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iTRAQ-coupled 2-D LC-MS/MS analysis of membrane protein profile in Escherichia coli incubated with apidaecin IB.

Zhou Y, Chen WN - PLoS ONE (2011)

Bottom Line: Cell division protease ftsH, an essential regulator in maintenance of membrane lipid homeostasis, was found to be overproduced in cells incubated with apidaecin IB.Its over-expression intensified the degradation of cytoplasmic protein UDP-3-O-acyl-N- acetylglucosamine deacetylase, which catalyzes the first committed step in the biosynthesis of the lipid A moiety of LPS, and thus leaded to the further unbalanced biosynthesis of LPS and phospholipids.Our findings suggested a new antibacterial mechanism of apidaecins and perhaps, by extension, for other proline-rich antimicrobial peptides.

View Article: PubMed Central - PubMed

Affiliation: School of Chemical and Biomedical Engineering, College of Engineering, Nanyang Technological University, Singapore, Singapore.

ABSTRACT
Apidaecins are a series of proline-rich, 18- to 20-residue antimicrobial peptides produced by insects. They are predominantly active against the gram-negative bacteria. Previous studies mainly focused on the identification of their internal macromolecular targets, few addressed on the action of apidaecins on the molecules, especially proteins, of bacterial cell membrane. In this study, iTRAQ-coupled 2-D LC-MS/MS technique was utilized to identify altered membrane proteins of Escherichia coli cells incubated with one isoform of apidaecins--apidaecin IB. Cell division protease ftsH, an essential regulator in maintenance of membrane lipid homeostasis, was found to be overproduced in cells incubated with apidaecin IB. Its over-expression intensified the degradation of cytoplasmic protein UDP-3-O-acyl-N- acetylglucosamine deacetylase, which catalyzes the first committed step in the biosynthesis of the lipid A moiety of LPS, and thus leaded to the further unbalanced biosynthesis of LPS and phospholipids. Our findings suggested a new antibacterial mechanism of apidaecins and perhaps, by extension, for other proline-rich antimicrobial peptides.

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Related in: MedlinePlus

Western blot analysis of LpxC in E. coli incubated with apidaecin IB.The relative densitometric intensity of LpxC to RpoA in cells without incubation of apidaecin IB was adjusted to 1 and that in cells incubated with apidaecin IB was normalized accordingly. Asterisk indicates p<0.05.
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pone-0020442-g005: Western blot analysis of LpxC in E. coli incubated with apidaecin IB.The relative densitometric intensity of LpxC to RpoA in cells without incubation of apidaecin IB was adjusted to 1 and that in cells incubated with apidaecin IB was normalized accordingly. Asterisk indicates p<0.05.

Mentions: Gram-negative bacteria have two membranes—IM and OM. The IM is a phospholipid bilayer, and the OM is an asymmetrical bilayer consisting of phospholipids and LPS in the inner and outer leaflet, respectively. The synthesis of LPS and phospholipids must be properly balanced, which is critical for cell viability. The same reaction precursor (R-3-hydroxymyristoyl-ACP) is used by LpxC for the biosynthesis of the lipid A moiety of LPS (LpxC catalyzes the first committed step) and by FabZ ((3R)-hydroxymyristoyl-[acyl-carrier-protein] dehydratase) for the synthesis of fatty acid (Figure 4) [19], [20], [21], [22], [23], [24]. Thus the balance of these enzymes is important to maintain a proper LPS/phospholipids ratio. FtsH is the sole, ATP-dependent, growth-essential protease of E. coli [25]. Its essentiality lies in its function in keeping a proper LpxC/FabZ ratio by degrading LpxC [18], [26]. The overproduction of FtsH in this study would probably lead to the changes in the cellular level of LpxC. We therefore analyzed LpxC by Western blotting. The results showed that no significant changes in the amount of LpxC in cells incubated with apidaecin IB for 1 h; however, the amount of LpxC markedly decreased in cells incubated with apidaecin IB for 2 h (Figure 5). We then did LPS and phospholipids analysis. The results showed that the amount of LPS markedly decreased in cells incubated with apidaecin IB for 2 h, in contrast, the amount of phospholipid significantly increased (Figure 6).


iTRAQ-coupled 2-D LC-MS/MS analysis of membrane protein profile in Escherichia coli incubated with apidaecin IB.

