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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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Effect of FtsH and LpxC overexpression on the growth of E. coli incubated with apidaecin IB.(A) Growth curve of E. coli separately harboring pET-24a, pET-24a/ftsH and pET-24a/lpxC plasmids without apidaecin IB incubation. Each value represents the mean OD readings from two cultures. (B) Growth curve of E. coli separately harboring pET-24a, pET-24a/ftsH and pET-24a/lpxC plasmids with apidaecin IB incubation. Each value represents the mean OD readings from two cultures. (C) Inhibition rate from the 6 h cultures. Inhibition rate was calculated from bacterial OD. Inhibition rate of E. coli harboring pET-24a plasmid was adjusted to 1 and those of cells harboring pET-24a/ftsH and pET-24a/lpxC plasmids were normalized accordingly. Asterisk indicates p<0.05.
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pone-0020442-g007: Effect of FtsH and LpxC overexpression on the growth of E. coli incubated with apidaecin IB.(A) Growth curve of E. coli separately harboring pET-24a, pET-24a/ftsH and pET-24a/lpxC plasmids without apidaecin IB incubation. Each value represents the mean OD readings from two cultures. (B) Growth curve of E. coli separately harboring pET-24a, pET-24a/ftsH and pET-24a/lpxC plasmids with apidaecin IB incubation. Each value represents the mean OD readings from two cultures. (C) Inhibition rate from the 6 h cultures. Inhibition rate was calculated from bacterial OD. Inhibition rate of E. coli harboring pET-24a plasmid was adjusted to 1 and those of cells harboring pET-24a/ftsH and pET-24a/lpxC plasmids were normalized accordingly. Asterisk indicates p<0.05.

Mentions: Moreover, we investigated the characterization of FtsH and LpxC in response to apidaecin IB incubation by using gene-overexpression strains. ftsH and lpxC were separately cloned into a pET-24a vector and expressed in E. coli BL21 (DE3) cells. Cells containing the pET-24a, pET-24a/ftsH and pET-24a/lpxC plasmid were then separately cultured in LB-kanamycin medium. After IPTG induction for 3 h, cell suspensions were diluted to obtain a concentration of 5×105 CFUs/ml and then incubated without and with 1/10 MIC of apidaecin IB. Cell growth was checked by measuring OD600 in interval of 1 h (Figure 7 A, B). The inhibition rate of these bacteria separately harboring pET-24a, pET-24a/ftsH and pET-24a/lpxC was obtained by comparing the OD600 of the 6 h cultures. The results showed that overexpression of FtsH enhanced the inhibition effect of apidaecin IB on cells; in contrast, overexpression of LpxC can significantly alleviate this effect (Figure 7 C). Cellular proteins of the 6 h cultures were also isolated; the LpxC level in ftsH overexpression cells was further analyzed by western blotting. The results indicated that only with the incubation of apidaecin IB, the overexpression of ftsH cause the decrease in the cellular level of LpxC (Figure 8). Collectively, the data suggest that apidaecin IB act against E. coli by overexpressing FtsH to intensify the degradation of LpxC. As R-3-hydroxymyristoyl-ACP is used by both LpxC for the synthesis of LPS and by FabZ for the synthesis of phospholipids, the over-degradation of LpxC will leave more R-3-hydroxymyristoyl-ACP to FabZ, and ultimately lead to the unbalance LPS/phospholipids ratio and the breaking of membrane lipid homeostasis (Figure 4).


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)

Effect of FtsH and LpxC overexpression on the growth of E. coli incubated with apidaecin IB.(A) Growth curve of E. coli separately harboring pET-24a, pET-24a/ftsH and pET-24a/lpxC plasmids without apidaecin IB incubation. Each value represents the mean OD readings from two cultures. (B) Growth curve of E. coli separately harboring pET-24a, pET-24a/ftsH and pET-24a/lpxC plasmids with apidaecin IB incubation. Each value represents the mean OD readings from two cultures. (C) Inhibition rate from the 6 h cultures. Inhibition rate was calculated from bacterial OD. Inhibition rate of E. coli harboring pET-24a plasmid was adjusted to 1 and those of cells harboring pET-24a/ftsH and pET-24a/lpxC plasmids were normalized accordingly. Asterisk indicates p<0.05.
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Related In: Results  -  Collection

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pone-0020442-g007: Effect of FtsH and LpxC overexpression on the growth of E. coli incubated with apidaecin IB.(A) Growth curve of E. coli separately harboring pET-24a, pET-24a/ftsH and pET-24a/lpxC plasmids without apidaecin IB incubation. Each value represents the mean OD readings from two cultures. (B) Growth curve of E. coli separately harboring pET-24a, pET-24a/ftsH and pET-24a/lpxC plasmids with apidaecin IB incubation. Each value represents the mean OD readings from two cultures. (C) Inhibition rate from the 6 h cultures. Inhibition rate was calculated from bacterial OD. Inhibition rate of E. coli harboring pET-24a plasmid was adjusted to 1 and those of cells harboring pET-24a/ftsH and pET-24a/lpxC plasmids were normalized accordingly. Asterisk indicates p<0.05.
Mentions: Moreover, we investigated the characterization of FtsH and LpxC in response to apidaecin IB incubation by using gene-overexpression strains. ftsH and lpxC were separately cloned into a pET-24a vector and expressed in E. coli BL21 (DE3) cells. Cells containing the pET-24a, pET-24a/ftsH and pET-24a/lpxC plasmid were then separately cultured in LB-kanamycin medium. After IPTG induction for 3 h, cell suspensions were diluted to obtain a concentration of 5×105 CFUs/ml and then incubated without and with 1/10 MIC of apidaecin IB. Cell growth was checked by measuring OD600 in interval of 1 h (Figure 7 A, B). The inhibition rate of these bacteria separately harboring pET-24a, pET-24a/ftsH and pET-24a/lpxC was obtained by comparing the OD600 of the 6 h cultures. The results showed that overexpression of FtsH enhanced the inhibition effect of apidaecin IB on cells; in contrast, overexpression of LpxC can significantly alleviate this effect (Figure 7 C). Cellular proteins of the 6 h cultures were also isolated; the LpxC level in ftsH overexpression cells was further analyzed by western blotting. The results indicated that only with the incubation of apidaecin IB, the overexpression of ftsH cause the decrease in the cellular level of LpxC (Figure 8). Collectively, the data suggest that apidaecin IB act against E. coli by overexpressing FtsH to intensify the degradation of LpxC. As R-3-hydroxymyristoyl-ACP is used by both LpxC for the synthesis of LPS and by FabZ for the synthesis of phospholipids, the over-degradation of LpxC will leave more R-3-hydroxymyristoyl-ACP to FabZ, and ultimately lead to the unbalance LPS/phospholipids ratio and the breaking of membrane lipid homeostasis (Figure 4).

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