Choose an application

• Reference Manager
• EndNote
• RefWorks (Direct export to RefWorks)

The discovery of antibiotics made it possible to overcome previously deadly infections. However, overuse and often misuse of these antibiotics led to a significant increase in (multi)drug resistant pathogens. One such pathogen, Pseudomonas aeruginosa, is one of the bacteria with the highest priority for which new drugs must be developed according to the World Health Organisation. P. aeruginosa is well-known for establishing biofilms; multicellular communities in which the bacteria are embedded in a matrix of extracellular polymeric substances. These substances include proteins, polysaccharides and extracellular DNA (eDNA) and fulfil several key roles in the biofilm way of life. To explore new strategies to combat P. aeruginosa infections, this biofilm aspect was taken into account. In this dissertation, the recently characterised miniDNase (mDNase), originating from the (bacterio)phage LUZ19, was used to target eDNA. This mDNase has interesting properties such as thermostability and is substantially smaller in comparison to commercially available nucleases. In the first part of this dissertation, alternative expression and purification strategies were tested in an attempt to increase the yield and/or reduce the time needed to produce purified mDNase. A heat shock was shown to effectively separate mDNase from its purification tag without any observed loss of specific activity. Even though this heat shock is time-efficient, the yield of this method should be increased in order to be competitive with the current purification protocol. On the other hand, it was not possible to express the mDNase in the periplasm of its expression host Escherichia coli using signal sequences. In the second part of this dissertation, the potential of the mDNase to target P. aeruginosa PAO1 biofilms was assessed. The mDNase was shown to inhibit biofilm formation in the same extent as the golden standard in this field, DNase I from bovine pancreas (bDNase). Furthermore, mDNase was also able to disperse 24 h old biofilms. Subsequently, an attempt was made to kill biofilm populations by combining the mDNase with enzyme-based antibacterials, called Artilysins. This indicated a potential synergy between these proteins with complementary properties, namely dispersion (mDNase) and killing (Artilysin) of bacterial populations. Finally, these two complementary modules were fused together by means of several peptide linkers. These fusion proteins were screened for nuclease, antibacterial and biofilm inhibitory activity. At least one of the assembled constructs was observed to possess all three activities in an initial screen and can therefore be considered as a promising combination. Further investigations will tell whether the promising combinations can be used to develop new antibacterial strategies.