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The effects of pinocembrin on Y-79 cell-cycle progression and TGF-β1-induced avβ3 integrin expression were determined by flow cytometry using FACScan (Becton Dickinson Immunocytometry Systems, UK). Firstly, to analyze the cell-cycle distribution, the cells were first treated with various concentrations of pinocembrin for 24 h, and then were collected by trypsinization, fixed in 75% absolute ethanol, washed in PBS, and resuspended in 1 ml of PBS containing 0.5 mg/ml RNase A and 0.01 mg/ml propidium iodide (PI) in the dark for 30 min at room temperature. The cell-cycle profiles were analyzed by a flow cytometer. The percentage of cells in the sub-G1, G0/G1, S, and G2/M phases of the cell cycle was analyzed by the ModFit LT 3.0 software (Verity Software, Topsham, ME). Furthermore, the cell surface expression of avβ3 integrin was determined using flow cytometry. Y-79 cells were plated in six-well dishes. The cells were then washed with PBS and detached with trypsin at 37C. Cells were fixed for 10 min in PBS containing 1% paraformaldehyde. After rinsing in PBS, the cells were incubated with a mouse anti-human antibody against avβ3 integrin (1:100) for 1 h at 4C. Cells were then washed again and incubated with fluorescein isothiocyanate-conjugated goat anti-rabbit secondary IgG (1:100; Leinco Tec. Inc., St. Louis, MO, USA) for 45 min and analyzed by flow cytometry.
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Because Kp52.145 is a highly virulent strain, our analyses were focused on comparing its genome with the genomes of K. pneumoniae strains SB2390, SB3193, NTUH-K2044 and MGH78578. According to SEED subsystems annotations [25], about 60% of protein-coding genes for each K. pneumoniae genome had predicted functions. More specifically, the largest percentage of annotated genes is involved in the metabolism of carbohydrates (approximately 20%), of amino acids and their derivatives (approximately 10%) and of cofactors, vitamins and prosthetic groups (approximately 8%) [see Additional file 1].
In order to demonstrate the phospholipase activity of PLD1 and characterize its involvement in lipid metabolism, the lipid composition of wild-type and mutant strains was analyzed by thin-layer chromatography (TLC). A remarkable lipid spot was absent from the pld1 mutant in comparison with the complemented strain, suggesting that the putative PLD1 is involved in lipid metabolism [see Additional file 1]. To reinforce this result, a plasmid carrying the pld1 gene was inserted into E. coli strain SD9 [79]. This strain is deficient in phosphatidylserine and cardiolipin, thus presenting a simpler lipid composition than its parental strain and Kp52.145. Lipid profiles of SD9 and complemented strains had a different lipid composition. Notably, the PLD1-expressing strain contained an additional lipid spot in comparison to the SD9 strain, suggesting that PLD1 is responsible for this difference (Figure 5A). SD9 wild-type strain also presented an extra lipid spot in comparison to the PLD1-expressing strain, possibly representing the PLD1 substrate (Figure 5A). Densitometric analysis of iodine-stained lipids on TLC plates revealed that this lipid spot corresponded to 21% of the total amount of lipids in SD9 strain, but only 6% of SD9 complemented with a plasmid expressing pld1. Mass spectrometry (MS) analysis of total lipid extract was carried out to identify such lipid. Comparing lipid profiles by MS, we found a lipid of mass 788.4 present only in the PLD1-deficient strain (Figure 5B) and identified it as phosphatidyl glycerol (PG) using the LipidMaps database.
Bacterial lipids were extracted by the method of Bligh and Dyer [87]. Briefly, bacterial stationary-phase cultures were concentrated 10 times and mixed with chloroform:methanol. After centrifugation at 1,000 rpm for five minutes, the organic phase was recovered. Lipid profiles were analyzed by two-dimensional TLC using TLC Silica gel 60 F254 plates (Merck, Whitehouse Station, NJ, USA). as the stationary phase and a chloroform 9:1 methanol mixture as the mobile phase in both dimensions. Staining was performed by iodine vapor. TLC calibrated images were aquired in ImageScanner using LabScan v5.0 software. The relative intensity of each spot was calculated in ImageMaster two-dimensional Platinum v7.0 software. Alternatively, lipid extracts were analyzed by MS and MS/MS in an ESI-Q-Tof Micro (Waters), in positive ion mode. Resolution was typically lower than 10 ppm.
Additional file 1: 1) Distribution of K. pneumoniae Kp52.145 genes, according to RAST categories. Each functional category is represented in a different color. The total number of genes per category is shown. 2) pld PCR assay. A collection of 42 virulent and non virulent clones was screened by PCR for pld gene. Strains containing pld gene are indicated by (+) and the absence of pld gene is (-). 3) TLC lipid profiles of K. pneumoniae Kp52.145 wild-type (left panel) and pld mutant strains (right panel). Black circles indicate differentially expressed lipids. 4) Bacterial competition assay. Anti-bacterial activity was measured as the number of E. coli cells recovered after the co-culture with K. pneumoniae Kp52.145 wild-type and pld mutant strains. S. marcesens was used as a positive control strain. 5) List of primers used for RT-PCR analysis. (PDF 128 KB)
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