Investigation of the bacterial uptake of fluorescently labeled peptides and motility studies using lab-on-a-chip technology

Abstract

Bacterial resistance in pathogens is a worldwide challenge associated with high mortality. The aim was to use microfluidic technology for two different applications in the fight with bacterial resistance. In the first part of the thesis the bacterial uptake of cell penetrating peptides (CPPs) was investigated with the motivation of following the internalization of peptide-based antibacterial drugs. In the second part, the changes in bacterial motility of Bacillus strains were investigated upon treatment with amiloride, with the motivation of live monitoring of drug effects. The outer membrane slows down the entry of available antibiotics into the periplasm. In this regard, CCPs are considered as promising alternatives to conventional antibiotics. Oligopeptide permease (Opp) is a transport system, which has an important role in peptide uptake. By following fluorescence intensity of pVEC and chimeric P4 and P2 peptides, the constructed microfluidic system, confirmed by confocal microscopy, has shown that peptides were able to pass through the inner membrane in wild type cells, while they are accumulated on the periplasm in mutant cells. This result suggested that Opp system can be used as a target to control delivery of antimicrobial peptides into the cytoplasm. Bacterial virulence is in part controlled by the flagellar structures on cellular membranes. The microfluidic system has confirmed that the swimming rate of neutrophilic Bacillus cells slightly decreased, while the alkaliphilic Bacillus cells were totally immotile with amiloride treatment. Reaction and rhombic chambers (zeonor) enabled continuous live cell imaging of the bacterial cells and to study the effects of the peptides/drugs in terms of growth, peptide uptake and flagellar motility.