The T6SS toxins are powerful weapons for Pseudomonas' antibacterial strategy

Bacteria have evolved plenty of strategies to thrive within harsh environments, to colonize hostile hosts and to compete with other microorganisms in a fierce race for goods and survival. Bacterial pathogens that infect humans can use various strategies. They develop acute infections, which turn fatal in a short period of time, but also establish chronic infections and persist within the host over a lifetime. That is the case for lung infections in cystic fibrosis (CF) patients. At early stages of infection, diverse bacteria are present, e.g. Staphylococcus, Burkholderia or Pseudomonas aeruginosa. At later stages in the patient life the sole microorganism left is P. aeruginosa, which is chronically established and will lead to patient death. The ability of P. aeruginosa to establish chronic infection is not exclusive to CF or pulmonary diseases but also results from contamination of medical devices used in clinical set up and on which P. aeruginosa establish a resilient biofilm barely sensitive to antibiotic treatments. From there it disseminates in the human body and causes high level of morbidity and mortality.
There are multiple reasons for which P. aeruginosa is such a successful colonizer and pathogenic organism. However, one recent discovery pointed to its ability to use a molecular weapon, which facilitates the elimination of bacterial competitors such as in the CF lung colonization. This weapon is the type VI secretion system (T6SS) and has the design of a puncturing device perforating the envelope of target organisms in order to allow injection of lethal toxins. To protect itself from T6SS toxins the bacterium produces an antidote, or immunity, thus preventing self-killing and only aiming at non-self organisms lacking these immunities.
The repertoire of T6SS toxins is currently undervalued and it will be invaluable to decipher the complete T6SS toxin armoury for several reasons. In the first place what makes the T6SS a potent weapon is not the gun but the toxin bullets it fires. How many and how distinct these toxins are will define how potent and efficient in colonization the bacterium is. We will use a completely randomized approach (TraDIS) to identify all T6SS toxin/immunity pairs. Using this knowledge we will be in a position to determine the T6SS profile of any clinical isolates and thus gain knowledge and understanding on why specific isolates may be worse than another. The cartography of the T6SS landscape could be an indicator of the context and severity of the infection. Secondly, the identification of toxins of unknown function could result in the development of new drugs that we desperately need considering the exhaustion of the antibiotic pipeline. In other words a T6SS toxin that kill bacteria by aiming at a yet unexplored target will provide pharmaceutical companies with a novel venue to explore and the possibility for drug design. Since P. aeruginosa possesses three distinct T6SSs, each potentially firing dozens of distinct toxins, the potential for new discoveries is huge and exciting.
In summary, not only this project aims at unraveling the repertoire of T6SS toxin/immunity pairs available to P. aeruginosa, but it also aims (i) at understanding whether these toxins are fired at once and simultaneously by all three T6SS machines, (ii) at visualizing how the T6SS docks onto prey cells and how toxins travels through it, (iii) at visualising the transport of the toxins using super resolution fluorescence microscopy, (iv) at applying this knowledge to clinical isolates from CF patients in monitoring a correlation between high T6SS potency and high capability of host colonisation and persistence. Basic knowledge about the T6SS mechanism is crucial since one can foresee the possibility to elaborate live innocuous organisms for human kind, which could be equipped with a specific T6SS weaponry able to eliminate bacterial pathogens, and this will only be possible by having such in depth understanding.

The T6SS kills bacteria and manipulates microbial populations. Pseudomonas aeruginosa is a Gram-negative pathogen colonizing the CF lungs where it establishes chronically after eliminating competitors. The P. aeruginosa T6SS delivers toxins of which 2 classes are characterized, peptidoglycan hydrolases, phospholipases. The strain producing the toxin (Tse) also makes immunity (Tsi). The ability to outcompete others in a T6SS-dependent manner relies on the diversity of these toxins. Hypothesis-driven approaches identified 6 toxins (Tse1-Tse6) but a larger reservoir exists. We use TraDIS to systematically identify immunity genes as Tn insertion in such genes is possible in a T6SS inactive but not active strain. We confirmed the identity of Tsi1-6 and pointed 4 new potential toxin/immunity pairs. One of 3 T6SSs has been studied extensively in P. aeruginosa, H1-T6SS, since induced in a retS mutant. We showed that H1-, H2- and H3-T6SS are induced in a rsmA mutant and will use this background in a TraDIS approach to characterize the full toxin repertoire. With this knowledge we can map the T6SS toxins content in P. aeruginosa clinical isolates and assess whether the T6SS landscape is a hallmark of infection strategy/disease severity. The TraDIS approach pointed that in a T6SS positive strain some genes are hit with higher frequency. These genes encode membrane proteins, which suggests they may be T6SS receptors. In their absence the strain is less sensitive to T6SS action. This is ignored in the field and source of novel concepts. Using super-resolution fluorescence microscopy we observed GFP-tagged T6SS inducing blebs in membranes suggesting puncturing. Using distinct fluorescent proteins we aim at visualising all 3 T6SSs simultaneously in one cell, imaging docking onto receptors of preys, following T6SS toxin translocation. Our proposal shall provide a global vision of the P. aeruginosa's antimicrobial strategy which could be mimicked in treatment of resistant bacteria.

The beneficiaries of this research are as follows:

1) The UK academic community with interests in molecular microbiology and infection. 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. Furthermore the diversity and distribution of T6SS toxins in a wide range of Pseudomonas aeruginosa isolates will be of benefit to evolutionary biologists by providing a clear example of lateral gene transfer and diversification.

2) Pharmaceutical industries and Biotech will also benefit form 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 P. aeruginosa. The identification of novel bacterial toxins with novel targets can obviously result in the development of new drugs. The identification of novel bacterial toxins and the way they are transported by the T6SS directly into bacterial targets to kill them may also form the basis for the development of live innocuous bacteria which may be used as competitor for human pathogens. Within the infected host such bacteria may serve as kind of "probiotics" contributing to improve human health. The potential development and manufacture of novel anti-infective strategies by European (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. The development of novel therapeutic approaches would improve quality of life and health in the UK. Specialist health care workers treating cystic fibrosis patients (e.g. our collaborators Profs Jane Davies and Eric Alton at the Royal Brompton Hospital) 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) The UK knowledge-based economy will benefit from the interdisciplinary training of the PDRA working in on the research project. Such trained RA (and associated PhD, masters and undergraduate students) are likely to benefit the biotechnology and pharmaceutical industries, as well as the academic base in the UK. 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.