The Sequential Analysis of GtfA and GtfB in SraP Operon and SraP Glycosylation in Staphylococcus Aureus
|School||Huazhong University of Science and Technology|
|Course||Clinical Laboratory Science|
|Keywords||Staphylococcus aureus SraP GtfA GtfB glycosyltransferase|
【Objective】 To study the functional contribution of the two putativeglycosyltransferases, GtfA and GtfB, to SraP glycosylation.【Methods】All clinical isolates of S. aureus (n=55) were collected. SraP, MecA(Methicillin determinant A) and PVL (panton valentine leukocidin) genes wereamplified using three independent real-time TaqMan PCR assays, respective. Thepurified SraP1-743fragment was ligated into the pETDuet-A, pETDuet-B andpETDuet-AB, respectively, to construct pETDuet-AS,pETDuet-BS andpETDuet-ABS. Construction of expression plasmid pETDuet-ABS andpETDuet-S.Coexpression of SraP1-743with Different Glycosyltransferases. Analysisof SraP1-743glycosylation by lectin blotting assays.【Results】Among55clinicalS.aureus isolates35(63.6%)isolates were considered asMRSA(methicillin-resistant S. aureus) according to the recommended standards fromCLSI. At80%similarity,14distinct RAPD profiles (I, II, III, IV, V, VI, VII, VIII, IX,X, XI, XII,XIII and XIV) were found. Structural homology searches revealed asignificant structural similarity of GtfA and GtfB with several glycosyltransferaseenzymes. Coexpression of GtfA and GtfB in E. coli lead to the production of aSraP1-743doublet that migrated at the95-and120-kD positions when probed withanti-S tag antibody (Fig.5C, lane4), whereas expression of GtfA or GtfB in E. coliled to the production of one band at95-kD position. The GlcNAc-specific lectinsWGA only reacted with the protein band at120-kDa positions.【Conclusion】SraP is present in all clinical S. aureus isolates showing wide genotypediversity. GtfA and GtfB are very highly conserved in clinical S. aureus isolates. BothGtfA and GtfB are involved in SraP glycosylation with GlcNAc-containingoligosaccharides, furthermore, the formation of the GtfA and GtfB protein complexmaybe required for SraP glycosylation.