Screening Of Biofilm-Forming Mutants And Effect Of Lamps On Apoptosis Of Chicken Embryo Fibroblast For Mycoplasma Gallisepticum

Mycoplasma Gallisepticum(MG) belongs to Mycoplasmatales,Mycoplasmataceae. It is causative agent of chronic respiratory disease in chicken, ducks, parrots, goose and turkey. The clinical signs of chicken were cough, sneezing, tears and dyspnoea. Furthermore, M.gallisepticum infection can cause the susceptibility in infection with other avian pathogen, such as Escherichia coli, avian influenza disease, Newcastle disease virus and infective bronchitis virus, and cause great economic loss.In order to adapt to the changed environments, many bacteria could form biofilms in vitro and in vivo. A plenty of evidence demonstrated that biofilm formation is involved in the immune escape and antibiotics resistance in many species of pathogenic bacteria. It plays an important role in the full infection and pathogenesis. In this assay, the biological function of will be studied.In addition, the random insertion vector has been successfully constructed, and the random insertion base has been established. To better understand the mechanisms involved in biofilm formation, mini-Tn4001-SGM, a novel transposon vector containing the gentamicin gene was constructed and electroporated into MG strain Riow. Of the 738 mutants obtained,12 had significantly reduced capacity to form biofilms in a polystyrene microtiter-plate biofilm assay. Ten different genes were identified as disrupted in these mutants. Lipoproteins (lipid associated membrane proteins, LAMPs) are significant structural of mycoplasma, which play an important role in the interactions with host. In recent years there have been many reports proved that lipoproteins may be importantly contributing in the pathogenesis of mycoplasma. In order to explore the role LAMPs play in the pathogenic of M. gallisepticum, following experiments were designed.1. Construction of random mutagenesis bank and screening of biofilm-producing mutants of inactivated genesMycoplasma gallisepticum (MG) is an important pathogen that can cause chronic respiratory disease in chickens and infectious sinusitis in turkeys. MG has the ability to form biofilms. The molecular mechanisms underlying MG biofilm formation are complex and poorly understood. To better understand the mechanisms involved in biofilm formation, mini-Tn4001-SGM, a novel transposon vector containing the gentamicin gene was constructed and electroporated into MG strain Rlow. Of the 738 mutants obtained,12 had significantly reduced capacity to form biofilms in a polystyrene microtiter-plate biofilm assay. Ten different genes were identified as disrupted in these mutants using genomic walking from the transposon insertion sites and Southern bolt hybridization with a transposon-based probe. Four genes were associated with cellular processes, especially synthesis of extracellular polysaccharide and several lipoproteins encoded. Other genes were associated with translation, metabolism and gene regulation, and one had unknown function. Seven genes identified in this study have been previously associated with biofilm formation in MG or other bacterial species. The other three have not been previously reported to play a role in biofilm formation in MG. In conclusion, a new transposon vector was shown to be a powerful tool for future studies of MG pathogenesis. This study adds to our understanding of the molecular mechanisms involved in MG biofilm formation and may shed light on the persistence of MG infections.2. Effect of LAMPs on apoptosis of chicken embryo fibroblast of mycoplasma gallisepticumLipoproteins (lipid associated membrane proteins, LAMPs) are significant structural of mycoplasma, which play an important role in the interactions with host. In recent years there have been many reports proved that lipoproteins may be importantly contributing in the pathogenesis of mycoplasma. Firstly, the interaction between LAMPs and chicken embryo fibroblast (DF1) was confirmed. To investigate the inhibition of LAMPs from MG on DF1 cells LAMPs were extracted by Trion-X114, the suppression ratio was detected throughMTT assay. Therefore, some experiments related to the typical characteristics of apoptosis were taken to detect whether LAMPs could induce DF1 cells apoptosis, including DAPI, TUNEL, AO-EB and Annexin-V-PI. To further demonstrate the regulatory mechanism of apoptosis in DF1 cells that leads to apoptosis induced by LAMPs, the activity of caspase was examined. The results indicated that caspase 3 has involvement in apoptotic pathway. Moreover, after treated with LAMPs, some proteins related in the pathway of apoptosis changed. PARP was cut into two fragments. The outcome of this study could contribute to quest the pathogenesis of mycoplasma, while the understanding of apoptosis pathway may help people find out right targets for treating mycoplasma infection.