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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 secretion of the proteins in Δrnc was inhibited at 1.5 h.A: qRT-PCR quantification of the level of efb mRNA. The level of efb mRNA in the different strains was detected at 6 h. The results showed that the expression of efb was not regulated by RNAIII. WT: wild type; ΔRNAIII: RNAIII deletion mutant; ΔRNAIIIR: the restoration of RNAIII in ΔRNAIII. B: Detection of the expression of Efb in the extracellular proteins from the different S. aureus strains by Western blot at 1.5 h. The extracellular proteins from same number of cells were extracted from different S. aureus strains. The expression of Efb was tested with the specific antibodies of Efb (prepared by ourselves) by Western blot. The result showed that Efb couldn't be detected in the supernatant of Δrnc. 1: WT, wild type, S. aureus 8325-4; 2: Δrnc, an RNase III inactivation mutant from 8325-4; 3: rncR, the restoration of RNase III activity in Δrnc. C: qRT-PCR quantification of the mRNA level of efb at 1.5 h. The quantity of efb mRNA from different strains was measured by qRT-PCR at 1.5 h. The result showed that the level of efb mRNA wasn't changed in Δrnc. 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. D: The schematic diagram of construction of the reporter vectors. Uefb::lacZ: the promoter and 5′UTR of efb were fused with Lacz; UefbSP: the promoter, 5′UTR and the signal peptides of efb were fused with LacZ. E: Detection of the β-galactosidase activity of different strains. The Uefb::lacZ reporter vector was separately transferred into Δrnc and its parent strains. Then the β-galactosidase activity of different strains was measured at 1.5 h and expressed by miller units. There was no significant difference observed. The results represented a mean of three independent experiments. WT: wild type, S. aureus 8325-4; Δrnc: an RNase III inactivation mutant from 8325-4. F: Detection of the β-galactosidase activity of the cultured medium from different strains.The UefbSP::lacZ reporter vector was separately transferred into Δrnc and its parent strains. Then the β-galactosidase activities of the cultured medium were measured and expressed by miller units. Comparing with it parent stain, the β-galactosidase activitiy of the cultured medium from the Δrnc was decreased at 1.5 h. The results represented a mean of three independent experiments. WT: wild type, S. aureus 8325-4; Δrnc: an RNase III inactivation mutant from 8325-4. (**: p<0.01).
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pone-0020554-g004: The secretion of the proteins in Δrnc was inhibited at 1.5 h.A: qRT-PCR quantification of the level of efb mRNA. The level of efb mRNA in the different strains was detected at 6 h. The results showed that the expression of efb was not regulated by RNAIII. WT: wild type; ΔRNAIII: RNAIII deletion mutant; ΔRNAIIIR: the restoration of RNAIII in ΔRNAIII. B: Detection of the expression of Efb in the extracellular proteins from the different S. aureus strains by Western blot at 1.5 h. The extracellular proteins from same number of cells were extracted from different S. aureus strains. The expression of Efb was tested with the specific antibodies of Efb (prepared by ourselves) by Western blot. The result showed that Efb couldn't be detected in the supernatant of Δrnc. 1: WT, wild type, S. aureus 8325-4; 2: Δrnc, an RNase III inactivation mutant from 8325-4; 3: rncR, the restoration of RNase III activity in Δrnc. C: qRT-PCR quantification of the mRNA level of efb at 1.5 h. The quantity of efb mRNA from different strains was measured by qRT-PCR at 1.5 h. The result showed that the level of efb mRNA wasn't changed in Δrnc. 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. D: The schematic diagram of construction of the reporter vectors. Uefb::lacZ: the promoter and 5′UTR of efb were fused with Lacz; UefbSP: the promoter, 5′UTR and the signal peptides of efb were fused with LacZ. E: Detection of the β-galactosidase activity of different strains. The Uefb::lacZ reporter vector was separately transferred into Δrnc and its parent strains. Then the β-galactosidase activity of different strains was measured at 1.5 h and expressed by miller units. There was no significant difference observed. The results represented a mean of three independent experiments. WT: wild type, S. aureus 8325-4; Δrnc: an RNase III inactivation mutant from 8325-4. F: Detection of the β-galactosidase activity of the cultured medium from different strains.The UefbSP::lacZ reporter vector was separately transferred into Δrnc and its parent strains. Then the β-galactosidase activities of the cultured medium were measured and expressed by miller units. Comparing with it parent stain, the β-galactosidase activitiy of the cultured medium from the Δrnc was decreased at 1.5 h. The results represented a mean of three independent experiments. WT: wild type, S. aureus 8325-4; Δrnc: an RNase III inactivation mutant from 8325-4. (**: p<0.01).

