Characterization of the antibacterial properties of human amyloid proteins and antimicrobial peptides based on bacterial membrane-binding domains.

Examensarbete för masterexamen
Master's Thesis
Biotechnology (MPBIO), MSc
Qvist, August
Antibiotic resistance is one of the largest current threats to public health and food security, being directly responsible for 1.27 million deaths worldwide in 2019 and is estimated to rise to 10 million deaths per year by 2050. Therefore, there is an urgent need to find new treatments against resistant infections. This can be done by finding new antibiotic compounds with new mechanisms of action or through optimizing and potentiating existing antibiotics. The antimicrobial mechanisms of action for the human proteins S100A12 and α-synuclein, and 18 peptides derived from peripheral membrane proteins of Mycobacterium tuberculosis were investigated. Antimicrobial activity of each protein and peptide was assayed by determining the minimal inhibitory concentration (MIC). The 18 peptides derived from M. tuberculosis were screened for outer membrane permeabilization effects on Gram-negative bacteria (Escherichia coli) and mycobacteria (Mycolicibacterium smegmatis and Mycobacterium marinum) using the fluorescence probe 1-N-phenylnaphtylamine (NPN). The NPN assay was adapted from the E. coli protocol and investigated for use in mycobacteria. The fluorescent probe 3,3′- dipropylthiadicarbocyanine (DiSC35) was used in a single cell fluorescence microscopy assay to investigate effects on membrane polarization, which can indicate activity toward the plasma membrane. To determine the resistance frequency of the peptides a disk diffusion assay was used. The disk diffusion plates were checked for colonies inside of the inhibition zone after 24 and 48 hours. For S100A12 and α-synuclein-treated Bacillus subtilis grown in a minimal medium, no MIC or significant change in growth could be observed at the tested concentrations. Addition of calcium ions or copper ions to the proteins did not change the growth or MIC. This suggests that these proteins do not have any antimicrobial effect on B. subtilis under the tested conditions. However, some older batches of the S100A12 protein showed a decrease in growth rate suggesting some antimicrobial activity, likely caused by a degradation product or toxic oligomer formation. The antimicrobial activity of the OMPPs ranged from no activity to relatively high activity. The NPN assay in E. coli showed that out of the eighteen peptides, six showed no outer membrane permeabilization, and six peptides showed outer membrane permeabilization of 20% or higher at 10 μg/ml. With the DiSC35 assay, treating E. coli with the peptides for 15 minutes before imaging, an increase in fluorescence emission could be seen for the peptides that also showed a higher outer membrane permeabilization. However, no significant change in fluorescence of the dye could be seen for most peptides. The increased fluorescence of DiSC35 is likely caused by an increase in uptake of the dye due to increased permeability of the outer membrane. The disk diffusion assay showed no colonies within the inhibition zone after 24 or 48 hours indicating low frequency of resistance development towards the peptides. The collective results of the screenings in E. coli showed that two peptides may be selective outer membrane permeabilizers and should be investigated further at different concentrations. The rest of the peptides showed other or unspecific modes of action that may be of interest as antimicrobials and should also be investigated further. In mycobacteria, using NPN to assess outer membrane permeabilization needs to be further developed and optimized to produce proper results, as no reliable positive control is known. Further assays should be used to evaluate the activity of these peptides in mycobacteria.
Antibiotics , mechanism of action , antimicrobial peptides , S100A12 , α-synuclein , outer membrane permeabilization , NPN , DiSC35 , Mycobacterium tuberculosis.
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