Dataset Information

Inactivation of Bacteria by ?-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 ?-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-?-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 bacteria-induced 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 (??) 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.

SUBMITTER: Correa W

PROVIDER: S-EPMC7019023 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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

Correa Wilmar W Brandenburg Julius J Behrends Jochen J Heinbockel Lena L Reiling Norbert N Paulowski Laura L Schwudke Dominik D Stephan Kerstin K Martinez-de-Tejada Guillermo G Brandenburg Klaus K Gutsmann Thomas T

Biophysical journal 20191018 10

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 act ...[more]

PMID: 31676134

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Project description:Ceragenins are a family of synthetic amphipathic molecules designed to mimic the properties of naturally occurring cationic antimicrobial peptides (CAMPs). Although ceragenins have potent antimicrobial activity, whether their mode of action is similar to that of CAMPs has remained elusive. Here, we reported the results of a comparative study of the bacterial responses to two well-studied CAMPs, LL37 and colistin, and two ceragenins with related structures, CSA13 and CSA131. Using transcriptomic and proteomic analyses, we found that Escherichia coli responded similarly to both CAMPs and ceragenins by inducing a Cpx envelope stress response. However, whereas E. coli exposed to CAMPs increased expression of genes involved in colanic acid biosynthesis, bacteria exposed to ceragenins specifically modulated functions related to phosphate transport, indicating distinct mechanisms of action between these two classes of molecules. Although traditional genetic approaches failed to identify genes that confer high-level resistance to ceragenins, using a Clustered Regularly Interspaced Short Palindromic Repeats interference (CRISPRi) approach we identified E. coli essential genes that when knocked down modify sensitivity to these molecules. Comparison of the essential gene-antibiotic interactions for each of the CAMPs and ceragenins identified both overlapping and distinct dependencies for their antimicrobial activities. Overall, this study indicated that, while some bacterial responses to ceragenins overlap those induced by naturally occurring CAMPs, these synthetic molecules target the bacterial envelope using a distinctive mode of action. IMPORTANCE The development of novel antibiotics is essential because the current arsenal of antimicrobials will soon be ineffective due to the widespread occurrence of antibiotic resistance. The development of naturally occurring cationic antimicrobial peptides (CAMPs) for therapeutics to combat antibiotic resistance has been hampered by high production costs and protease sensitivity, among other factors. The ceragenins are a family of synthetic CAMP mimics that kill a broad spectrum of bacterial species but are less expensive to produce, resistant to proteolytic degradation, and seemingly resistant to the development of high-level resistance. Determining how ceragenins function may identify new essential biological pathways of bacteria that are less prone to the development of resistance and will further our understanding of the design principles for maximizing the effects of synthetic CAMPs.

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Dataset Information

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

Publications

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

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