Mentions: In above result there was no significant difference observed in the extracellular proteins production between ΔRNAIII and its parent strain at 1.5 h (figure 3B). The reason should be that RNAIII was a cell density-dependent regulator [19] and the level of RNAIII was too low to regulate its targets at the lag phase. However, the extracellular proteins in Δrnc decreased at 1.5 h (figure 2). It suggested that the reduction of extracellular proteins in Δrnc was not completely due to the RNAIII level decreasing. And the reduction of extracellular proteins in Δrnc at 1.5 h should have no relation with RNAIII. To discover other factors involved in this process, we chose Efb (extracellular fibrinogen binding protein) as the indicator of extracellular proteins [20], [21], [22] because the expression of Efb was not influenced by RNAIII (figure 4A) and the level of Efb in Δrnc supernatant decreased at 1.5 h (figure 4B).Then we analyzed the mRNA level, translation and secretion of Efb to discover the mechanism of reducing the extracellular proteins in Δrnc at 1.5 h. Firstly, the mRNA level of the efb gene was determined by qRT-PCR. It showed that the mRNA level of efb in Δrnc did not alter at 1.5 h (figure 4C). Secondly, we constructed the lacZ fusion vectors to analyze the translation of Efb in Δrnc and its parent strains (figure 4D). The upstream region of efb containing its promoter and 5′UTR was fused with lacZ (named as Uefb::lacZ). The constructed vector was transformed to Δrnc and its parent strain respectively. The results of β-galactosidase activity detection showed that Δrnc did not exhibit significant difference comparing with its parent strain (figure 4E). This suggested that the inactivation of RNase III did not influence the transcription and translation of Efb. And thirdly, we checked if the secretion of Efb was affected in Δrnc. The upstream of efb containing its promoter, 5′UTR and the signal peptide fused with lacZ was termed as UefbSP::lacZ (figure 4D). The β-galactosidase activity of cultured medium in the two different strains was detected. Our results showed that the β-galactosidase activity of the cultured medium from Δrnc was significantly lower than that from its parent strain (figure 4F). It suggested that the reduction of extracellular proteins of Δrnc at 1.5 h was because the secretion of extracellular proteins was suppressed.


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 secretion of the proteins in Δrnc was inhibited at 1.5 h.A: qRT-PCR quantification of the level of efb mRNA. The level of efb mRNA in the different strains was detected at 6 h. The results showed that the expression of efb was not regulated by RNAIII. WT: wild type; ΔRNAIII: RNAIII deletion mutant; ΔRNAIIIR: the restoration of RNAIII in ΔRNAIII. B: Detection of the expression of Efb in the extracellular proteins from the different S. aureus strains by Western blot at 1.5 h. The extracellular proteins from same number of cells were extracted from different S. aureus strains. The expression of Efb was tested with the specific antibodies of Efb (prepared by ourselves) by Western blot. The result showed that Efb couldn't be detected in the supernatant of Δrnc. 1: WT, wild type, S. aureus 8325-4; 2: Δrnc, an RNase III inactivation mutant from 8325-4; 3: rncR, the restoration of RNase III activity in Δrnc. C: qRT-PCR quantification of the mRNA level of efb at 1.5 h. The quantity of efb mRNA from different strains was measured by qRT-PCR at 1.5 h. The result showed that the level of efb mRNA wasn't changed in Δrnc. 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. D: The schematic diagram of construction of the reporter vectors. Uefb::lacZ: the promoter and 5′UTR of efb were fused with Lacz; UefbSP: the promoter, 5′UTR and the signal peptides of efb were fused with LacZ. E: Detection of the β-galactosidase activity of different strains. The Uefb::lacZ reporter vector was separately transferred into Δrnc and its parent strains. Then the β-galactosidase activity of different strains was measured at 1.5 h and expressed by miller units. There was no significant difference observed. The results represented a mean of three independent experiments. WT: wild type, S. aureus 8325-4; Δrnc: an RNase III inactivation mutant from 8325-4. F: Detection of the β-galactosidase activity of the cultured medium from different strains.The UefbSP::lacZ reporter vector was separately transferred into Δrnc and its parent strains. Then the β-galactosidase activities of the cultured medium were measured and expressed by miller units. Comparing with it parent stain, the β-galactosidase activitiy of the cultured medium from the Δrnc was decreased at 1.5 h. The results represented a mean of three independent experiments. WT: wild type, S. aureus 8325-4; Δrnc: an RNase III inactivation mutant from 8325-4. (**: p<0.01).
