Determination of the mode of action of the antibacterial peptide ApoEdp

• Main Author: Okechuku, Adaora
• Other Authors: Roberts, Ian
• Published: University of Manchester 2011
• Subjects: antimicrobial peptides
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.747990

The emergence of multidrug resistant strains of bacteria has resulted in the need for novel therapeutic agents. The ApoEdp peptide, derived from the receptor-binding region of the human apolipoprotein E, had previously been shown to have activity against herpes simplex viruses, human immunodeficiency virus and certain bacterial species. However, its antibacterial mode of action was not elucidated, therefore the present study aimed to determine this mechanism. The susceptibility of several different strains, including Pseudomonas aeruginosa, Staphylococcus aureus, Mycobacterium smegmatis, Staphylococcus epidermidis and Escherichia coli, to ApoEdp was investigated. No significant difference was observed between the minimal inhibitory concentrations (MICs) of ApoEdp against a range of Gram positive and Gram negative bacteria. The presence of E. coli K5 capsular polysaccharide in the growth medium led to a decrease in ApoEdp susceptibility of the non-capsulated E. coli MS101 DeltakfiC strain. Bacteria with non-functioning multidrug efflux pumps showed no difference in susceptibility. A mutation in the phoP gene of Salmonella enterica Serovar Typhimurium LT2, which regulates cell surface modifications led to an increase in ApoEdp susceptibility. Transmission electron microscopy (TEM) images showed changes in the membrane and internal structures of strains incubated with a minimal bactericidal concentration (MBC) of ApoEdp for 5 min. ApoEdp was able to depolarise the cytoplasmic membrane. The ability of ApoEdp to induce cell lysis was assessed by the release of β-galactosidase into the supernatant. There was no significant difference in the supernatant β-galactosidase levels of ApoEdp treated and unlysed cells. ApoEdp, however was able to form pores in artificial lipid bilayers and decrease intracellular ATP levels. The effect of ApoEdp on transcription and translation was determined using an in vitro transcription/translation system. Results showed that ApoEdp did not affect protein synthesis. ApoEdp also worked in synergy with rifampicin, chloramphenicol, ampicillin and ciprofloxacin against bacteria. Overall, the results showed that ApoEdp acts by targeting the cytoplasmic membrane, although it may also have intracellular targets. Its ability to work in combination with conventional antibiotics and antibacterial activity against a range of different bacteria species demonstrates its therapeutic potential.