Experimental Study On The Mechanism Of Nitric Oxide In The Regulation Of Staphylococcus. Aureus Biofilm Formation

Nitric Oxide (NO) is an important active molecule in biology, it is involved in many important physiological processes in vivo, such as cell differentiation and apoptosis, the relaxation of blood vessels, defense during immune response and seed germination, iron metabolism,regulation of stress resistance and anti-aging etc. It is described as a multifunctional second messenger. NO has attracted a great deal of attention from researchers in animals and high plants, however, the research about NO in bacteria especially its role in the biofilm is rarely reported. Biofilms are multicellular, matrix-enclosed microbial communities that are attached to an environmental surface throughout the biological world. Cells within a biofilm have a number of advantages over their planktonic counterparts, including protection against the immune system and enhanced resistance to antimicrobial agents and other stresses, but the molecular mechanism is relatively complex, involving many signaling pathway and regulatory proteins, to ascertain the mechanism of biofilm formation is important for treatment of biofilm infections. In the previous study, we found NO can enhancement biofilm formation of Staphylococcus. aureus. This thesis is aimed at experimental study on the mechanism of NO in the regulation of S.aureus biofilm formation.The compact cell wall and abundant extracellular polysaccharide (PIA) were the main barrier for S.aureus gene manipulation. In this studyβ-1,6-N-acetyglu-cosaminidase (DSPB) was used to further optimize the method of electroporation of S.aureus. DSPB is a specific hydrolase enzyme of PIA. The result indicated that DSPB can increase the quality and quantity of plasmid purified from RN4220. The transformation efficiency of DSPB pretreated competent cells is more than 1000 folds compared to the unpretreated one and it's also more effective than lysozyme and lysostaphin.To further verified the effect of nitric oxide synthase (NOS) in the regulation of biofilm formation of S.aureus, nos gene of RN6390△nos is complemented, and then biofilm formation of RN6390 RN6390△nos and RN6390Anos-PMGS100-nos are compared. The result verified that endogenous NOS plays important role in S. aureus biofilm formation. The NO donor SNP was also used in the biofilm forming assay in 96 well and flow-cell biofilm model. The results showed that SNP could enhance the growth of biofilm, which suggested that exdogenous NO also can promote biofilm formation of S. aureus.Extracellular polysaccharide was an important component of bacteria biofilm. It was reported that Staphylococcus biofilm formation was divided into PIA-dependent and PIA-independent types. Whether NO can increase PIA synthesis was inspected from in vivo transcription and in vitro extracellular polysaccharide detection. The results showed that NO donor SNP reduced both the transcription of ica and the production of PIA which indicated that the promotion of S.aureus biofilm formation by NO was PIA-independent.It is reported that the two component system ResDE of Bacillus subtilis can detect NO by NsrR. S. aureus has the two-component system srrAB which was highly identity with ResDE. To test the relationship of srrAB with NO srrAB deficient strain was construted via homologous recombination. It inicated that deletion mutant has a poor biofilm formation ability compared to the wild type, but NO was still able to promote its biofilm formation. Thus the regulation mechanism of NO on biofilm formation has no relation with the srrAB.In conclusion,this thesis was based on former studies to further proof that NO can promote S.aureus biofilm formation,this promotive effect does not depend on ica which can encode polysaccharide intercellular adhesin and two two-component system srrAB.This study revealed the effect of NO on biofilm of S. aureus and the mechanisms were preliminarily investigated,laid the foundation for further investigation.Furthermore, this study aslo has important value in the investigation of the regulation mechanism of biofilm and the control of biofilm related infection.