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pone-0020554-g004: The secretion of the proteins in Δrnc was inhibited at 1.5 h.A: qRT-PCR quantification of the level of efb mRNA. The level of efb mRNA in the different strains was detected at 6 h. The results showed that the expression of efb was not regulated by RNAIII. WT: wild type; ΔRNAIII: RNAIII deletion mutant; ΔRNAIIIR: the restoration of RNAIII in ΔRNAIII. B: Detection of the expression of Efb in the extracellular proteins from the different S. aureus strains by Western blot at 1.5 h. The extracellular proteins from same number of cells were extracted from different S. aureus strains. The expression of Efb was tested with the specific antibodies of Efb (prepared by ourselves) by Western blot. The result showed that Efb couldn't be detected in the supernatant of Δrnc. 1: WT, wild type, S. aureus 8325-4; 2: Δrnc, an RNase III inactivation mutant from 8325-4; 3: rncR, the restoration of RNase III activity in Δrnc. C: qRT-PCR quantification of the mRNA level of efb at 1.5 h. The quantity of efb mRNA from different strains was measured by qRT-PCR at 1.5 h. The result showed that the level of efb mRNA wasn't changed in Δrnc. 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. D: The schematic diagram of construction of the reporter vectors. Uefb::lacZ: the promoter and 5′UTR of efb were fused with Lacz; UefbSP: the promoter, 5′UTR and the signal peptides of efb were fused with LacZ. E: Detection of the β-galactosidase activity of different strains. The Uefb::lacZ reporter vector was separately transferred into Δrnc and its parent strains. Then the β-galactosidase activity of different strains was measured at 1.5 h and expressed by miller units. There was no significant difference observed. The results represented a mean of three independent experiments. WT: wild type, S. aureus 8325-4; Δrnc: an RNase III inactivation mutant from 8325-4. F: Detection of the β-galactosidase activity of the cultured medium from different strains.The UefbSP::lacZ reporter vector was separately transferred into Δrnc and its parent strains. Then the β-galactosidase activities of the cultured medium were measured and expressed by miller units. Comparing with it parent stain, the β-galactosidase activitiy of the cultured medium from the Δrnc was decreased at 1.5 h. The results represented a mean of three independent experiments. WT: wild type, S. aureus 8325-4; Δrnc: an RNase III inactivation mutant from 8325-4. (**: p<0.01).
Mentions: In above result there was no significant difference observed in the extracellular proteins production between ΔRNAIII and its parent strain at 1.5 h (figure 3B). The reason should be that RNAIII was a cell density-dependent regulator [19] and the level of RNAIII was too low to regulate its targets at the lag phase. However, the extracellular proteins in Δrnc decreased at 1.5 h (figure 2). It suggested that the reduction of extracellular proteins in Δrnc was not completely due to the RNAIII level decreasing. And the reduction of extracellular proteins in Δrnc at 1.5 h should have no relation with RNAIII. To discover other factors involved in this process, we chose Efb (extracellular fibrinogen binding protein) as the indicator of extracellular proteins [20], [21], [22] because the expression of Efb was not influenced by RNAIII (figure 4A) and the level of Efb in Δrnc supernatant decreased at 1.5 h (figure 4B).Then we analyzed the mRNA level, translation and secretion of Efb to discover the mechanism of reducing the extracellular proteins in Δrnc at 1.5 h. Firstly, the mRNA level of the efb gene was determined by qRT-PCR. It showed that the mRNA level of efb in Δrnc did not alter at 1.5 h (figure 4C). Secondly, we constructed the lacZ fusion vectors to analyze the translation of Efb in Δrnc and its parent strains (figure 4D). The upstream region of efb containing its promoter and 5′UTR was fused with lacZ (named as Uefb::lacZ). The constructed vector was transformed to Δrnc and its parent strain respectively. The results of β-galactosidase activity detection showed that Δrnc did not exhibit significant difference comparing with its parent strain (figure 4E). This suggested that the inactivation of RNase III did not influence the transcription and translation of Efb. And thirdly, we checked if the secretion of Efb was affected in Δrnc. The upstream of efb containing its promoter, 5′UTR and the signal peptide fused with lacZ was termed as UefbSP::lacZ (figure 4D). The β-galactosidase activity of cultured medium in the two different strains was detected. Our results showed that the β-galactosidase activity of the cultured medium from Δrnc was significantly lower than that from its parent strain (figure 4F). It suggested that the reduction of extracellular proteins of Δrnc at 1.5 h was because the secretion of extracellular proteins was suppressed.

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