In vitro and in vivo analysis of the mode of action of a new Pseudomonas aeruginosa antipersister molecule

The increasing rate of antibiotic resistance, together with the limited number of novel antibacterial compounds, is causing a true public health crisis where treatment options for patients infected with multidrug-resistant bacteria are down to only a few last-line antibiotics. Greatly contributing to the difficult treatment of these bacterial infections is the presence of a small fraction of persister cells; phenotypical variants which are highly tolerant to antibiotic treatment. Persister cells are responsible for the recalcitrant nature of chronic infections, contribute significantly to the antibiotic tolerance of biofilms and evidence is mounting that persister cells may serve as a reservoir for the development of antibiotic resistance. Clearly, there is an urgent need for novel therapies that allow the treatment of multidrug-resistant infections while effectively clearing the antibiotic-tolerant persister fraction. Despite the increasing insights into the molecular mechanisms behind persistence, their apparent redundancy and species-specific mechanisms are hampering the rational development of targeted anti-persister therapies.

One possible way to circumvent this is the use of screenings specifically directed towards non-dividing cells, allowing the identification of novel anti-persister compounds from large chemical libraries. Previous research within the SPI group successfully used this top-down approach to identify SPI009, a propanol-amine derivative capable of decreasing the persister fraction of Pseudomonas aeruginosa in combination with the conventional antibiotic ofloxacin.

This doctoral research project focused on the further characterization of this novel compound and the determination of its mechanism of action. While originally identified in a screening against P. aeruginosa, extensive in vitro testing revealed the ability of SPI009 to directly kill persister cells of several clinically relevant pathogens, including the ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, P. aeruginosa and Enterobacter spp.), Burkholderia cenocepacia and the model organism Escherichia coli. Additionally, the observed antibiotic-independent effect of SPI009 allows successful combination with antibiotics from mechanistically distinct classes to further increase antibacterial activity. SPI009 showed capable of sensitizing bacteria to antibiotic treatment and, strikingly, restores antibiotic sensitivity even in (multidrug) resistant strains. The use of several in vitro set-ups revealed a potent anti-biofilm activity and ability of SPI009 to eradicate intracellular infections while the first in vivo tests demonstrated a significant increase in Caenorhabditis elegans survival upon treatment with the combination therapy of SPI009 and ciprofloxacin. These characteristics greatly increase the clinical potential of our compound as SPI009-based combination therapies have the potential to be employed in a great variety of bacterial infections.

In a second part of this project, we aimed to unravel the mechanism of action of SPI009. This is not only essential for future clinical development but may also increase our knowledge about persister cells and ways to eradicate them, thereby contributing to the development of future anti-persister therapies. A combination of molecular-genetic and cellular approaches was used to demonstrate that SPI009 is capable of directly killing both persister and non-persister cells through extensive membrane damage. High levels of membrane damage result in cell lysis while intermediate permeabilization can significantly increase the sensitivity towards different antibiotics, confirming the potential of SPI009 as a candidate for antibacterial combination therapies.

Within the frame of this PhD, we also explored the anti-persister effects of Art-175 in P. aeruginosa and A. baumannii. Artilysin®s are comprised of a bacteriophage-produced endolysin coupled to a lipopolysaccharide-destabilizing peptide, allowing lysis of Gram-negative cells and, as such, possessing potential anti-persister activity. The demonstrated ability of Art-175 to quickly kill both normal and persister cells makes it an attractive alternative anti-persister therapy to treat bacterial infections in a highly specific manner.

Overall, this PhD project contributes to the development of novel anti-persister therapies through the exploration of the previously described Art-175 as a potential anti-persister strategy and the detailed and extensive characterization and determination of the mechanism of action of the novel anti-persister molecule SPI009. While there are still some hurdles to overcome in the further clinical development of SPI009, this novel compound shows good potential and may serve as a scaffold for the development of future anti-persister therapies.