The T6SS as a search engine for naturally validated antibacterial targets

Bacterial resistance has emerged to all clinically relevant antibiotics. Despite the widespread recognition of this as a global crisis the majority of the strategies implemented thus far have been disappointingly unsuccessful, notably those based on rational drug design and target identification approaches. One fundamental and key issue is indeed to define what a good antimicrobial target is so that it would effectively be amenable to drug design. Over billions of years microbes have evolved the best ways to out-compete others to gain access to space and nutrients. They have identified bacterial Achille's heels and designed strategies to inactivate corresponding molecular pathways that would right away challenge bacterial growth and survival. This is what one can consider as naturally validated drug targets.
The bacterial Type VI Secretion System (T6SS) is the ultimate weapon killing competitors by toxins injection. Most characterized T6SS toxins work against cell wall, membrane, nucleic acids, which are all classic targets for most of our current antibiotics. We are at the beginning of understanding how this system works and the characterized T6SS toxins represent only the tip of the iceberg. It is also important to realise that the bacteria that produces T6SS toxins would also produce specific immunity proteins that specifically protect against the activity of each individual T6SS toxin. One key observation is that the gene encoding the toxin and the gene encoding the immunity are found on the bacterial genome as tandem gene pairs next to each other.
This project aims at exploiting the largely overlooked reservoir of natural antibacterials, namely the plethora of T6SS toxins that bacteria have evolved to effectively kill bacterial competitors and which we believe exist in a much larger number and have an unexpected broad range of diverse biochemical activity. While T6SS toxins themselves are unlikely to be effective therapeutics in the short term, the naturally validated antibacterial targets they point us towards would be of great value.
A potent gram-negative bacterium using the T6SS to outcompete and kill foes is Pseudomonas aeruginosa, an organism which is high on the WHO list of pathogens that are critical for Anti-Microbial Resistance (AMR). Here, we will use P. aeruginosa as model to perform a systematic search for T6SS toxins, which are not identifiable using standard genomic and bioinformatic analysis. In previous work my laboratory implemented a genetic screen (TraDIS) to identify transposon mutants sensitive to the T6SS. These mutants are affected in distinct immunity genes and the characterization of the adjacent gene would lead to the identification of a novel T6SS toxin. Our screen was fully validated since it allowed to identify the toxin/immunity pairs which were already known and proved to be very effective at the identification of unsuspected T6SS toxins and their immunities. One of the T6SS toxin which we propose to fully characterize in the present proposal is called Tse8 and is suspected to interfere with protein synthesis, notably by hampering function of the transamidosome complex which leads to a shortage in Asn- and Gln-tRNA.
Finding new toxins and determining their mechanisms of action is going to offer a gold mine of usable antibacterial targets that pharmaceutical companies would be able to consider in the future. Furthermore, understanding how bacteria fight each other in ways we might not suspect is what is needed to prepare translational impact.

The type VI secretion system (T6SS) is an antimicrobial weapon used by Gram-negative bacteria to inject toxins into competitors, restricting their growth to benefit scarce resources. The T6SS toxins, and their cognate immunities, identified so far are phospholipase, DNase or peptidoglycan hydrolase. We consider the T6SS as a gold mine for the search of novel antimicrobial activities since bacteria competed for billions of years and evolved unexpected strategies. Since toxin/immunity (TI) pair genes are only occasionally found genetically linked to T6SS genes, we developed an unbiased approach using transposon mutagenesis and sequencing (TraDIS) to search for T6SS immunity genes in Pseudomonas aeruginosa and characterize the associated T6SS toxins. This was possible using a mutant systematically activating its 3 T6SSs and all associated TI genes as we published recently (Allsopp et al., PNAS, 2017). Our screen is validated by the identification 12 of the known TI pairs, e.g. Tse2/Tsi2; Tle5a/Tli5a. We started to characterize Tse8/Tsi8, which we propose targets the transamidosome and impairs protein synthesis in organisms using this complex to load Asn/Gln on tRNA. We also point at an extra 7 putative novel TI pairs which we believe have original biochemical activity and would direct us toward novel targets. Our proposal shall provide a global vision of the P. aeruginosa's T6SS toxins and antimicrobial strategy which could be mimicked in treatment of resistant bacteria and thus be in the future considered for antimicrobial research.
We took the opportunity of having implemented the TraDIS approach to screen for mutants resistant/sensitive to T6SS attack in an immunity-independent manner. The exploitation of these data combined with a "resistance" evolution approach where serial contact of a T6SS-immunity deficient strain with a T6SS attacker result in acquired resistance would take us into an unexplored and ground-breaking area of the T6SS bacterial warfare.

The beneficiaries of this research are as follows:

1) The UK academic community in the field of molecular microbiology and infection, but also biochemistry and structural biology. The insights gained will be of use to academic researchers interested in developing and applying the general principles of bacterial secretion systems to understanding how they contribute to the molecular basis of human infections and antimicrobial resistance. Furthermore, the broad range of T6SS toxins will be of benefit to evolutionary biologists by providing a clear example of diversification.

2) Pharmaceutical industries and Biotech will also benefit from this research as it will potentially provide novel insight to develop antimicrobial strategies. The T6SS system is conserved within a number of human pathogens and the mechanistic information produced in the research will be applicable to other human pathogens in addition to Pseudomonas aeruginosa. The identification of novel bacterial toxins with novel targets can obviously result in the development of new drugs. The identification of mechanisms which make a bacterium hypersensitive (sensitizers) or resistant (blockers) to the T6SS would also be of interest to further understand the evolution of microbial population within human microbiota which is currently a fast moving and exciting topic with huge impact on human health. Overall and in the long term, the potential development and manufacture of novel anti-infective strategies by European (e.g. Sanofi-Aventis) and UK-based Pharma or Biotech based on T6SS-dependent toxin delivery will be of direct benefit to the European and UK economy as such therapeutics if successful would have a world-wide market.

3) Public sector health care professionals will benefit from the research in terms of an improved knowledge about the cause of P. aeruginosa infections and possible new treatment plans. P. aeruginosa is the 3rd most commonly-isolated nosocomial pathogen accounting for 10% of hospital-acquired infections, with 10,000 cases each year in UK. It has also been ranked by WHO in the top 3 of bacteria which are critical in terms of antibiotic resistance and need immediate attention. The development of novel therapeutic approaches would improve quality of life and health in the UK. Specialist health care workers treating cystic fibrosis (CF) patients would particularly benefit from the work as in late stage CF, the sole microorganism left in CF patient lungs is P. aeruginosa, which is firmly and chronically established and will lead to the patient death.

4) In terms of capacity building and training the research proposal offers Research Co-I, technician, MRes and PhD student an interdisciplinary training in cutting-edge research that will benefit their professional development. As such these staff will be able to apply these skills in the Pharma and Biotech industries as well as in teaching/training or in further higher education research. We therefore anticipate medium term economic benefits arising from a well-trained UK and international research base, reflected in maintaining internationally competitive research-intensive universities and associated industries.

5) The proposed research also provides a compelling narrative for school children and lay persons in relation to the T6SS as an exemplar of a bacterial molecular killing machine involved in bacterial warfare. In fact, our research has been used to benefit a greater public understanding of microbes and their extraordinary ability to compete with each other in a world of trillions of microbes.