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The antimicrobial activity and mechanism of action of recombinant Ib-AMP4, an antimicrobial peptide from Impatiens balsamina

Fan, Xiaobo

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Abstract

Drug resistant microbes have become widely prevalent and are a large clinical burden. Infectious diseases have again become life threatening and affect millions of people each year because of the rapid evolution of drug resistance. Therefore, the development of antibiotics with novel antimicrobial mechanisms of action is highly prioritized. AMPs are antimicrobials with potential to fulfill this need because of their differences from traditional antibiotics. Ib-AMP4 has been isolated from Impatiens balsamina and exhibits broad antimicrobial activity against plant pathogens. Large amounts of Ib-AMP4 were prepared by expressing the peptide in Escherichia coli. Bacteria showing resistance to current antibiotics are susceptible to AMPs. No cross-resistance was observed between present antibiotics and AMPs. Ib-AMP4 has shown promising antimicrobial effects against human pathogens, including the most prevalent MDR species of MRSA and ESBL-producing E. coli. When combined with other antimicrobials, Ib-AMP4 restored the susceptibility of MDR strains and greatly reduced the amount of antimicrobial agents required for efficacy. However, the antimicrobial efficiency of Ib-AMP4 was also largely affected by cations, especially divalent cations. To determine the mechanism of action underlying the antimicrobial activity of Ib-AMP4, LPS from bacterial cell walls and lipids from biomembranes were used to construct an in vitro model system. We found that in the presence of Ca2+, our model system of the LPS-Re monolayer absorbed Ca2+ and became increasingly compact. Bacterial pathogens used Ca2+ to repel Ib-AMP4 electrically, thereby reducing cell wall permeability to Ib-AMP4. The cytoplasmic membrane is the primary target of Ib-AMP4’s rapid bactericidal effect. Liposome leakage assays and QCM-D indicate that Ib-AMP4 may intercalate into the DOPC bilayer membrane, thereby resulting in liposomal leakiness, surface swelling, and wrinkling. The development of pores develop within the lipid bilayer is still unknown. However, pore development most likely follows the “sinking raft” model because the Ib-AMP4 structure includes a large hydrophilic headgroup coupled to a short and small hydrophobic tail. After the initial insertion of Ib-AMP4, a rapid and intensive leakage occurred for approximately 20 min. Along with this leakage, the insertion of Ib-AMP4 continued in a “self-promoting” pattern, in which the initial insertion of Ib-AMP4 induced further insertions of Ib-AMP4. Surface pressure, line tension, and curvature strain were likely involved in the formation and resealing of the pores. Studies using single DOPC and DOPC/DOPG binary monolayers in calcium-loaded and -free buffers indicated that the charge properties of the lipids and cations in the buffer affect the insertion of Ib-AMP4 regardless of surface pressure. Cholesterol may inhibit calcein leakage caused by Ib-AMP4 by changing the membrane’s compressibility and curvature. Cations may also inhibit Ib-AMP4 insertion into membranes either by interrupting the conformational change of Ib-AMP4 during the insertion stage or by electrically repelling Ib-AMP4. Such inhibition is similar to that with cell wall LPS.

Item Type: Dissertation
Supervisor: Wink, Prof. Dr. Michael
Date of thesis defense: 16 December 2014
Date Deposited: 19 Dec 2014 06:53
Date: 2014
Faculties / Institutes: The Faculty of Bio Sciences > Institute of Pharmacy and Molecular Biotechnology
Subjects: 570 Life sciences
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