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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 Δrnc was less pathogenic compared with its parent strain.A: Analysis of apoptosis and necrosis of MDBK cell after treatment with the supernatant from different strains. Flow cytometric analysis was prepared to observe the apoptosis and necrosis of MDBK cell after treated with the supernatant of S. aureus. Comparing with its parent stain, the percentage of apoptosis and necrosis induced by the supernatant of Δrnc was significantly decreased. Data were from a representative experiment repeated for three times. (**: p<0.01). B: Analysis of the production of the heat-stable toxins from different stains. The heat-stable toxins were obtained as the described method and incubated with MDBK cell. The survival cell number was determined by MTT method. Comparing with its parent strain, the heat-stable toxins of Δrnc supernatant was decreased. Data were from a representative experiment repeated for three times. (**: p<0.01). C: The detection of the pathogenicity of the different strains with the acute peritonitis animal model. Groups of 10 Balb/c mice were injected intra-abdominally with 500 µl of Δrnc and its parent strain (1×108 CFU). The number of the survival mice was recorded at different time points. The survival rate was calculated. The result showed that the pathogenicity of Δrnc was decreased.
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pone-0020554-g007: The Δrnc was less pathogenic compared with its parent strain.A: Analysis of apoptosis and necrosis of MDBK cell after treatment with the supernatant from different strains. Flow cytometric analysis was prepared to observe the apoptosis and necrosis of MDBK cell after treated with the supernatant of S. aureus. Comparing with its parent stain, the percentage of apoptosis and necrosis induced by the supernatant of Δrnc was significantly decreased. Data were from a representative experiment repeated for three times. (**: p<0.01). B: Analysis of the production of the heat-stable toxins from different stains. The heat-stable toxins were obtained as the described method and incubated with MDBK cell. The survival cell number was determined by MTT method. Comparing with its parent strain, the heat-stable toxins of Δrnc supernatant was decreased. Data were from a representative experiment repeated for three times. (**: p<0.01). C: The detection of the pathogenicity of the different strains with the acute peritonitis animal model. Groups of 10 Balb/c mice were injected intra-abdominally with 500 µl of Δrnc and its parent strain (1×108 CFU). The number of the survival mice was recorded at different time points. The survival rate was calculated. The result showed that the pathogenicity of Δrnc was decreased.

Mentions: In the further investigation, we compared the cytotoxicity induced by the supernatant between Δrnc and its parent strain. The supernatant of the cultured cells at 6 h was collected and incubated with MDBK cells. And then the cytotoxicity of the supernatant was tested with flow cytometric analysis. It was found that the percentage of apoptosis and necrosis induced by the Δrnc supernatant was significantly lower compared with its parent strain (figure 7A). At the same time, we also detected the cytotoxicity induced by the heat-stable toxins in the supernatant using MTT assay. In line with expectations, the heat-stable toxins of Δrnc were decreased (figure 7B). Then the pathogenicity of Δrnc was assessed in a murine peritonitis model. The same numbers of cells of Δrnc and its parent strain were delivered intraperitoneally to mice. As shown in figure 7C, the survival rate of the mice in the Δrnc group was significantly higher than that of its parent strain group at the different time points (8 h, 16 h, and 24 h), which was in accordance with the cell toxicity results. It suggested that the RNase III played an important role in the pathogenicity of S. aureus.


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 Δrnc was less pathogenic compared with its parent strain.A: Analysis of apoptosis and necrosis of MDBK cell after treatment with the supernatant from different strains. Flow cytometric analysis was prepared to observe the apoptosis and necrosis of MDBK cell after treated with the supernatant of S. aureus. Comparing with its parent stain, the percentage of apoptosis and necrosis induced by the supernatant of Δrnc was significantly decreased. Data were from a representative experiment repeated for three times. (**: p<0.01). B: Analysis of the production of the heat-stable toxins from different stains. The heat-stable toxins were obtained as the described method and incubated with MDBK cell. The survival cell number was determined by MTT method. Comparing with its parent strain, the heat-stable toxins of Δrnc supernatant was decreased. Data were from a representative experiment repeated for three times. (**: p<0.01). C: The detection of the pathogenicity of the different strains with the acute peritonitis animal model. Groups of 10 Balb/c mice were injected intra-abdominally with 500 µl of Δrnc and its parent strain (1×108 CFU). The number of the survival mice was recorded at different time points. The survival rate was calculated. The result showed that the pathogenicity of Δrnc was decreased.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3105085&req=5

pone-0020554-g007: The Δrnc was less pathogenic compared with its parent strain.A: Analysis of apoptosis and necrosis of MDBK cell after treatment with the supernatant from different strains. Flow cytometric analysis was prepared to observe the apoptosis and necrosis of MDBK cell after treated with the supernatant of S. aureus. Comparing with its parent stain, the percentage of apoptosis and necrosis induced by the supernatant of Δrnc was significantly decreased. Data were from a representative experiment repeated for three times. (**: p<0.01). B: Analysis of the production of the heat-stable toxins from different stains. The heat-stable toxins were obtained as the described method and incubated with MDBK cell. The survival cell number was determined by MTT method. Comparing with its parent strain, the heat-stable toxins of Δrnc supernatant was decreased. Data were from a representative experiment repeated for three times. (**: p<0.01). C: The detection of the pathogenicity of the different strains with the acute peritonitis animal model. Groups of 10 Balb/c mice were injected intra-abdominally with 500 µl of Δrnc and its parent strain (1×108 CFU). The number of the survival mice was recorded at different time points. The survival rate was calculated. The result showed that the pathogenicity of Δrnc was decreased.
Mentions: In the further investigation, we compared the cytotoxicity induced by the supernatant between Δrnc and its parent strain. The supernatant of the cultured cells at 6 h was collected and incubated with MDBK cells. And then the cytotoxicity of the supernatant was tested with flow cytometric analysis. It was found that the percentage of apoptosis and necrosis induced by the Δrnc supernatant was significantly lower compared with its parent strain (figure 7A). At the same time, we also detected the cytotoxicity induced by the heat-stable toxins in the supernatant using MTT assay. In line with expectations, the heat-stable toxins of Δrnc were decreased (figure 7B). Then the pathogenicity of Δrnc was assessed in a murine peritonitis model. The same numbers of cells of Δrnc and its parent strain were delivered intraperitoneally to mice. As shown in figure 7C, the survival rate of the mice in the Δrnc group was significantly higher than that of its parent strain group at the different time points (8 h, 16 h, and 24 h), which was in accordance with the cell toxicity results. It suggested that the RNase III played an important role in the pathogenicity of S. aureus.

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