Cellular Uptake of Apidaecin 1b and Related Analogs in Gram-negative Bacteria Reveals Novel Antibacterial Mechanism for Proline-rich Antimicrobial Peptides

Author(s): Nicole Berthold, Ralf Hoffmann.

Journal Name: Protein & Peptide Letters

Volume 21 , Issue 4 , 2014

Abstract:

Proline-rich antimicrobial peptides (PrAMPs) freely penetrate through the outer membrane into the periplasm of Gram-negative bacteria, before they are actively translocated by a permease/transporter-mediated uptake into the cytoplasm where they are reported to inhibit chaperone DnaK. Here we have studied the PrAMP apidaecin 1b, which is produced in honey bees in response to bacterial infections, and optimized apidaecin analogs for their bacterial uptake. The peptides were labeled with 5(6)-carboxyfluorescein and their internalization in Escherichia coli and Klebsiella pneumoniae was visualized by fluorescence microscopy and quantified by flow cytometry for four different time points over an incubation period of 4 h. Apidaecin 1b entered only 40% to 50% of the cells at detectable quantities, whereas designer peptides Api88, Api134 and Api155 entered more than 95% of the bacteria within 30 min at around fourfold higher quantities than the native peptide. Interestingly, a shortened version designated as 1-17Api88, bound DnaK as efficiently as the 18-residue long Api88 and entered the bacteria at similar kinetics as Api88, but was unable to inhibit the bacterial growth. Similar conflicts with currently proposed mechanisms of PrAMPs were also obtained for some Ala-substituted analogs and reverse apidaecin sequences. Although peptides with C-terminal amides enter the cells much more efficiently than homologous C-terminal acids, this improved cell penetration does not improve the antibacterial activities. These studies suggest that PrAMPs utilize additional modes of action to kill sensitive organisms.

Keywords: Antimicrobial peptide (AMP), confocal fluorescence microscopy, Escherichia coli, Klebsiella pneumoniae, proline-rich peptides.

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Article Details

VOLUME: 21
ISSUE: 4
Year: 2014
Page: [391 - 398]
Pages: 8
DOI: 10.2174/09298665113206660104
Price: $58

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