| Staphylococcus aureus is an extensive gram-positive pathogen, which can cause a wide variety of diseases, ranging from locolized infections of skin and soft tissue and toxic shock to life-threatening systemic infections, such as septicemia, endocarditis, pneumonia, meningitis. And it also can cause urinary tract infection, osteomyelitis, septic arthritis and enteritis. Now, the infectious dieaseas caused by S. aureus usually are treated with antibiotics. But the public health is being threatened by S. aureus with multidrug-resistance, such as MRSA and VRSA. There are many kinds of proteins involved in the infection caused by S. aureus. The investigation on these molecules will be helpful for the treatment of the infection caused by S. aureus.Autolysins can be expressed by many kinds of bacteria, which are considered to be involved in the physiological processes. LytM, one of autolysins, is expressed and secreted by S. aureus. LytM can specifically cleave glycyl–glycine peptide bond, so it might be used to treat the infections caused by Staphylococcus in future. LytM has a significant amino acid sequence homology with Lysostaphin of Staphylococcus simulans. It is the only crystallized Lysostaphin-type metalloprotease and is Zn2+-dependent. In recent years, LytM has been studied extensively for its lytic properties. The studies in vitro have verified that full length LytM is inactive, while its C terminal domain can hydrolyze the peptide bonds. But the native form with lystic activites is still not clear. In addition, the regulation of the expression of LytM has not been investigated.Here, LytM was expressed and the native form with lystic activites was investigated. We also studied how RNAⅢcould regulate the expression of LytM. At the same time, the protein interacting with LytM was studied. Our work is divided into three parts:1,Investigation of the native active form of LytM. The recombinant proteins of LytM and its C terminal(185-316aa) were expressed in Escherichia coli. And the activities of the recombinant proteins were detected. It was found that only C terminal of LytM could hydrolyze the substrates. Anti-LytM polyclonal antibodies were prepared by immunization of animals, which could also specifically bind with its C terminal. The native active form of LytM was investigated by Western Blot and immunoprecipitation. Unfortunately, we could not identify the exactly native active form of LytM, although we had tried various methods.2,The interaction between LytM and TRAP. Our previous studies showed that Lysostaphin could specifically bind with TRAP, which was considered as the signal transductor molecule in S. aureus. The interaction between LytM and TRAP was assayed by ELISA and Western Blot. At the same time, the lytM mutant was constructed from S. aureus 8325-4. In the further study, we found the similar phenotypes of lytM mutant and traP mutant, and tried to investigate the potential biological significance of the interaction.3,The expression of LytM was regulated by RNAⅢ. Here, we had demonstrated that RNAⅢcould down-regulate the expression of LytM by RT-PCR and Western Blot. In further study, LytM was verified as one of the targets of RNAⅢby the lacZ reporter vector system. Morover, the RNAⅢbinding site of lytM mRNA were identified by the site sepecific mutation.In summary, our study confirmed that the C terminus but not full length of LytM could hydrolyze the substrates. And we had tried to search the native form of LytM in vivo. Here, we revealed that LytM was one of the targets of RNAⅢ, which was negatively regulated by RNAⅢ. We also find that LytM could specifically bind with TRAP. |
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