Identification Of A LPS Structural Gene WaaC In Cronobacter Sakazakii And Its Effect On Biofilm Formation

Cronobacter sakazakii is a ubiquitous, food-borne Gram-negative opportunistic pathogen, mainly detected in powdered infant formula. Because the clear evidence of causality, C. sakazakii was designated as Category A. C. sakazakii can cause meningitis, sepsis, bacteremia, and necrotizing enterocolitis in neonates. The mortality rate caused by the severe infection of C. sakazakii could be up to 80%, but its virulence mechanism has not been well understood. The microorganism caused illnesses presumably by propagatiing in the intestinal tract, then attaching, and invading through the intestinal epithelial layer. Biofilm formation may play an important role in these processes. Lipopolysaccharide (LPS) is the main pathogenic macromolecule on the cell surface of C. sakazakii. LPS is closely related to biofilm formation in several Gram-negative bacteria such as Escherichia coli, Salmonella, and Campylobacter jejuni. However, the majority of the genes in LPS biosynthetic pathway and their relationship with biofilm formation are not known.In this study, the gene waaC in Cronobacter sakazakii ATCC BAA-894 related to LPS biosynthesis was identified. To explore the relationship between LPS structure and biofilm formation in C. sakazakii, the important role of waaC gene in biofilm formation was analyzed.The main conclusions were as follows:(1) It was conformed that the gene ESA₀4107 in C. sakazakii ATCC BAA-894 encodes the heptosyltransferase I (WaaC). The BLASTp analysis showed that C. sakazakii ESA₀4107 has the highest homology with waaC gene in E. coli W3110. Therefore, C. sakazakii mutant strain LWW02 was constructed by inactivating the gene ESA₀4107. Lipopolysaccharides were extracted from LWW02, and analyzed by SDS-PAGE and thin layer chromatography. The lipopolysaccharides were further purified by DEAE-cellulose anion exchange chromatography and their structures were analyzed by electrospray ionization mass spectrometry. The results suggest that the gene ESA₀4107 in C. sakazakii ATCC BAA-894 encodes the heptosyltransferase I.(2) The biofilm-forming abilitiy of LWW02 was studied. Compared with the wildtype strain BAA-894, the growth of LWW02 was affected. However, LWW02 formed more biofilms on both hydrophilic and hydrophobic surfaces, suggesting that biofilm formation is closely related to the LPS structure.(3) The effect of LPS structure on the key properties of C. sakazakii was investigated. Cell surface hydrophobicity, membrane permeability, autoaggregation and exocellular secretions of LWW02 were all stronger than those of BAA-894, suggesting that these factors might be the main reason for more biofilm formation of LWW02.Based on the waaC in C. sakazakii ATCC BAA-894, the relationship between LPS and biofilm formation in C. sakazakii was investigated. The study was important in understanding the pathogenic mechanism of C. sakazakii and in establishing methods to efficiently detect and control C. sakazakii contamination.