Zhou Y, Chen WN - PLoS ONE (2011)

Western blot analysis of LpxC in E. coli incubated with apidaecin IB.The relative densitometric intensity of LpxC to RpoA in cells without incubation of apidaecin IB was adjusted to 1 and that in cells incubated with apidaecin IB was normalized accordingly. Asterisk indicates p<0.05.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3105997&req=5

pone-0020442-g005: Western blot analysis of LpxC in E. coli incubated with apidaecin IB.The relative densitometric intensity of LpxC to RpoA in cells without incubation of apidaecin IB was adjusted to 1 and that in cells incubated with apidaecin IB was normalized accordingly. Asterisk indicates p<0.05.
Mentions: Gram-negative bacteria have two membranes—IM and OM. The IM is a phospholipid bilayer, and the OM is an asymmetrical bilayer consisting of phospholipids and LPS in the inner and outer leaflet, respectively. The synthesis of LPS and phospholipids must be properly balanced, which is critical for cell viability. The same reaction precursor (R-3-hydroxymyristoyl-ACP) is used by LpxC for the biosynthesis of the lipid A moiety of LPS (LpxC catalyzes the first committed step) and by FabZ ((3R)-hydroxymyristoyl-[acyl-carrier-protein] dehydratase) for the synthesis of fatty acid (Figure 4) [19], [20], [21], [22], [23], [24]. Thus the balance of these enzymes is important to maintain a proper LPS/phospholipids ratio. FtsH is the sole, ATP-dependent, growth-essential protease of E. coli [25]. Its essentiality lies in its function in keeping a proper LpxC/FabZ ratio by degrading LpxC [18], [26]. The overproduction of FtsH in this study would probably lead to the changes in the cellular level of LpxC. We therefore analyzed LpxC by Western blotting. The results showed that no significant changes in the amount of LpxC in cells incubated with apidaecin IB for 1 h; however, the amount of LpxC markedly decreased in cells incubated with apidaecin IB for 2 h (Figure 5). We then did LPS and phospholipids analysis. The results showed that the amount of LPS markedly decreased in cells incubated with apidaecin IB for 2 h, in contrast, the amount of phospholipid significantly increased (Figure 6).

Bottom Line: Cell division protease ftsH, an essential regulator in maintenance of membrane lipid homeostasis, was found to be overproduced in cells incubated with apidaecin IB.Its over-expression intensified the degradation of cytoplasmic protein UDP-3-O-acyl-N- acetylglucosamine deacetylase, which catalyzes the first committed step in the biosynthesis of the lipid A moiety of LPS, and thus leaded to the further unbalanced biosynthesis of LPS and phospholipids.Our findings suggested a new antibacterial mechanism of apidaecins and perhaps, by extension, for other proline-rich antimicrobial peptides.

View Article: PubMed Central - PubMed

Affiliation: School of Chemical and Biomedical Engineering, College of Engineering, Nanyang Technological University, Singapore, Singapore.

ABSTRACT
Apidaecins are a series of proline-rich, 18- to 20-residue antimicrobial peptides produced by insects. They are predominantly active against the gram-negative bacteria. Previous studies mainly focused on the identification of their internal macromolecular targets, few addressed on the action of apidaecins on the molecules, especially proteins, of bacterial cell membrane. In this study, iTRAQ-coupled 2-D LC-MS/MS technique was utilized to identify altered membrane proteins of Escherichia coli cells incubated with one isoform of apidaecins--apidaecin IB. Cell division protease ftsH, an essential regulator in maintenance of membrane lipid homeostasis, was found to be overproduced in cells incubated with apidaecin IB. Its over-expression intensified the degradation of cytoplasmic protein UDP-3-O-acyl-N- acetylglucosamine deacetylase, which catalyzes the first committed step in the biosynthesis of the lipid A moiety of LPS, and thus leaded to the further unbalanced biosynthesis of LPS and phospholipids. Our findings suggested a new antibacterial mechanism of apidaecins and perhaps, by extension, for other proline-rich antimicrobial peptides.

Show MeSH
Related in: MedlinePlus