The Structural Analysis Of LPS And Functional Research Of CslpxL Gene In Cronobacter Sakazakii

The Gram-negative food borne pathogen Cronobacter, found in powdered milk formula, can induce necrotizing enterocolitis, severe meningitis and sepsis in neonates. The mortality is high and ranging from 40 to 80 %. Lipopolysaccharide (LPS), anchors in the outer membrane of Gram-negative strain, is an important structural and functional molecule. As one component of LPS, lipid A, also referred to as endotoxin, is an important virulence factor. However, there is little information available for lipid A structure and genes related to its biosynthesis in Cronobacter.We firstly studied the antibiotics sensitivity of ampicillin, chloramphenicol and kanamycin to Cronobacter sakazakii BAA-894 and Cronobacter muytjensii ATCC 51329, and the replication capacity and transformation efficiency of three E. coli vectors pWSK29, pACYC184 and pET28a, which contain different replical origins, have been studied. Then by characterization of LPS in C. sakazakii BAA-894 and C. muytjensii ATCC 51329, and mostly analysis of lipid A and related gene CslpxL in C. sakazakii BAA-894, we concluded as follows. (a) By SDS-PAGE analysis of isolated LPS, we found that O-antigen can be synthesized in C. sakazakii BAA-894, but not in C. muytjensii ATCC 51329; (b) By mild acid hydrolysis, DEAE-cellulose anion exchange chromatography and preparative thin-layer chromatography, the purified lipid A was analyzed by electrospray ionization/mass spectrometry. Two lipid A species have been detected in C. sakazakii BAA-894. The structural difference, identified further by ESI/MS and MS/MS of alkaline hydrolyzed lipid A, is secondary fatty acid residue at C2′-position, which is myristate or laurate; (c) Based on the structural difference of lipid A in C. sakazakii BAA-894, the gene CslpxL, which is responsible for the difference of secondary fatty acid chain lengths at C2′-position, was studied. By expression of CslpxL in E. coli mutant MKV15, we found the protein, encoded by this gene, has the activity of myristoyl acyltransferase.This is the first time for characterization of the structure of lipid A and the function of CslpxL gene in C. sakazakii. This information would shed light on pathogenesis research of this food borne pathogen.