Targeted Genome Editing of Virulent Pseudomonas Phages Using CRISPR-Cas3.

The vast number of unknown phage-encoded ORFan genes and limited insights into the genome organization of phages illustrate the need for efficient genome engineering tools to study bacteriophage genes in their natural context. In addition, there is an application-driven desire to alter phage properties, which is hampered by time constraints for phage genome engineering in the bacterial host. We here describe an optimized CRISPR-Cas3 system in Pseudomonas for straightforward editing of the genome of virulent bacteriophages. The two-vector system combines a broad host range CRISPR-Cas3 targeting plasmid with a SEVA plasmid for homologous directed repair, which enables the creation of clean deletions, insertions, or substitutions in the phage genome within a week. After creating the two plasmids separately, a co-transformation to P. aeruginosa cells is performed. A subsequent infection with the targeted phage allows the CRISPR-Cas3 system to cut the DNA specifically and facilitate or select for homologous recombination. This system has also been successfully applied for P. aeruginosa and Pseudomonas putida genome engineering. The method is straightforward, efficient, and universal, enabling to extrapolate the system to other phage-host pairs.

A SEVA-based, CRISPR-Cas3-assisted genome engineering approach for Pseudomonas with efficient vector curing.

IMPORTANCE: The CRISPR-Cas3 editing system as presented here facilitates the creation of genomic alterations in Pseudomonas putida and Pseudomonas aeruginosa in a straightforward manner. By providing the Cas3 system as a vector set with Golden Gate compatibility and different antibiotic markers, as well as by employing the established Standard European Vector Architecture (SEVA) vector set to provide the homology repair template, this system is flexible and can readily be ported to a multitude of Gram-negative hosts. Besides genome editing, the Cas3 system can also be used as an effective and universal tool for vector curing. This is achieved by introducing a spacer that targets the origin-of-transfer, present on the majority of established (SEVA) vectors. Based on this, the Cas3 system efficiently removes up to three vectors in only a few days. As such, this curing approach may also benefit other genomic engineering methods or remove naturally occurring plasmids from bacteria.

The phage-encoded PIT4 protein affects multiple two-component systems of Pseudomonas aeruginosa.

IMPORTANCE: More and more Pseudomonas aeruginosa isolates have become resistant to antibiotics like carbapenem. As a consequence, P. aeruginosa ranks in the top three of pathogens for which the development of novel antibiotics is the most crucial. The pathogen causes both acute and chronic infections, especially in patients who are the most vulnerable. Therefore, efforts are urgently needed to develop alternative therapies. One path explored in this article is the use of bacteriophages and, more specifically, phage-derived proteins. In this study, a phage-derived protein was studied that impacts key virulence factors of the pathogen via interaction with multiple histidine kinases of TCSs. The fundamental insights gained for this protein can therefore serve as inspiration for the development of an anti-virulence compound that targets the bacterial TCS.

The phage-encoded protein PIT2 impacts Pseudomonas aeruginosa quorum sensing by direct interaction with LasR.

In recent decades, there has been a notable increase in antibiotic-resistant Pseudomonas aeruginosa isolates, necessitating the development of innovative treatments to combat this pathogen. This manuscript explores the potential of different phage proteins to attenuate virulence factors of P. aeruginosa, particularly the type II secretion system (T2SS). PIT2, a protein derived from the lytic Pseudomonas phage LMA2 inhibits the T2SS effectors PrpL and LasA and attenuates the bacterial virulence toward HeLa cells and Galleria mellonella. Using RNAseq-based differential gene expression analysis, PIT2's impact on the LasR regulatory network is revealed, which plays a key role in bacterial quorum sensing. This discovery expands our knowledge on phage-encoded modulators of the bacterial metabolism and offers a promising anti-virulence target in P. aeruginosa. As such, it lays the foundation for a new phage-inspired anti-virulence strategy to combat multidrug resistant pathogens and opens the door for SynBio applications.

Bacteriophage-antibiotic combination therapy against extensively drug-resistant Pseudomonas aeruginosa infection to allow liver transplantation in a toddler.

Post-operative bacterial infections are a leading cause of mortality and morbidity after ongoing liver transplantation. Bacteria causing these infections in the hospital setting can exhibit high degrees of resistance to multiple types of antibiotics, which leads to major therapeutic hurdles. Alternate ways of treating these antibiotic-resistant infections are thus urgently needed. Phage therapy is one of them and consists in using selected bacteriophage viruses - viruses who specifically prey on bacteria, naturally found in various environmental samples - as bactericidal agents in replacement or in combination with antibiotics. The use of phage therapy raises various research questions to further characterize what determines therapeutic success or failure. In this work, we report the story of a toddler who suffered from extensively drug-resistant Pseudomonas aeruginosa sepsis after liver transplantation. He was treated by a bacteriophage-antibiotic intravenous combination therapy for 86 days. This salvage therapy was well tolerated, without antibody-mediated phage neutralization. It was associated with objective clinical and microbiological improvement, eventually allowing for liver retransplantation and complete resolution of all infections. Clear in vitro phage-antibiotic synergies were observed. The occurrence of bacterial phage resistance did not result in therapeutic failure, possibly due to phage-induced virulence tradeoffs, which we investigated in different experimental models.

Bacteriophages as drivers of bacterial virulence and their potential for biotechnological exploitation.

Bacteria-infecting viruses (phages) and their hosts maintain an ancient and complex relationship. Bacterial predation by lytic phages drives an ongoing phage-host arms race, whereas temperate phages initiate mutualistic relationships with their hosts upon lysogenization as prophages. In human pathogens, these prophages impact bacterial virulence in distinct ways: by secretion of phage-encoded toxins, modulation of the bacterial envelope, mediation of bacterial infectivity and the control of bacterial cell regulation. This review builds the argument that virulence-influencing prophages hold extensive, unexplored potential for biotechnology. More specifically, it highlights the development potential of novel therapies against infectious diseases, to address the current antibiotic resistance crisis. First, designer bacteriophages may serve to deliver genes encoding cargo proteins which repress bacterial virulence. Secondly, one may develop small molecules mimicking phage-derived proteins targeting central regulators of bacterial virulence. Thirdly, bacteria equipped with phage-derived synthetic circuits which modulate key virulence factors could serve as vaccine candidates to prevent bacterial infections. The development and exploitation of such antibacterial strategies will depend on the discovery of other prophage-derived, virulence control mechanisms and, more generally, on the dissection of the mutualistic relationship between temperate phages and bacteria, as well as on continuing developments in the synthetic biology field.