Membrane permeability and antimicrobial kinetics of cecropin P1 against Escherichia coli

Abstract

The interaction of cecropin P1 (CP1) with Escherichiacoli was investigated to gain insight into the time-dependent antimicrobial action. Biophysical characterizations of CP1 with whole bacterial cells were performed using both fluorescent and colorimetric assays to investigate the role of membrane permeability and lipopolysaccharide (LPS) binding in lytic behavior. The kinetics of CP1 growth inhibition assays indicated a minimal inhibitory concentration (MIC) of 3 microM. Bactericidal kinetics at the MIC indicated rapid killing of E.coli (<30 min). Membrane permeability studies illustrated permeation as a time-dependent event. Maximum permeability at the MIC occurred within 30 min, which correlates to the bactericidal action. Further investigation showed that the immediate permeabilizing action of CP1 is concentration-dependent, which correlates to the concentration-dependent nature of the inhibition assays. At the MIC and above, the immediate permeability was significant enough that the cells could not recover and exhibit growth. Below the MIC, immediate permeability was evident, but the level was insufficient to inhibit growth. Dansyl polymyxin B displacement studies showed LPS binding is essentially the same at all concentrations investigated. However, it does appear that only the immediate interaction is important, because binding continued to increase over time beyond cell viability. Our studies correlated CP1 bactericidal kinetics to membrane permeability suggesting CP1 concentration-dependent killing is driven by the extent of the immediate permeabilizing action of the peptide.

The structure and behavior of the NA-CATH antimicrobial peptide with liposomes

“…The region encompassing these charged residues adopts a helical confirmation. The NA-CATH structure appears to be similar to previous straight α-helical AMPs and there is strong evidence supporting a membrane disruption behavior via membrane thinning, defects, transient pores, and carpet model dissolution [20,23,32,88]. Our fluorescence and microscopy data indicate that NA-CATH functions in a similar manner.…”

Transmission Electron Microscopic Morphological Study and Flow Cytometric Viability Assessment ofAcinetobacter baumanniiSusceptible toMusca domesticacecropin

“…The results revealed that Mdc can mediate rapid killing of MDR- A. baumannii in 30 min and resulted in log orders of cell complete lysis at a concentration as low as 4  μ g/mL (Figure 2). These observations were in agreement with previous results obtained in other multidrug-resistant strains of bacteria with antimicrobial peptides [17]. …”

“…Many of these belong to the family of cecropin peptide [16]. Previous studies in cecropin and its related peptides demonstrate that formation of membrane-spanning pores that disrupt the cell membrane of the bacteria seems to be the most likely mechanism [17, 18]. However, the details surrounding the mechanism of bactericidal still need to be elucidated.…”

A New Synthetic Peptide Having Two Target of Antibacterial Action in E. coli ML35

“…Figure 4 shows that dead bacteria and those treated with peptide (4x MIC) incorporated PI (63.5%), whilst bacteria without treatment did not incorporate PI. The AMP cecropin, which has been reported to induce inner membrane perturbation (Chen et al, 2003; Arcidiacono et al, 2009), induced high PI incorporation (83.75%) ( Figure 4 ).…”

“…Maximum permeability was determined by evaluating cells pre-treated with cecropin (3 μM). Permeability rate was evaluated by ONPG hydrolysis, measuring absorbance at 405 nm in 30 min intervals for up to 3 h (Arcidiacono et al, 2009). …”