June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
High affinity LPS-binding branched peptides display synergistic activity with other antibiotics
Author Affiliations & Notes
  • Rajamani Lakshminarayanan
    SINGAPORE EYE RESEARCH INSTITUTE, Singapore, Singapore
    SRP Iin Neuroscience and Behavioral Disorders, Duke-NUS Graduate Medical School, Singapore, Singapore
  • Shouping Liu
    SINGAPORE EYE RESEARCH INSTITUTE, Singapore, Singapore
    SRP Iin Neuroscience and Behavioral Disorders, Duke-NUS Graduate Medical School, Singapore, Singapore
  • Roger Beuerman
    SINGAPORE EYE RESEARCH INSTITUTE, Singapore, Singapore
    SRP Iin Neuroscience and Behavioral Disorders, Duke-NUS Graduate Medical School, Singapore, Singapore
  • Footnotes
    Commercial Relationships Rajamani Lakshminarayanan, None; Shouping Liu, None; Roger Beuerman, Allergan (F), SERI (P), Santen (R)
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 4292. doi:
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    • Get Citation

      Rajamani Lakshminarayanan, Shouping Liu, Roger Beuerman, ; High affinity LPS-binding branched peptides display synergistic activity with other antibiotics. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4292.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: The purpose of this study is to understand the relationship between outermembrane permeability and synergism in two branched peptides, B2088 and B2088_99 which carry two copies of putative sequence RGRKVVRR and RGRKGGRR, respectively.

Methods: The outermembrane permeability of the peptides were analyzed by N-1-phenyl naphthalene (NPN) fluorescence assay. We used various biophysical techniques such as Circular Dichroism spectropolarimetry, fluorescence spectrometry, isothermal titration calorimetry and Surface plasmon resonance to probe the interactions between lipopolysaccharide and peptides.

Results: Both the peptides displayed potent antimicrobial activity against Gram-negative pathogens (2.7 μM). NPN assay suggested that both the peptides permeabilized the outer membrane of P. aeruginosa. B2088 permeabilized more effectively compared to B2088_99. E. coli mutants which lack O-antigen, outer core and inner core polysaccharides were hypersensitive to peptides. BODIPY-cadaverine assay and TEM studies indicated that the peptides bind to Lipid A and disrupt the supramolecular structure of LPS. ITC studies indicated that both the peptides bind to LPS with moderate affinity. Both the peptides were non-haemolytic (~3% haemolysis) up to ~8.5 mM (20 mg/mL). To probe if the permeability of peptides allows other antibiotics to enhance the antibacterial action, we performed checker board assay and estimated the fractional inhibitory concentration index (FICI) against multi-drug resistant P. aeruginosa. Both the peptides displayed synergism/additive action against 8 different commercially available antibiotics.

Conclusions: The results showed that moderate affinity and disruption of LPS supramolecular structure are important for rapid bactericidal action and synergism with other antibiotics.

Keywords: 422 antibiotics/antifungals/antiparasitics • 573 keratitis • 765 wound healing  
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