Abstract
Objective To screen the factors that can affect α-toxin expression of CA-MRSA except for quorum-sensing system and to investigate the regulative mechanism of the interesting genes. Methods S. aureus CA-MRSA transposon mutagenesis library was constructed by using mariner based transposon mutagenesis system. The clones with significantly changed level of hemolysis were selected, the location of erm insertion in a gene was confirmed by arbitrary primed (inverse) PCR and nucleotide sequence. Genetic complementation, mice bacteremia and skin abscess models and real time RT-PCR were used to study the function of the interesting gene. Results Twenty-five mutants with down-expression of α-toxin were selected by screening about 104 isolates of transposon mutagenesis library. The hemolytic diameter of CA-MRSA wild type was about 212 mm, no clear hemolysis was found in AraC-, The hemolytic diameter of AraC-pT181 araC was about 197 mm. Real time RT-PCR results showed that compared to the expression of the virulence factors in CA-MRSA wild type( PSMα 257. 30 ±37. 33 ;agr 115. 60 ±0. 81 and α-toxin 3.23 ±0. 21), in AraC-, α-toxin, PSMα and agr were significantly down regulated(α-toxin 1.09 ±0.01 :t = 10. 18, P 0.05;agr:t= 1.563, P>0.05;α-toxm: t = 1. 923, P > 0. 05 ). There were no significant difference of the expression of ClpP in AraC-(0. 21 ±0.01) and in AraC-pT181araC(0.17 ±0.03)compared to the expression of ClpP in CA-MRSA wild type (0. 20 ± 0.01) (t=0.555, P>0.05 and t=0. 851, P>0.05). The result of mice skin abscess model showed that the dermonecrosis area caused by CA-MRSA was (136. 5 ±21.45) mm2, the dermonecrosis area caused by AraC- was (55. 69 ± 13. 81) mm2, the different was significant (t = 3.169, P < 0. 05). Conclusion In CA-MRSA, AraC-type transcriptional regulator controlled the pathogenesis of CA-MRSA by regulating the expression of the most important virulence factors such as hla, PSMα and agr. Key words: Methicillin resistance; Staphylococcus aureus; AraC transcription factor; Virulence factors; Bacterial toxins
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