Interaction of cyclotides and bacteria : A study of the cyclotide action and the bacterial reaction

• The growing problem of antibiotic resistance and the lack of promising prospective antibiotics have forced us to search for new classes of antibiotics. Among the candidates to develop into future antibacterials are antimicrobial peptides (AMPs). Thesepotent, broad spectrum compounds are important components of innate immunity of organism from all kingdoms of life. One such family of mini-proteins from plants is called cyclotides, whose members are defines by cyclic backbone and a cystine knot (CCK), which confers to them extreme stability in the face of biological, chemical and physical insults.    Some cyclotides possess Gram-negative specific antibacterial activity; the purpose of this thesis was to characterize how these molecules kill bacteria, and how bacteria would respond to treatment with cyclotides. For this purpose, Salmonella enterica and Escherichia coli mutants resistant to the cyclotides cycloviolacin O2 and cycloviolacin O19, respectively, were selected. These mutants were characterized by whole genome sequencing, genetic reconstitution, fitness measurements, and cross-resistance studies. These studies identified a number of genetic pathways for resistance development to cyclotides. These mutants displayed variable fitness profiles in laboratory growth media and in mice competition experiments, with some mutants possessing a fitness advantage in mice. Cross-resistance studies resulted in the identification of several cases of cross-resistance and collateral sensitivity between cyclotides and other AMPs/antibiotics.     Antimicrobial effects of cyclotides were assayed in different conditions and in bacterial organisms with different surface characteristics. In addition, immunolocalization experiments were performed to explore the biological distribution of cyclotides in plants and to determine the mechanism of action of cyclotides in bacteria, respectively. Antibodies raised against cyO2 were used for this purpose. Immunohistochemical techniques applied to plant cells, tissues and organs provided the information that cyclotides were distributed in all plant organs, and were found in tissues vulnerable to pathogen attack, and that cyclotides were stored in the vacuoles of plant cells. Immunogold staining of cyclotide treated cells of S. typhimurium, showed effects of cyclotide treatment on the cell envelope components as well as cytoplasm. A higher number of cyclotide molecules was associated with the cell envelope, but a considerable fraction of them penetrated into the cytoplasm.

Keyword

Cyclotides

cycloviolacin O2

cycloviolacin O3

cycloviolacin O19

antimicrobial peptide resistance

Salmonella enterica

Eschericia coli

Viola odorata

Farmakognosi

Pharmacognosy

Publication and Content Type

vet (subject category)

dok (subject category)