Polydim-I antimicrobial activity against MDR bacteria and its model membrane interaction

<div><p>The rapid spread of multi-drug resistant pathogens represents a serious threat to public health, considering factors such as high mortality rates, treatment restrictions and high prevalence of multi-drug resistant bacteria in the hospital environment. Antimicrobial peptides (AMPs) may exhibit powerful antimicrobial activity against different and diverse microorganisms, also presenting the advantage of absence or low toxicity towards animal cells. In this study, the evaluation of the antimicrobial activity against multi-drug resistant bacteria of a recently described AMP from wasp, Polydim-I, was performed. Polydim-I presented activity against standard strains (non-carriers of multi-resistant genes) that are susceptible to commercial antimicrobials, and also against multi-drug resistant strains at concentrations bellow 1μg/ml (0.41 μM). This is a rather low concentration among those reported for AMPs. At this concentration we found out that Polydim-I inhibits almost 100% of the tested pathogens growth, while with the ATCC strains the minimum inhibitory concentration (MIC₁₀₀) is 400 times higher. Also, in relation to <i>in vitro</i> activity of conventional drugs against multi-drug resistant bacteria strains, Polydim-I is almost 10 times more efficient and with broader spectrum. Cationic AMPs are known as multi-target compounds and specially for targeting the phospholipid matrix of bacterial membranes. Exploring the interactions of Polydim-I with lipid bilayers, we have confirmed that this interaction is involved in the mechanism of action. Circular dichroism experiments showed that Polydim-I undergoes a conformational transition from random coil to a mostly helical conformation in the presence of membrane mimetic environments. Zeta potential measurements confirmed the binding and partial charge neutralization of anionic asolectin vesicles, and also suggested a possible aggregation of peptide molecules. FTIR experiments confirmed that some peptide aggregation occurs, which is minimized in the presence of strongly anionic micelles of sodium dodecyl sulfate. Also, Polydim-I induced channel-like structures formation to asolectin lipid bilayers, as demonstrated in the electrophysiology experiments. We suggest that cationic Polydim-I targets the membrane lipids due to electrostatic attraction, partially accumulates, neutralizing the opposite charges and induces pore formation. Similar mechanism of action has already been suggested for other peptides from wasp venoms, especially mastoparans.</p></div

Inhibition of multi-resistant bacteria strains by Polydim-I and commercial antibiotics, in percentage ± SEM.

<p>Experimental details as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178785#pone.0178785.t002" target="_blank">Table 2</a>. Three independent experiments were performed in triplicate.</p

CD spectra of Polydim-I, obtained at 20 μM concentration, at 25^°C, in different environments.

<p>Spectra are the average of 10 accumulations obtained at 50 nm/min. No smooth has been applied.</p

Polydim-I induced pores in anionic (AZO) bilayers according to the applied voltage (V_hold).

<p>Mean (± SEM), minimum and maximum conductance of pores obtained in 3–6 different experiments for each V_hold condition. Dwell time (time duration of channel aperture) and P_open of pores (probability of pore aperture during a recording, calculated by the sum of time of all apertures under a V_hold divided by the total time of recording in that same V_hold condition) averaged after 3 experiments. Measurements of the single channels currents and duration of apertures were performed using PatchMaster software. Other analyses were performed in GraphPad Prism 5.0 software.</p

Representative recordings of Polydim-I (0.7 to 2 μM) single channel incorporation in AZO bilayers using a Nanion Port-a-Patch device and PatchControl software.

<p>Solution: 150 mM HCl (symmetrical). Arrows indicate some channel apertures or closings. A) V_hold = +100mV, Pore conductances = 265 and 629 pS; B) V_hold = -100mV, Pore conductance = 161 pS; C) Representative current-voltage linear relation of the pores formed by Polydim-I peptide in AZO bilayers (R² = 0,9850; slope of 0.2088). Measurements of the single channels currents and duration of apertures were performed using PatchMaster software. Other analyses were performed in GraphPad Prism 5.0 Software. Six independent experiments were performed.</p

Zeta-potential isotherm obtained at 25^°C, in the absence (ζ₀) and presence of 250 μg/μL AZO vesicles.

<p>A series of buffered peptide solutions, pH 5.5, at increasing concentrations were mixed with an aliquot of AZO vesicles suspension and left for 30 minutes to equilibrate. Error bars represent the standard deviation of 3 measurements.</p

Secondary structure elements (%) of Polydim-I as determined by the deconvolution of CD and FTIR spectra.

<p>Secondary structure elements (%) of Polydim-I as determined by the deconvolution of CD and FTIR spectra.</p

Deconvolution of the amide I band (1600–1700 cm^-1) of the FTIR spectra of Polydim-I in different deuterated environments.

<p>The component peaks were obtained from the second derivatives (not shown) and result from a Gaussian curve fitting. The dashed lines are the experimental FTIR spectra (averaged over 20 scans) after the 13-data point Savitzky-Golay smoothing. The colored lines correspond to the sum of the components. The grey lines represent the deconvolution of the spectra in the different wave numbers. Spectra were acquired at 1 mM Polydim-I, at 25^°C.</p

Inhibition of ATCC bacteria strains by commercial antibiotics, in percentage ± SEM.

<p>Each well received 90 μl of antibiotic solutions plus 90 μl of broth. In each well 20 μl of bacterial suspension was added, resulting in a final volume of 200 μl at the concentration of 10⁵ CFU/ml. The plates were incubated (37°C, 18 h) and turbidity was measured at 595 nm to assess bacterial growth. The results were expressed as inhibition percentage of optical density (OD) against a control in the absence of the control antibiotics. Three independent experiments were performed in triplicate.</p

Polydim-I activity against ATCC bacteria strains (in μg/ml, with 95% confidence limits for MIC₅₀ presented in parentheses).

<p>MICs were determined by 2-fold serial micro dilution in Müeller-Hinton broth in 96-well plates. Each well received 90 μl of peptide plus 90 μl of broth. In each well 20 μl of bacterial suspension was added, resulting in a final volume of 200 μl at the concentration of 10⁵ CFU/ml. The plates were incubated (37°C, 18 h) and turbidity was measured at 595 nm to assess bacterial growth. The results were expressed as inhibition percentage of optical density (OD) against a control in the absence of the peptide. Three independent experiments were performed in triplicate.</p