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Molecular elucidation of three antibacterial proteins from Pseudomonas phage LUZ19 for biotechnological applications

Book - Dissertation

Bacterial viruses or 'bacteriophages' have been proven to be valuable research objects. Modern molecular biology is founded on phage research, providing key insights in fundamental microbiology and resulting in commonly used phage-derived biotechnological tools for research purposes. As effective bacterial killing machines, phages have potential applications to control bacterial growth with applications in health, industrial, food and agricultural settings. Also, phage proteins and derived products have shown promise in the treatment of bacterial infections. Since phages are the most abundant and diversified biological entities on earth, outnumbering bacteria by a factor 10, the phage pool is virtually limitless. Therefore, it is assumed that phages represent a goldmine for novel molecular tools for antimicrobial and biotechnological applications.Pseudomonas aeruginosa is an opportunistic pathogen that can cause life-threatening diseases in immunocompromised patients and persons with burn wounds or cystic fibrosis. Due to its high antibiotic resistance, the bacterium is categorized as 'superbug' and listed by the World Health Organization as top-ranked bacterium with the highest need for new antibiotics. One innovative strategy to develop novel antibacterial drugs against P. aeruginosa is by studying phage proteins that are toxic to the host. These proteins are lethal or very deleterious to the host cells, targetting key bacterial processes in function of host take-over for phage progeny production. However, the functional mechanisms of these proteins are largely unknown and since they show little or no sequence similarity to other proteins, sequence-based predictions are absent. In this regard, this dissertation focuses on the functional genomics of three hypothetical proteins of phage LUZ19 which display antibacterial activity against P. aeruginosa growth.LUZ19 gp4, renamed as 'Qst', was identified to modulate the P. aeruginosa specific quorum sensing system PQS, resulting in toxicity and metabolic reprogramming. This function could be associated with the acetyl-Co metabolism and is achieved by a complex interaction network, in which Qst interacts with both enzymes of cofactor biosynthesis pathways (ThiD and CoaC, for the synthesis of cofactors thiamine pyrophosphate en coenzyme A) and enzymes of two signaling molecule biosynthesis pathways: i) the well-known quorum sensing molecule PQS (biosynthesis enzyme PqsD) and ii) a predicted non-ribosomal peptide (biosynthesis enzyme PA1217). The interacting protein of the latter pathway was also able to neutralize the antibacterial effect of Qst, hinting to a phage-mediated effect on cell signaling. Indeed, it was found that Qst decreased the metabolite levels of quorum sensing molecules PQS and its precursor 2-heptyl-4(1H)-quinolone. Additionaly, its interaction partner PqsD was shown to be essential for normal LUZ19 infection, hinting to a novel host take-over mechanism by phage LUZ19 via quorum sensing interference.LUZ19 gp21 was identified as a nucleic acid binding protein that assembles into mobile foci in bacterial cells. By studying loss-of-toxicity mutant proteins, both features could be directly linked to its antibacterial activity. Although preliminary results of DNA binding tests did not show substrate or sequence specificity in vitro, foci formation might hint to transcription regulation similar to foci formation by transcriptional repressors. This hypothesis is consistent with the observed lack of protein interaction partner and previously observed interference of gp21 in bacterial transcription and translation without affecting replication. However, more research is needed for conclusive evidence towards functional elucidation within its biological context.The small phage protein LUZ19 gp5 (< 9 kDa) shows nuclease activity in vitro, making it the smallest nuclease identified to date. Consequently, the protein was renamed to Pmd, referring to phage-encoded mini-DNase. Its nuclease activity was qualitatively demonstrated by degrading all tested DNA substrate, even when it was fused to another protein, such as GST tag. Pmd is shown to be extremely stable, being active in a broad pH (pH 1-10) and temperature (10-100°C) range and possessing higher tolerance to reducing agents, denaturants and metal chelators compared to the commercially available DNase I. Neverthless, Pmd can be inactivated by metal chelator citrate, hinting to its dependency on metal ions for its activity. Despite the characterized in vitro activity, the biological role of Pmd during phage infection could not be determined yet. Loss-of-toxicity mutant proteins could not be produced. Moreover, the bacteriostatic activity of Pmd calls into question its function in bacterial genome degradation in vivo. In this regard, a viral interaction partner within phage LUZ19 was identified, hypothetical protein gp11, which might help to resolve the biological role of Pmd in the future.From a fundamental biology point of view, interaction partners of all three studied phage proteins were identified, providing first clues towards biological function. Therefore, this research contributes to one of the main challenges in phage biology, namely bridging the gap between the massive amount of annotated phage proteins and their functional annotation. Moreover, this dissertation provides the first identification of a viral protein which is targeting the bacterial quorum sensing system to support lytic phage infection. This is in accordance with the recently reported importance of quorum sensing during viral attack. Additionaly, a completely novel interaction network has been identified in P. aeruginosa, linking quorum sensing to an uncharacterized non-ribosomal peptide synthetase and two cofactor biosynthesis pathways. This shows that the impact of research of hypothetical phage proteins goes well beyond phage biology. Furthermore, gp21 was found to phenotypically resemble transcriptional repressors under the microscope, which would be, to our knowledge, the first transcriptional repressor encoded by a strictly lytic phage. Finally, the characterized nuclease Pmd does not show any sequence similarity to other nucleases and seems to be the smallest identified nuclease so far, providing novel insights into the widely studied biological activity of DNA degradation.Further exploration of the identified phage-host interactions may lead to insights that could aid in the development of new antibacterial therapies. Both Qst and Pmd are interesting study tools for novel antibiofilm drugs, which are applied complementary to antibiotics to enhance bacterial infection treatment. Qst possesses quorum quenching activity, inhibiting signaling pathways involved in biofilm formation. In contrast, Pmd has nuclease activity, implicating that it might be used to digest the extracellular matrix of biofilms. Besides biofilm degradation in medicine, Pmd migth have applications in industrial and laboratory settings. Indeed, its unique combination of properties make it a biotechnologically interesting tool. Therefore, first steps towards biotechnological use of Pmd has been taken during this dissertation, by patenting Pmd for above-mentioned applications. This shows that the tailored research of toxic phage proteins not only provides novel insights into phage biology, but also might provide a basis for innovative phage-derived antibacterials and biotechnological tools.
Publication year:2019