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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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The decrease of secY2 resulted in the inhibition of extracellular protein secretion in Δrnc at 1.5 h.A: Detection of the mRNA level of secA1, secY1,secA2 and secY2. The mRNA levels of secA1, secY1,secA2 and secY2 were detected at 1.5 h by qRT-PCR. The results showed that the level of secY2 was decreased in Δrnc. (**: P<0.01). Then the decrease of secY2 mRNA was confirmed 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 profile of extracellular proteins and the expression of Efb in the different strains. The pOS1-secY2 plasmid was constructed and transferred to Δrnc to recover the level of SecY2. At the same time, the double mutant Δrnc/secY2 was constructed. And then the extracellular proteins were extracted. The results showed that the production of the extracellular proteins was significantly increased at 1.5 h after the recovery of the level of secY2. The mRNA level of secY2 was measured by RT-PCR. 16s rRNA was used as the internal control. At the same time, the expression of Efb was determined by Western blot. The result showed that Efb was restored after the level of secY2 was recovered in Δrnc. 1: wild type; 2: Δrnc; 3: secY2r(the Δrnc strain transferred with the plasmid pOS1-secY2); 4, Δrnc/secY2; 5. ΔsecY2.
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pone-0020554-g005: The decrease of secY2 resulted in the inhibition of extracellular protein secretion in Δrnc at 1.5 h.A: Detection of the mRNA level of secA1, secY1,secA2 and secY2. The mRNA levels of secA1, secY1,secA2 and secY2 were detected at 1.5 h by qRT-PCR. The results showed that the level of secY2 was decreased in Δrnc. (**: P<0.01). Then the decrease of secY2 mRNA was confirmed 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 profile of extracellular proteins and the expression of Efb in the different strains. The pOS1-secY2 plasmid was constructed and transferred to Δrnc to recover the level of SecY2. At the same time, the double mutant Δrnc/secY2 was constructed. And then the extracellular proteins were extracted. The results showed that the production of the extracellular proteins was significantly increased at 1.5 h after the recovery of the level of secY2. The mRNA level of secY2 was measured by RT-PCR. 16s rRNA was used as the internal control. At the same time, the expression of Efb was determined by Western blot. The result showed that Efb was restored after the level of secY2 was recovered in Δrnc. 1: wild type; 2: Δrnc; 3: secY2r(the Δrnc strain transferred with the plasmid pOS1-secY2); 4, Δrnc/secY2; 5. ΔsecY2.

Mentions: The general secretory (sec) pathway is the most commonly used one for bacterial protein transport [12]. In addition, S. aureus contains an accessory Sec2 pathway involving the SecA2 and SecY2 proteins [13], [14]. However, there were few reports on the sec pathway of S. aureus [12]. We analyzed the genome of S. aureus and detected the mRNA level of the genes which were involved in the general and accessory sec pathway (secA1, secY1, secA2 and secY2) by qRT-PCR. It was found that only the expression of secY2 decreased significantly in Δrnc at 1.5 h (figure 5A). Then the decline of secY2 mRNA level was confirmed by Northern blot (figure 5A). In additon, the production of extracellular proteins of ΔsecY2 (SecY2 inactivation mutant) was decreased at 1.5 h compared with its parent strain (figure 5B). In the further study, we constructed the plasmid of pOS1-secY2 using the promoter of ssrA, which is a tmRNA in S. aureus. It was found that the level of ssrA in Δrnc was not altered (data not shown). And then the plasmid was transferred to Δrnc to recover the expression level of secY2. The result showed the extracellular proteins increased after the expression level of secY2 recovered in Δrnc (figure 5B). At the same time, the level of Efb in the supernatant was correspondingly restored (figure 5B). In the further investigation, the RNase III inactivated strain was constructed from the ΔsecY2 strain, named as Δrnc/secY2, it was found that the profile of extracellular proteins of the Δrnc/secY2 was the same as that of ΔsecY2 (figure 5B).


