New virulence factor CSK29544_02616 as LpxA binding partner in Cronobacter sakazakii

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.

pMOD <MCS> reverse-sequencing primer). 49 All plasmids used in this study are listed in Table S1. To generate pSK01 50 producing CSK29544_02616 under its putative intrinsic promoter, CSK29544_02616 gene 51 containing its upstream DNA sequences was PCR-amplified using primers of 52 4 CSK29544_02616-F-SphI and CSK29544_02616-R-BamHI. PCR products were 53 introduced into pACYC184 digested with SphI and BamHI. For the construction of pSK02 54 expressing His-CSK29544_02616 under arabinose-inducible promoter, CSK29544_02616 55 was PCR-amplified with His-CSK29544_02616-F and His-CSK29544_02616-R primers 56 and PCR products were inserted into pBAD24 cut with EcoRI and SalI. CSK29544_02616, 57 lpxA, and lpxD genes were amplified using primers of CSK29544_02616-F-BamHI and 58 CSK29544_02616-R-EcoRI, lpxA-F-SalI and lpxA-R-BamHI, and lpxD-F-SalI and lpxD-59 R-BamHI, respectively. These PCR products were inserted between BamHI and EcoRI 60 sites of pKT25 vector or SalI and BamHI sites of pUT18C vector. In the construction of 61 pSK05 and pSK06 for GST pull-down assay, pETDuet-1 and pGST parallel 1 vectors were 62 used to express His-LpxA and GST-CSK29544_02616, respectively. Plasmids pSK07, 63 pSK08, and pSK09 were generated for protein purification and used for LpxA enzymatic 64 assay. Plasmids expressing CSK29544_02616 derivatives with W29L and V94A 65 substitutions were constructed using the following primers: CSK29544_02616-W29L-F 66 and CSK29544_02616-W29L-R for CSK29544_02616 W29L, and CSK29544_02616-67 V94A-F and CSK29544_02616-V94A-R for CSK29544_02616 V94A. For the 68 construction of pSK17 expressing mCherry protein, pQE30 harboring gfp 3 was digested 69 with BamHI and SacI and ligated with mCherry-encoding sequences PCR-amplified using 70 5 mCherry-F-BamHI and mCherry-R-SacI primers. Primers used in this study for plasmids 71 construction are listed in Supplementary Table S3.

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Hydrophobicity assay 73 The hydrophobicity of bacterial surface was measured as described previously. 4

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Briefly, bacterial cells were grown overnight, harvested, washed twice with PBS (pH 7.4), 75 and resuspended in 2 ml of PBS. OD600 value was measured and recorded as H0. were selected starting with the most intense ion using a selection isolation width of ~ 4 Da.

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Rolling collision-energy feature was used to determine collision energy based on precursor 99 value and charge state. Dynamic exclusion time for precursor-ion m/z values was 20 s.   (20 mM Tris-Cl, 300 mM NaCl, and 250 mM imidazole, adjusted to pH 8.0) and 142 9 concentrated using Amicon R Ultra-4 (Millipore, USA) according to the manufacturer's 143 instructions. The buffer was changed to storage buffer (20 mM Tris-Cl, 300 mM NaCl, and 144 50% glycerol, adjusted to pH 8.0) using PD midiTrap TM G-25 (GE Healthcare, UK).

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Aliquots of protein were then stored at -20°C until further use.  washed with ACP buffer containing gradient NaCl concentrations from 0 mM to 500 mM.

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SDS-PAGE analysis revealed that holo-ACP was eluted by 300 mM NaCl. The eluent 167 containing holo-ACP was desalted with ACP buffer using PD midiTrap TM G-25 and stored 168 at 4°C. Protein concentration was measured using Bio-Rad protein assay.