. 1,5: WT, wild type; 2,6: Δrnc; 3,7: RNAIIIr(the Δrnc strain transferred with the plasmid pOS1-RNAIII); 4,8, Δrnc/RNAIII. The experiment has been repeated for three times.
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pone-0020554-g003: RNAIII regulates the levels of extracellular proteins at 6 h and 12 h.A: The expression level of RNAIII was analyzed by Northern blot. The level of RNAIII in different strains at 6 h and 12 h was detected by Northern blot. 16s rRNA was used as the internal control. WT: wild type, S. aureus 8325-4; Δrnc: an RNase III inactivation mutant from 8325-4; rncR: the restoration of RNase III activity in Δrnc. B: Detection of the extracellular proteins of WT and ΔRNAIII at the different time points. The extracellular proteins from the equal number of cells were extracted at the indicated time points. The results of SDS-PAGE showed that the extracellular proteins of ΔRNAIII were decreased in comparing with WT at 6 h and 12 h. 1,4,7: wild type; 2,5,8: ΔRNAIII (RNAIII deletion mutant); 3,6,9: ΔRNAIIIR (the restoration of RNAIII in ΔRNAIII). C: Detection of the extracellular proteins from different strains. The pOS1-RNAIII plasmid was constructed to recover the level of RNAIII in Δrnc. At the same time, the double mutant Δrnc/RNAIII was constructed. Then the extracellular proteins were extracted. The results showed that the extracellular proteins were increased at 6 h and 12 h after the level of RNAIII was recovered in Δrnc. The level of RNAIII was measured by RT-PCR. 16s rRNA was used as the internal control. 1,5: WT, wild type; 2,6: Δrnc; 3,7: RNAIIIr(the Δrnc strain transferred with the plasmid pOS1-RNAIII); 4,8, Δrnc/RNAIII. The experiment has been repeated for three times.

Mentions: As RNAIII is a positive regulator of extracellular virulence [18] and RNase III can mediate the interaction between RNAIII and its target mRNAs [7], [8], we checked the level of RNAIII in Δrnc by Northern blot. Compared with its parent strain, the expression of RNAIII in Δrnc decreased at 6 h and 12 h (figure 3A). In order to avoid the unintended mutation in agr system during we constructed the Δrnc, we analyzed the sequence of agrA and agrC of Δrnc. No mutated nucleotide was observed in the genome of Δrnc strain (data not shown).


The production of extracellular proteins is regulated by ribonuclease III via two different pathways in Staphylococcus aureus.

Liu Y, Dong J, Wu N, Gao Y, Zhang X, Mu C, Shao N, Fan M, Yang G - PLoS ONE (2011)

RNAIII regulates the levels of extracellular proteins at 6 h and 12 h.A: The expression level of RNAIII was analyzed by Northern blot. The level of RNAIII in different strains at 6 h and 12 h was detected by Northern blot. 16s rRNA was used as the internal control. WT: wild type, S. aureus 8325-4; Δrnc: an RNase III inactivation mutant from 8325-4; rncR: the restoration of RNase III activity in Δrnc. B: Detection of the extracellular proteins of WT and ΔRNAIII at the different time points. The extracellular proteins from the equal number of cells were extracted at the indicated time points. The results of SDS-PAGE showed that the extracellular proteins of ΔRNAIII were decreased in comparing with WT at 6 h and 12 h. 1,4,7: wild type; 2,5,8: ΔRNAIII (RNAIII deletion mutant); 3,6,9: ΔRNAIIIR (the restoration of RNAIII in ΔRNAIII). C: Detection of the extracellular proteins from different strains. The pOS1-RNAIII plasmid was constructed to recover the level of RNAIII in Δrnc. At the same time, the double mutant Δrnc/RNAIII was constructed. Then the extracellular proteins were extracted. The results showed that the extracellular proteins were increased at 6 h and 12 h after the level of RNAIII was recovered in Δrnc. The level of RNAIII was measured by RT-PCR. 16s rRNA was used as the internal control. 1,5: WT, wild type; 2,6: Δrnc; 3,7: RNAIIIr(the Δrnc strain transferred with the plasmid pOS1-RNAIII); 4,8, Δrnc/RNAIII. The experiment has been repeated for three times.
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Related In: Results  -  Collection

