Figure 1.
Detection of RNase III inactive mutant.
A: Verification of RNase III inactive mutant by RT-PCR. Total RNA of cells was extract and used as the template to amplify the rnc gene. In Δrnc strain, the rnc mRNA could not be detected like WT and rncR because the kana gene was inserted into the rnc gene of Δrnc genome. 16s rRNA was used as the internal control. WT (wild type, S. aureus 8325-4), Δrnc(an RNase III inactivation mutant from 8325-4) and rncR (the restoration of RNase III activity in Δrnc). B: The growth curves of S. aureus strains. There is no significant difference between WT and Δrnc. WT: wild type, S. aureus 8325-4; Δrnc: an RNase III inactivation mutant from 8325-4. The experiment has been repeated for three times. C: qRT-PCR quantification of the expression level of spa. The total RNA of the cells cultured for 6 h was extracted and the mRNA level of spa was detected by qRT-PCR. In the Δrnc strain, the level of spa mRNA was significantly increased compared with WT. 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. (**: P<0.01).
Table 1.
Sequences of forward and reverse primers used in this study.
Figure 2.
Detection of the protein profile from different phases of WT and Δrnc.
Equal number of S. aureus cells was harvested at the indicated time points. The total proteins of whole-cell and supernatant proteins were extracted. The results showed that the supernatant proteins of the Δrnc were decreased significantly compared with WT, while the total proteins of whole-cell did not show the same change as the supernatant proteins. The experiment has been repeated for three times. 1,4,7: WT, wild type, S. aureus 8325-4; 2,5,8: Δrnc; 3,6,9: rncR.
Figure 3.
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.
Figure 4.
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).
Figure 5.
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.
Figure 6.
Half-lives of secY2 mRNA and RNAIII were determined in the presence of rifampicin (500 µg ml-1) in the WT and Δrnc strains. Percentage of RNA was calculated normalizing with 5s rRNA.
Figure 7.
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.
Table 2.
Bacterial strains and plasmids.