Analytical and Bioanalytical Chemistry

, Volume 409, Issue 23, pp 5581–5592 | Cite as

Correlating uptake and activity of proline-rich antimicrobial peptides in Escherichia coli

Research Paper
First Online:
Received:
Revised:
Accepted:

Abstract

Increasing death tolls accounted for by antimicrobial drug resistance demand novel antibiotic lead compounds. Among different promising candidate classes, proline-rich antimicrobial peptides (PrAMPs) are very favorable due to their intracellular mechanism, i.e., binding to the 70S ribosome and DnaK, after active uptake relying on bacterial transporters like SbmA and MdtM. Studies on peptide internalization as the first step of their complex mode of action rely typically on fluorophore or radioactive labeling and quantification using microscopy, flow cytometry, or radioactivity. Here, a liquid chromatography based assay was applied to quantify the unlabeled internalized full-length peptides and their proteolytic degradation products (metabolites) using UV absorbance and mass spectrometry. Knockout mutants lacking transporter proteins showed reduced PrAMP uptakes, explaining their reduced susceptibility against PrAMPs. Interestingly, major metabolites produced by bacterial proteases still bound to the 70S ribosome provide evidence that degradation by cytosolic proteases as a possible resistance mechanism is not very efficient.

Graphical abstract

The uptake of unlabeled proline-rich antimicrobial peptides (PrAMPs) is analyzed in Escherichia coli BW25113 wild-type and transporter knockout mutants ΔsbmA and BS2 (ΔsbmA yjiL::Tn10) by reversed-phase chromatography and quantified by UV detection or mass spectrometry with multi-reaction monitoring (scheme right). Internalized peptide amounts correlated to minimal inhibitory concentrations and bacterial transport activities based on the present transporter proteins (scheme left).

Keywords

Apidaecin Label-free quantification Mass spectrometry Oncocin Proline-rich antimicrobial peptide (PrAMP) Reversed-phase chromatography 
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Notes

Acknowledgements

Financial support by the Federal Ministry of Education and Research (BMBF; project no. 01GU1104A), Deutsche Forschungsgemeinschaft (DFG; project no. INST 268/289-1 FUGG), and the European Fund for Regional Structure Development (EFRE; European Union and Free State of Saxony; Nos. 100105139 and 100127675) is gratefully acknowledged. The authors thank Dr. Daniela Volke for acquiring MALDI-MS, Natalja Kabankov for assistance when establishing peptide quantification by RP-HPLC, and Tina Goldbach for technical support in peptide synthesis and fluorescence polarization.

Compliance with ethical standards

Conflict of interest

R.H. is a cofounder of AMP Therapeutics (Leipzig, Germany) and a member of its scientific advisory board. D.K. was a part-time coworker of AMPT. L.H. has no conflict of interest to declare.

Supplementary material

216_2017_496_MOESM1_ESM.pdf (337 kb)
ESM 1 (PDF 337 kb)

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Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  1. 1.Institute of Bioanalytical Chemistry, Faculty of Chemistry and MineralogyUniversität LeipzigLeipzigGermany
  2. 2.Center for Biotechnology and BiomedicineUniversität LeipzigLeipzigGermany

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