Lysostaphin gene cloned from Staphylococcus simulans and its functional study in immortal caprine mammary epithelial cells
Date Issued
2008
Date
2008
Author(s)
Chung, Pei-Hsuan
Abstract
Mastitis is an inflammatory reaction of mammary gland usually caused by a microbial infection and is the most prevalent disease of dairy cows and goats. However, Staphylococcus aureus (S. aureus), which currently accounts for 15-30% of mastitis, has been proved more difficult to be controlled using standard management practices. This disease, in addition to causing animal distress, dairy processors also incur losses from detrimental changes in milk composition that accompany mammary gland inflammation. These changes include: decrease of milk production and milk quality. But, the cure rate for treatment of S. aureus mastitis with beta-lactam antibiotics is often less than 15%. Therefore, to develop a novel antibacterial protein is required to cure mastitis infections. Lysostaphin, a 25 kDa glycylglycine endopeptidase produced by Staphylococcus simulans, can specifically cleave bond between the third and fourth glycines of the pentaglycine cross bridge of the staphylococcal cell wall, and cause the rapid lysis of actively growing staphylococci. It has been demonstrated that lysostaphin treatment significantly reduced viable S. aureus in a mouse and a bovine mastitis model. A lysostaphin transgenic goat was established and showed resistant to mastitis infection, but the detail biological characterization of the recombinant lysostaphin expression in mammary epithelial cells is still unknown. In order to investigate whether the recombinant lysostaphin could follow the common protein secretory pathway and perform its normal biological function after posttranslational modification in goat mammary epithelial cells, a telomerase-immortalized caprine mammary epithelial cell line (CMEC) was used as a platform to study the biological characteristics of recombinant lysostaphin protein. In an attempt to observe production and secretory pathway of lysostaphin, three plasmids containing recombinant lysostaphins separately fused with beta-casein, lactoferrin and prokaryotic signal peptides were constructed to examine the efficiency of secretion into culture medium. Also, to investigate whether lysostaphin is modified after translation in CMEC, total proteins and mediums were obtained from the CMEC transfected with recombinant plasmids to examine the expression of lysostaphin in CMEC. The beta-casein and lactoferrin signal peptides fused recombinant lysostaphins were detected both in medium and cell extract, indicated both of them could be secreted into medium. The localizations of recombinant lysostaphin proteins in CMEC were examined by immunofluorescence staining after transfections, data revealed the eukaryotic signal peptides directed secretion of the lysostaphin through the endoplasmic reticulum and Golgi apparatus in the CMEC, but prokaryotic signal peptide could not. A large shift in molecular weight were also observed in these recombinant proteins expressing in CMEC, suggesting that lysostaphin might be glycosylated in CMEC before secretion. To determine whether the modification was glycosylation, site-directed mutagenesis was performed to remove N-linked glycosylation sites from the recombinant lysostaphin-expression plasmids. The N-linked glycosylation sites mutated recombinant protein decreased shift in electrophoretic mobility compared to that of unmodified recombinant proteins, indicated the recombinant proteins were glycosylated in CMEC. It seemed that these mutated recombinant proteins secreted in culture medium were proved to possess staphylolytic activity based on bacterial plate assay, but not detected in wild type recombinant proteins, indicating that glycosylation was responsible for the inactivity of lysostaphin in CMEC. Together these results, suggest that the mutated recombinant proteins may possess normal biological function of antibacterial in caprine mammary epithelial cells. The detailed biological effect on caprine mammary epithelial cells needs further investigation.
Subjects
lysostaphin
immortal caprine mammary epithelial cells
Type
thesis
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