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pone-0020554-g003: RNAIII regulates the levels of extracellular proteins at 6 h and 12 h.A: The expression level of RNAIII was analyzed by Northern blot. The level of RNAIII in different strains at 6 h and 12 h was detected by Northern blot. 16s rRNA was used as the internal control. WT: wild type, S. aureus 8325-4; Δrnc: an RNase III inactivation mutant from 8325-4; rncR: the restoration of RNase III activity in Δrnc. B: Detection of the extracellular proteins of WT and ΔRNAIII at the different time points. The extracellular proteins from the equal number of cells were extracted at the indicated time points. The results of SDS-PAGE showed that the extracellular proteins of ΔRNAIII were decreased in comparing with WT at 6 h and 12 h. 1,4,7: wild type; 2,5,8: ΔRNAIII (RNAIII deletion mutant); 3,6,9: ΔRNAIIIR (the restoration of RNAIII in ΔRNAIII). C: Detection of the extracellular proteins from different strains. The pOS1-RNAIII plasmid was constructed to recover the level of RNAIII in Δrnc. At the same time, the double mutant Δrnc/RNAIII was constructed. Then the extracellular proteins were extracted. The results showed that the extracellular proteins were increased at 6 h and 12 h after the level of RNAIII was recovered in Δrnc. The level of RNAIII was measured by RT-PCR. 16s rRNA was used as the internal control. 1,5: WT, wild type; 2,6: Δrnc; 3,7: RNAIIIr(the Δrnc strain transferred with the plasmid pOS1-RNAIII); 4,8, Δrnc/RNAIII. The experiment has been repeated for three times.
Mentions: As RNAIII is a positive regulator of extracellular virulence [18] and RNase III can mediate the interaction between RNAIII and its target mRNAs [7], [8], we checked the level of RNAIII in Δrnc by Northern blot. Compared with its parent strain, the expression of RNAIII in Δrnc decreased at 6 h and 12 h (figure 3A). In order to avoid the unintended mutation in agr system during we constructed the Δrnc, we analyzed the sequence of agrA and agrC of Δrnc. No mutated nucleotide was observed in the genome of Δrnc strain (data not shown).

Bottom Line: It was found that the extracellular proteins of Δrnc were decreased.We found during the lag phase of the bacterial growth cycle RNase III could influence the extracellular protein secretion via regulating the expression of secY2, one component of accessory secretory (sec) pathway.Our results suggest that RNase III could regulate the pathogenicity of S. aureus by influencing the level of extracellular proteins via two different ways respectively at different growth phases.

View Article: PubMed Central - PubMed

Affiliation: Beijing Institute of Basic Medical Sciences, Beijing, People's Republic of China.

ABSTRACT
Staphylococcus aureus ribonuclease III belongs to the enzyme family known to degrade double-stranded RNAs. It has previously been reported that RNase III cannot influence cell growth but regulates virulence gene expression in S. aureus. Here we constructed an RNase III inactivation mutant (Δrnc) from S. aureus 8325-4. It was found that the extracellular proteins of Δrnc were decreased. Furthermore, we explored how RNase III regulated the production of the extracellular proteins in S. aureus. We found during the lag phase of the bacterial growth cycle RNase III could influence the extracellular protein secretion via regulating the expression of secY2, one component of accessory secretory (sec) pathway. After S. aureus cells grew to exponential phase, RNase III can regulate the expression of extracellular proteins by affecting the level of RNAIII. Further investigation showed that the mRNA stability of secY2 and RNAIII was affected by RNase III. Our results suggest that RNase III could regulate the pathogenicity of S. aureus by influencing the level of extracellular proteins via two different ways respectively at different growth phases.

Show MeSH
Related in: MedlinePlus