Combating multidrug-resistant Gram-negative bacteria with structurally nanoengineered antimicrobial peptide polymers

Abstract

With the recent emergence of reports on resistant Gram-negative ‘superbugs’, infections caused by multidrug-resistant (MDR) Gram-negative bacteria have been named as one of the most urgent global health threats due to the lack of effective and biocompatible drugs. Here, we show that a class of antimicrobial agents, termed ‘structurally nanoengineered antimicrobial peptide polymers’ (SNAPPs) exhibit sub-μM activity against all Gram-negative bacteria tested, including ESKAPE and colistin-resistant and MDR (CMDR) pathogens, while demonstrating low toxicity. SNAPPs are highly effective in combating CMDR Acinetobacter baumannii infections in vivo, the first example of a synthetic antimicrobial polymer with CMDR Gram-negative pathogen efficacy. Furthermore, we did not observe any resistance acquisition by A. baumannii (including the CMDR strain) to SNAPPs. Comprehensive analyses using a range of microscopy and (bio)assay techniques revealed that the antimicrobial activity of SNAPPs proceeds via a multimodal mechanism of bacterial cell death by outer membrane destabilization, unregulated ion movement across the cytoplasmic membrane and induction of the apoptotic-like death pathway, possibly accounting for why we did not observe resistance to SNAPPs in CMDR bacteria. Overall, SNAPPs show great promise as low-cost and effective antimicrobial agents and may represent a weapon in combating the growing threat of MDR Gram-negative bacteria.

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Figure 1: Synthesis of SNAPPs.
Figure 2: In vivo efficacy of SNAPP S16 in a mouse peritonitis model.
Figure 3: Optical Microscope eXperimental 3D-SIM images of E. coli before and after treatment with AF488-tagged SNAPP S16 in MHB.
Figure 4: Morphological studies of E. coli before and after treatment with S16 in MHB.
Figure 5: A comparison between the antimicrobial mechanism(s) of typical membrane-disrupting cationic AMPs and the possible mechanism of SNAPPs against Gram-negative bacteria.

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Acknowledgements

G.G.Q. acknowledges financial support from the Australian Research Council under the Future Fellowship (FT110100411) scheme. E.C.R. acknowledges financial support from the Australian Government, Department of Industry, Innovation and Science. S.J.L. acknowledges the Australian Government for providing an International Postgraduate Research Scholarship (IPRS) and an Australian Postgraduate Award (APAInt). The authors thank the Advanced Fluorescence Imaging Platform at the Materials Characterisation and Fabrication Platform (The University of Melbourne) for instrument access. The authors thank B. Hibbs for assistance with the Delta Vision OMX V4 BLAZE, S. Lowe for technical laboratory assistance and J. Li for the CMDR bacterial strains.

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G.G.Q., N.M.O'B.-S., A.B. and E.C.R. oversaw the project. S.J.L. synthesized and characterized the polymers, performed the in vitro and imaging experiments and wrote the paper, with intellectual input from E.H.H.W., A.S. and A.B. N.P. contributed to the in vitro experiments. J.C.L. contributed to the in vivo animal models and immune cell phenotyping and in vitro experiments. J.A.H. contributed to the RT-PCR and in vitro experiments. Y.-Y.C. contributed to the cryo-TEM experiments. All authors gave suggestions to improve the presentation of the paper.

Corresponding authors

Correspondence to Eric C. Reynolds or Greg G. Qiao.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Materials and Instrumentation, Supplementary Figures 1–36, legends for Supplementary Videos 1 and 2, Supplementary Tables 1–11, Supplementary References. (PDF 5572 kb)

Supplementary Video 1

A sample 3D view of an E. coli cell that demonstrates the membrane association of SNAPPs after treatment. (MOV 1499 kb)

Supplementary Video 2

A sample 3D view of an E. coli cell that demonstrates the cell internalization of SNAPPs after treatment. (MOV 1648 kb)

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Lam, S., O'Brien-Simpson, N., Pantarat, N. et al. Combating multidrug-resistant Gram-negative bacteria with structurally nanoengineered antimicrobial peptide polymers. Nat Microbiol 1, 16162 (2016). https://doi.org/10.1038/nmicrobiol.2016.162

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