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)

The decrease of secY2 resulted in the inhibition of extracellular protein secretion in Δrnc at 1.5 h.A: Detection of the mRNA level of secA1, secY1,secA2 and secY2. The mRNA levels of secA1, secY1,secA2 and secY2 were detected at 1.5 h by qRT-PCR. The results showed that the level of secY2 was decreased in Δrnc. (**: P<0.01). Then the decrease of secY2 mRNA was confirmed 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 profile of extracellular proteins and the expression of Efb in the different strains. The pOS1-secY2 plasmid was constructed and transferred to Δrnc to recover the level of SecY2. At the same time, the double mutant Δrnc/secY2 was constructed. And then the extracellular proteins were extracted. The results showed that the production of the extracellular proteins was significantly increased at 1.5 h after the recovery of the level of secY2. The mRNA level of secY2 was measured by RT-PCR. 16s rRNA was used as the internal control. At the same time, the expression of Efb was determined by Western blot. The result showed that Efb was restored after the level of secY2 was recovered in Δrnc. 1: wild type; 2: Δrnc; 3: secY2r(the Δrnc strain transferred with the plasmid pOS1-secY2); 4, Δrnc/secY2; 5. ΔsecY2.
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getmorefigures.php?uid=PMC3105085&req=5

pone-0020554-g005: The decrease of secY2 resulted in the inhibition of extracellular protein secretion in Δrnc at 1.5 h.A: Detection of the mRNA level of secA1, secY1,secA2 and secY2. The mRNA levels of secA1, secY1,secA2 and secY2 were detected at 1.5 h by qRT-PCR. The results showed that the level of secY2 was decreased in Δrnc. (**: P<0.01). Then the decrease of secY2 mRNA was confirmed 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 profile of extracellular proteins and the expression of Efb in the different strains. The pOS1-secY2 plasmid was constructed and transferred to Δrnc to recover the level of SecY2. At the same time, the double mutant Δrnc/secY2 was constructed. And then the extracellular proteins were extracted. The results showed that the production of the extracellular proteins was significantly increased at 1.5 h after the recovery of the level of secY2. The mRNA level of secY2 was measured by RT-PCR. 16s rRNA was used as the internal control. At the same time, the expression of Efb was determined by Western blot. The result showed that Efb was restored after the level of secY2 was recovered in Δrnc. 1: wild type; 2: Δrnc; 3: secY2r(the Δrnc strain transferred with the plasmid pOS1-secY2); 4, Δrnc/secY2; 5. ΔsecY2.
Mentions: The general secretory (sec) pathway is the most commonly used one for bacterial protein transport [12]. In addition, S. aureus contains an accessory Sec2 pathway involving the SecA2 and SecY2 proteins [13], [14]. However, there were few reports on the sec pathway of S. aureus [12]. We analyzed the genome of S. aureus and detected the mRNA level of the genes which were involved in the general and accessory sec pathway (secA1, secY1, secA2 and secY2) by qRT-PCR. It was found that only the expression of secY2 decreased significantly in Δrnc at 1.5 h (figure 5A). Then the decline of secY2 mRNA level was confirmed by Northern blot (figure 5A). In additon, the production of extracellular proteins of ΔsecY2 (SecY2 inactivation mutant) was decreased at 1.5 h compared with its parent strain (figure 5B). In the further study, we constructed the plasmid of pOS1-secY2 using the promoter of ssrA, which is a tmRNA in S. aureus. It was found that the level of ssrA in Δrnc was not altered (data not shown). And then the plasmid was transferred to Δrnc to recover the expression level of secY2. The result showed the extracellular proteins increased after the expression level of secY2 recovered in Δrnc (figure 5B). At the same time, the level of Efb in the supernatant was correspondingly restored (figure 5B). In the further investigation, the RNase III inactivated strain was constructed from the ΔsecY2 strain, named as Δrnc/secY2, it was found that the profile of extracellular proteins of the Δrnc/secY2 was the same as that of ΔsecY2 (figure 5B).

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