Antimicrobial and Amyloidogenic Activity of Peptides Synthesized on the Basis of the Ribosomal S1 Protein from Thermus Thermophilus

Controlling the aggregation of vital bacterial proteins could be one of the new research directions and form the basis for the search and development of antibacterial drugs with targeted action. Such approach may be considered as an alternative one to antibiotics. Amyloidogenic regions can, like antibacterial peptides, interact with the “parent” protein, for example, ribosomal S1 protein (specific only for bacteria), and interfere with its functioning. The aim of the work was to search for peptides based on the ribosomal S1 protein from T. thermophilus, exhibiting both aggregation and antibacterial properties. The biological system of the response of Gram-negative bacteria T. thermophilus to the action of peptides was characterized. Among the seven studied peptides, designed based on the S1 protein sequence, the R23I (modified by the addition of HIV transcription factor fragment for bacterial cell penetration), R23T (modified), and V10I (unmodified) peptides have biological activity that inhibits the growth of T. thermophilus cells, that is, they have antimicrobial activity. But, only the R23I peptide had the most pronounced activity comparable with the commercial antibiotics. We have compared the proteome of peptide-treated and intact T. thermophilus cells. These important data indicate a decrease in the level of energy metabolism and anabolic processes, including the processes of biosynthesis of proteins and nucleic acids. Under the action of 20 and 50 μg/mL R23I, a decrease in the number of proteins in T. thermophilus cells was observed and S1 ribosomal protein was absent. The obtained results are important for understanding the mechanism of amyloidogenic peptides with antimicrobial activity and can be used to develop new and improved analogues.

According to fluorescence spectroscopy data: the fluorescence intensity of thioflavin T (ThT) increases from 50 to 80 au. This indicates the presence of amyloids in the peptide preparation GlyGlySarGlyVTDFGVFVEI (G14I). According to electron microscopy data: the (G14I) peptide GlyGlySarGlyVTDFGVFVEI make many thin prefibrills/fibrils, amorphous aggregates of peptides of different sizes. According to fluorescence spectroscopy data: the initial fluorescence intensity of ThT, which decreased from 9 to 4 a.u. indicate the presence of amyloids (which tend to disaggregate) in the peptide preparation GlyGlySarGlyVVEGTVVEVT (G14T). According to electron microscopy data: the (G14T) peptide GlyGlySarGlyVVEGTVVEVT make short dense fibrils.

ThT intensity for peptide R23T
According to fluorescence spectroscopy data: the initial low intensity of ThT fluorescence is about 2 a.u. does not change significantly, which indicates the absence of R23T amyloids.
According to electron microscopy data: aggregates of different sizes and complexes of aggregates are present. There are no fibrils. Table 2. Experimental results of studying the kinetics of coaggregation of samples of amyloidogenic peptides based on the ribosomal S1 protein and the whole S1 protein of T. thermophilus. 1 S1 (0.5 mg/ml) According to fluorescence spectroscopy data: upon joint incubation of the S1 protein and V10T peptide, the fluorescence intensity of Thioflavin T (ThT) increases from 6 to 6 in a 1:1 ratio and from 6 to 120 a.u. in a ratio of 1:5. This indicates the presence of amyloids, which tend to disaggregate.

Supplementary
According to electron microscopy data: The V10T peptide and the S1 protein form aggregates. Thus, it is noticeable that these aggregates have morphological differences (the components of the aggregates of the V10T peptide are slightly smaller than those of the S1 protein).
In the mixture of the V10T peptide and S1 protein in a 1:1 ratio, only aggregates are present.
In the mixture of the V10T peptide and S1 protein in a ratio of 5:1, both aggregates and fibrils are present.