Abstract
Cronobacter sakazakii is an opportunistic pathogen that can cause meningitis and necrotizing enterocolitis in premature infants, but its virulence determinants remain largely unknown. In this study, a transposon-mediated random-mutant library of C. sakazakii was used to identify new virulence factors. Compared to wild-type bacteria, a mutant lacking CSK29544_02616 (referred to as labp) was defective in invasion into intestinal epithelial cells (by at least 1000-fold) and showed less phagocytosis by macrophages (by at least 50-fold). The lack of labp in C. sakazakii changed the profile of outer membrane proteins, decreased the production of lipopolysaccharides, and increased the production of membrane phospholipids. Bacterial physiological characteristics including surface hydrophobicity and motility were also altered in the absence of labp, presumably because of changes in the bacterial-envelope structure. To systematically determine the role of labp, ligand fishing was conducted using Labp as a bait, which revealed LpxA as a binding partner of Labp. LpxA is UDP-N-acetylglucosamine (GlcNAc) acyltransferase, the first enzyme in the pathway of lipid A biosynthesis. Labp increased the enzymatic activity of LpxA without influencing lpxA expression. Considering multifaceted roles of lipopolysaccharides in virulence regulation, Labp is a novel virulence factor that promotes the production of lipid A by LpxA in Cronobacter.
Highlights
Cronobacter sakazakii is an opportunistic food-borne pathogen that causes bacteremia, meningitis, and necrotizing enterocolitis, in premature infants[1,2,3]
When bacterial persistence inside macrophage cells was evaluated using murine macrophage RAW264.7 cells, the ΔCSK29544_02616 strain showed 50-fold lower internalization by macrophages compared to the WT strain and was cleared out completely at 72 h post-infection, whereas WT bacteria persisted inside macrophages even at 96 h after phagocytosis (Fig. 1)
Because the invasion of C. sakazakii into host cells is mediated by the interaction between host receptors and bacterial surface constituents[34], we investigated whether deletion of CSK29544_02616 caused structural alterations in bacterial surface appendages including outer membrane proteins (OMPs), LPS, and flagella, which are known as key virulence determinants in C. sakazakii infection[6,7,8,35]
Summary
Cronobacter sakazakii is an opportunistic food-borne pathogen that causes bacteremia, meningitis, and necrotizing enterocolitis, in premature infants[1,2,3]. The core oligosaccharide attached to a glucosamine unit of lipid A is a short chain of sugars such as Kdo (3-deoxy-D-manno-oct-2-ulosonic acid), heptose, and hexose and is decorated with diverse substituents including phosphate, ethanolamine, and amino acids. This core structure is connected to O-antigen, a hyper-variable polysaccharide region among bacterial species and different strains within the same species[20,21]. A pentadecapeptide known as peptide 920 directly bound to LpxA, resulting in bacterial growth inhibition[31] In this context, peptide 920 has been exploited as a promising control agent against gram-negative pathogenic bacteria[20]
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