Identification and characterization of interactions between bacteriophage proteins and protein complexes of Pseudomonas aeruginosa

Pseudomonas aeruginosa is a rod-shaped, Gram-negative bacterium which is able to colonize a very wide range of ecological niches, because of its large genome which contains a broad regulatory network to allow fast adaptations to changing environments. This capacity and the production of various virulence factors, allows this opportunistic pathogen to infect humans and cause severe, life-threatening diseases, especially in immunocomprized persons. Its increasing resistance against antibiotics because of both intrinsic and acquired mechanisms, makes this bacterium one of the six members of the ESKAPE-pathogens, which are able to escape current antibiotic therapies. Since the efforts to discover new antibiotic classes experienced an ‘innovation gap’ over the last decennia, there is an urgent need for new strategies to develop novel antibacterial therapies to combat these superbugs.

This search has renewed the interest in bacteriophages, which are the natural enemies of bacteria. Bacterial viruses are widely spread all over the world and are present in a five- to ten-fold excess over their bacterial hosts, making them the most abundant entities on earth (app. 10^30). Through billion years of co-evolution with their specific host, they have developed mechanisms to inhibit, activate and redirect the host metabolism towards efficient phage production. In these processes, protein-protein interactions between phage proteins and bacterial key complexes plays a crucial. Since some of these interactions can be lethal for the bacterial cell, their investigation can be useful in the search for new antibacterial targets.

However, because of the revolution in high-throughput sequencing methods and the ease to isolate new phages, there is an increasing gap between the number of annotated phage genes and their functional annotation. Currently, about 70 % of the annotated phage genes are hypothetical genes of unknown function. Since it has become clear that proteins exert their biological function through interactions and their place in an interaction network, we developed a strategy to identify phage proteins through their interaction with bacterial proteins, likewise revealing hints towards the function of these proteins. Hence, the main goal of this dissertation was to identify and characterize host-phage protein-protein interactions. A strategy that contributes to global phage biology and that is useful is the search for new antibacterial agents.

This project shows that affinity purifications linked to mass spectrometry analyses (AP-MS) is an efficient method to identify host-phage interactions. P. aeruginosa target proteins belonging to eight key complexes which play a role in transcription, DNA replication, fatty acid biosynthesis, RNA degradation and regulation, energy metabolism and cell division, were successfully equipped with a genomically introduced affinity tag. After infecting the engineered P. aeruginosa strains with seven distinct clades of lytic phages, AP-MS allowed the identification of 39 host complex-associated phage proteins. Eight of these showed an inhibitory effect on bacterial growth upon episomal expression, suggesting that these phage proteins are potentially involved in hijacking the host complexes, consequently making them interesting towards drug discovery.

Proteins which bind to the RNA polymerase complex, were purified for five of the seven phages. This demonstrates that the transcription machinery is an important target for bacteriophages. Two proteins were previously confirmed to interact to a specific subunit of the RNA polymerase, providing a proof of concept for the AP-MS strategy. For two other phage proteins, the interaction with the RNA polymerase was proven during this research. Gp8 of phage YuA was found to interact within the first 500 amino acids of the β’ subunit. In contrast, gp12 of phage 14-1 interacts with the α subunit of the RNA polymerase. First in vitro transcription assays predict that this protein inhibits the transcriptional function of the RNA polymerase by its binding. Although the precise mode of action still remains to be determined, this data suggest that gp12 might play a role in the switch from host to phage transcription or from early phase to late phase transcription.

Secondly, this dissertation provided the first identification of a viral protein which is interacting with the RNA degradosome. This protein of giant phage φKZ was therefore termed Dip, ‘degradosome interacting protein’. The tertiary structure of Dip was determined by crystallography, and revealed to be a dimer forming protein with an open claw like structure and a groove which possibly serves as a binding site for its interaction partner. The interaction of Dip with the RNA degradosome was confirmed by several protein-protein interaction techniques and was specified to a very small region of the scaffold domain of the endoribonuclease subunit RNase E (residues 756-775). Since this region is predicted to be involved in RNA binding, the phage protein is probably influencing the binding of RNA to the degradosome and subsequently the catalytic activity of the RNA degradosome on its substrate. In vitro degradation assays indicate that the binding of Dip to RNase E is inhibiting the cleaving activity of the RNA degradosome complex on both bacterial and phage RNA transcripts. Therefore, we hypothesize that a first step in the infection by phage φKZ is the active degradation of the host RNA. In a second step, Dip is produced to inhibit the activity of the RNA degradosome which stabilizes the newly transcribed phage RNA.

This research demonstrates that AP-MS is a good strategy to screen for interspecies interactions between phages and bacteria: (1) by screening a high amount of samples, the method is able to exclude most false positives, (2) AP-MS is able to identify true interactions as was proven with complementary assays for five phage proteins, (3) because the studied interactions are not limited to binary interactions, a wide screen which investigates many interactions by only few manipulations is provided, (4) the screen is unbiased towards annotated genes, and (5) the method also provides information on the intracellular interactions of P. aeruginosa itself.

 In the search for new antibacterial drugs, the AP-MS strategy can serve as a platform to discover new targets to combat drugresistant bacteria or to find new mechanisms to influence currently approved targets. Indeed, this dissertation shows that phages possess a wide range of different strategies to influence their host and that their modes of action seem to be independent of these used for drug therapy. Narrowing down the host target and subsequent site of interaction of an inhibitory phage protein can therefore help focusing the search and rational design of small molecules that can mimic the effect of the phage protein.

Moreover, this research contributes to the functional annotation of the huge amount of hypothetical phage proteins of unknown function, which is a main challenge in phage biology. By revealing their interaction partner, a first hint towards their biological function is provided. Three proteins were characterized in more detail, suggesting totally new functions and mechanisms. This shows that many interesting and unexplored proteins and their function are still waiting to be discovered, and that AP-MS is a successful strategy to do so.