Inactivation of Bacteria by γ-Irradiation to Investigate the Interaction with Antimicrobial Peptides

Abstract

The activity of antimicrobial peptides (AMPs) has been investigated extensively using model membranes composed of phospholipids or lipopolysaccharides in aqueous environments. However, from a biophysical perspective, there is a large scientific interest regarding the direct interaction of membrane-active peptides with whole bacteria. Working with living bacteria limits the usability of experimental setups and the interpretation of the resulting data because of safety risks and the overlap of active and passive effects induced by AMPs. We killed or inactivated metabolic-active bacteria using g-irradiation or sodium azide, respectively. Microscopy, flow cytometry, and SYTOX green assays showed that the cell envelope remained intact to a high degree at the minimal bactericidal dose. Furthermore, the tumor-necrosis-factor-a-inducing activity of the lipopolysaccharides and the chemical lipid composition was unchanged. Determining the binding capacity of AMPs to the bacterial cell envelope by calorimetry is difficult because of an overlapping of the binding heat and metabolic activities of the bacteriainduced by the AMPs. The inactivation of all active processes helps to decipher the complex thermodynamic information. From the isothermal titration calorimetry (ITC) results, we propose that the bacterial membrane potential (Dj) is possibly an underestimated modulator of the AMP activity. The negative surface charge of the outer leaflet of the outer membrane of Gram-negative bacteria is already neutralized by peptide concentrations below the minimal inhibitory concentration. This proves that peptide aggregation on the bacterial membrane surface plays a decisive role in the degree of antimicrobial activity. This will not only enable many biophysical approaches for the investigation between bacteria and membrane-active peptides in the future but will also make it possible to compare biophysical parameters of active and inactive bacteria. This opens up new possibilities to better understand the active and passive interaction processes between AMPs and bacteria.

Application of Biophysical Techniques to Investigate the Interaction of Antimicrobial Peptides With Bacterial Cells

“…Fluorescence spectroscopy using labeled peptides is used to determine the binding stoichiometry and binding constant of peptides to cells. Studies reported for TBKKG6A labeled with nitrobenzodiazole show that this peptide binds to E. coli cells in a 1 × 10 6 :1 (peptide:cell) stoichiometry; similar values are reported in the literature for other peptides ( 29 – 31 ). The binding constant of TBKKG6A to cells, expressed as a dissociation constant, reveals a very tight binding.…”

“…Solid state 31 P NMR is used to examine the interaction of peptides with the phospholipids of live bacteria. As the number of phosphate groups in a bacterial membrane is estimated to overwhelm that found in bacterial RNA, 31 P is a suitable probe of membrane dynamics. The first paper on this topic dates back to 2000 and reports on the interactions of caerin 1.1, caerin 4.1, and maculatin 1.1 with the Gram-positive bacteria B. cereus and S. epidermidis.…”

“…Using labeled peptides, it is possible to correlate permeabilization and internalization kinetics. Time-resolved FCAs performed on Rhodamine B labeled Ctn and Ctn (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34) show that binding and internalization processes occur until an equilibrium is reached; Ctn(15-34) uptake is faster and precedes bacterial membrane damage, suggesting that permeabilization occurs only upon achievement of a threshold surface concentration (33). In line with this, single cell images show that both peptides are mostly localized on the E. coli surface, but a small percentage co-localizes with the dye SYTOX green, suggesting a partial internalization.…”

Irradiated Non-replicative Lactic Acid Bacteria Preserve Metabolic Activity While Exhibiting Diverse Immune Modulation

“…We observed an interesting, varied mosaic of statistically significant differences in the regulation of some of the key genes involved in immune modulation. This variability can be justified by the strain-dependent response to inactivation methods among LAB, as for bacteria in general, documented in previous studies, especially on radiation resistance ( 28 ), structure composition ( 31 ), immune stimulation ( 25 ), and adhesion abilities ( 23 ), just to name a few. For example, IFNα, mostly involved in antiviral activity ( 44 ), was downregulated by heat-killed Lc.…”

“…This leads to inactivated bacteria which is defined as “metabolically-active non-replicative,” potentially preserving all (or most of) the characteristics of the live bacterium while guaranteeing a high level of safety. Evidence showed that gamma irradiation successfully protected surface antigens and cell composition of bacteria compared to other means of inactivation (e.g., heat treatment) ( 31 ). Despite the great potential of this technology, few studies in the literature explored the application of gamma irradiation to generate paraprobiotics.…”