PAT6SS · Bacterial warfare: Investigation into the Type VI secretion system-associated toxin repertoire and mechanism of target cell puncture in Pseudomonas aeruginosa

Bacterial warfare: Investigation into the Type VI secretion system-associated toxin repertoire and mechanism of target cell puncture in Pseudomonas aeruginosa

Publishable summary (wide audience) Pseudomonas aeruginosa is a human pathogen which causes life-threatening infections. While this bacterium possesses a wide range of virulence mechanisms, the secretion systems play a key role in delivering effectors, toxins and other virulence factors from the bacteria into the environment or target host cells. In recent years, a new secretion system, the Type VI secretion system (T6SS), was shown to be important for P. aeruginosa interbacterial competition. P. aeruginosa has three T6SSs encoded in its genome. One of these systems, the H1-T6SS, is important for P. aeruginosa to compete with other bacteria by either killing or inhibiting their growth. To do this the H1-T6SS is involved in the secretion of at least seven bacterial effectors, Type-VI secreted effectors 1-7 (Tse1-7). Identification and characterisation of additional T6SS-associated toxins, as well as the precise mechanisms of T6SS target-cell recognition, membrane puncturing and toxin delivery will be important in understanding P. aeruginosa virulence. Project objectives This project will identify novel H1-T6SS toxins, as well as investigate the mode of T6SS action using three approaches: Objective 1. To identify and characterise additional H1-T6SS toxins and immunity proteins in P. aeruginosa. Objective 2. To understand how the T6SS exports effector proteins Objective 3. To examine the puncture and delivery of effectors by the H1-T6SS into target cells in situ Project Results Achieved Objective 1: High density transposon mutant libraries were generated in T6SS active and inactive backgrounds in P. aeruginosa strain PAK. These were then deep sequenced to identify known and novel T6SS toxins. Of the novel toxins identified we have followed up on one PA4163, now re-named Tse8, and shown that this toxin interacts with the GatABC transamidosome complex, to selectively inhibit protein synthesis in bacteria which rely solely upon this enzyme complex for tRNA loading of asparagine and glutamine. We are finalizing the characterization of the interaction of Tse8 with the transamidosome to complete to work for publication in a high impact journal. The mode and target of action of Tse8 points to a novel antimicrobial target. With this in mind we are in communication with Imperial Innovations regarding the IP aspects of this project. Additionally, due to the high density and saturation of the transposon insertions in the generated libraries we have also been able to identify three putative T6SS receptor targets. As there are currently no known T6SS receptor targets this adds an additional dimension to the already highly successful TraDIS-based approach we have used. To obtain more a more significant fold change in transposon insertions we will use a library background which is more sensitive to T6SS killing (PAKretStsei1-3) to lead us to the best putative receptor targets. Objective 2: As mentioned in the last periodic report, due to the outstanding success of the global approach in objective 1 to identify novel T6SS toxin/immunity pairs and the success of objective 3 in visualising the membrane puncturing action of the T6SS, this objective is not a key contributor to providing us with novel insight into the T6SS toxin repertoire nor into the role of different T6SS components in toxin secretion. Hence, our focus in this past year has been on finalising objectives 1 and 3. Objective 3: Fluorescent microscopy has been successfully utilised to visualise the action of the P. aeruginosa T6SS in situ. This has involved using a fluorescently-tagged T6SS-sfgfp protein fusion in a number of different P. aeruginosa strain backgrounds to visualise T6SS membrane puncturing and secretion in situ. We have been utilising an extremely powerful state-of-the-art technique, 3D-structured illumination microscopy (3D-SIM), in conjunction with super-resolution microscopy to visualise these aspects of P. aeruginosa biology. This work has allowed us to achieve a previously unexplored level of visual resolution into the mechanism of T6SS membrane puncture. We submitted a manuscript based upon these results to Nature Microbiology which was sent out for review. Unfortunately, the manuscript was not accepted and we are currently in the process of finalising a few experiments and reformatting the manuscript for re-submission. Expected final results and their potential impact and use The results obtained in the project will, once published, have a high impact within the field. We have utilised a cutting edge technique to identify novel T6SS toxin-immunity pairs, as well as putative T6SS receptor components. The target and mode of action of our Tse8 toxin is extremely novel and may lead to development of novel antimicrobials. Additionally, we have been able to observe, in super-resolution detail, the potential mode of action of the T6SS in P. aeruginosa. Understanding how the T6SS acts will allow us to not only potentially target an important virulence factor of this pathogen, but also utilise this knowledge to potentially harness the T6SS for delivery of therapeutics. As mentioned above, one high impact publication is in preparation based upon the Tse8 work, and an additional high impact paper is being reworked based upon the T6SS mode of membrane puncture. Additionally, this work has been presented at a number of international and national meetings to disseminate the results. Potential Impact As the three T6SS of the human pathogen P. aeruginosa have been implicated in virulence of humans, mice, rats and plant models this is an exciting area of research that will have many fruitful discoveries. In addition these systems appear to be involved in interbacterial competition. This is particularly timely given the threat of antibiotic resistant microbes. If these systems can be correctly harnessed, these bacteria and their T6SS could be deployed as nano-soldiers in microbial warfare enabling the treatment of resistant infections.

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