Type VI secretion in Pseudomonas species: bacterial competition and biocontrol

Pseudomonas aeruginosa is a gram-negative bacterium that thrives in a multitude of environmental niches, e.g. soil, moist surfaces or plants. It has the ability to become a successful pathogen and infects a wide range of hosts, e.g. amoeba, worms, insects, plants or animals. It is also a dreadful human pathogen and is known for incurable infections in the lungs of cystic fibrosis patients. Because of this versatility of environments and hosts, P. aeruginosa encounters numerous microorganisms colonizing the same niche and competing for the same resources. It was found that a molecular weapon, called the type VI secretion system (T6SS), plays an important part in the resulting bacterial warfare.
The T6SS is akin a bacteriophage tail, but instead of injecting DNA in prey cells, it injects bacterial toxins. These toxins degrade essential bacterial components, such as the cell wall/envelope or DNA, which results in irreversible damages and cell death. P. aeruginosa has been instrumental in T6SS research and numbers of breakthroughs in the field were made upon studying this organism. The development of T6SS-dependent killing assay showed that the P. aeruginosa T6SS is potent and kills most gram-negative preys. The killing assay allows the monitoring of surviving prey cells in a drop of co-culture deposited on an agar plate. The T6SS attack by P. aeruginosa is not systematic and often occurs when the encountered organism is threatening, for example by exhibiting a T6SS armory, which engages the duel.
The number of T6SS toxins is far more comprehensive than anticipated. Many T6SS toxins have function that could not even be predicted. Yet the injection of these molecules in target bacteria results in death, suggesting that new antibacterial activities are to be found. We begun to unfold the T6SS toxin repertoire in P. aeruginosa and discovered that other Pseudomonas species have an active T6SS and a unique T6SS toxins repertoire. This is the case for Pseudomonas syringae, a plant pathogen, or Pseudomonas putida, a plant beneficial organism. All three Pseudomonas outcompete Escherichia coli in the in vitro killing assay, while P. aeruginosa outcompetes both P. putida and P. syringae. We showed that even though P. aeruginosa outcompetes the plant pathogen Agrobacterium tumefaciens in vitro, upon co-inoculation in planta the power relations in between these organisms is changed and A. tumefaciens prevails. These studies revealed a complex inter-bacterial relationship, in which the T6SS is central but is influenced by the specificity of interactions between organisms and the conditions and environments in which they met.
In our project we investigate how the T6SS influences evolution of bacterial population and composition of polymicrobial communities. We want to build on our expertise and pioneering work in the identification of new T6SS toxins such as the DNase Tde, the characterization of T6SSs in Pseudomonas species and the use of in planta competition assays between T6SS-proficient organisms. We want to monitor the evolution of mixed bacterial populations in a dynamic environment, such as mixed biofilms using flow cells and fluorescent-tagged organisms. Biofilm is a natural lifestyle of complex bacterial populations. Each bacterial strain carries a distinct T6SS repertoire thus giving access to the role/importance of individual T6SS feature in the outcome of biofilm composition.
In conclusion, we will investigate the role of the T6SS in distinct Pseudomonas species using various environmental set-ups, i.e. in vitro, mixed biofilms and in planta assays. The knowledge acquired should result in the characterization of novel antibacterial activities, understanding in the evolution of bacterial population and engineering of plant beneficial P. putida strains for the control of crops diseases. This study will have implications in areas including, Ecology and Agriculture (root colonization), Medicine (antibacterial) and Basic Sciences.

Bacteria developed strategies to adapt to changes while colonizing their niche. They face the encounter of microorganisms competing for the same resources. This either results in one prevailing species or the establishment of a polymicrobial community, such as in the gut.
The type VI secretion system (T6SS) emerged as the ultimate molecular weapon for this warfare. It delivers antibacterial toxins with a broad range of activity, e.g. peptidoglycan hydrolase, phospholipase or nuclease. This results in the deterioration of essential cell components and prey's death.
Pseudomonas aeruginosa thrives in most environments, and is a human and plant bacterial pathogen. P. aeruginosa's T6SS armory is comprehensive and studies with this organism contributed major advances in the field. The T6SS is akin a bacteriophage tail. Toxins can be placed at the tip of the device or loaded in the T6SS tube. Contraction injects them into target cells and siblings are protected by immunity proteins.
We suggested that the P. aeruginosa T6SS toxin repertoire is broader than anticipated. We identified novel T6SS toxins such as the DNase Tde. We showed that the outcome of the T6SS-dependent competition is different when comparing in vitro situation or co-infection of a host, e.g. P. aeruginosa and Agrobacterium tumefaciens in planta. We showed T6SS functionality in the plant pathogen Pseudomonas syringae and the plant growth promoter Pseudomonas putida.
We aim at understanding the T6SS-dependent interactions occurring when distinct species are thriving in the same niche or host. We will study the T6SS toxin repertoire and the specificity of T6SS contact in between species. We will use all three Pseudomonas species as a case study to investigate the evolution of mixed bacterial populations using in vitro, biofilm or in planta set up. We shall further establish basic principles on the role of the T6SS and new ideas to engineer bacterial strains used as biocontrol agent in crops protection.

Pseudomonas aeruginosa is a major cause of hospital-acquired infection, with an estimated 10,000 cases each year in UK. Infection is life-threatening, leading to pneumonia or septicaemia. P. aeruginosa is dreaded by cystic fibrosis (CF) patients. Up to 80% carry the bacteria in their lungs. Whereas patients in their early life carry a polymicrobial population, adults have P. aeruginosa as most exclusive colonizing organism. The establishment of P. aeruginosa in the CF lungs is actually a dramatic example of an unassailable chronic infection during which P. aeruginosa has eliminated other bacterial colonizers, established a resistant biofilm, which ultimately induces the self-inflammatory destruction of the lungs. Studies with P. aeruginosa established that the type VI secretion system (T6SS) is induced in CF patients. The T6SS is an antibacterial weapon used to kill competitors thriving in the same niche. The T6SS is conserved in most gram-negative bacteria and delivers antibacterial toxins into prey cells. The T6SS-dependent tactic is thus instrumental in the process of colonization and can be used as a source of discovery for new antimicrobial strategies. Several T6SS toxins have still an unknown function, which opens the door to the discovery of novel antimicrobial targets.
The T6SS is an intense field of research involving top class laboratories all over the world and Alain Filloux's laboratory made significant contribution in this area using P. aeruginosa as model organism. P. aeruginosa not only is a human pathogen but also a plant pathogen. Our recent work published in Cell Host & Microbes demonstrates that P. aeruginosa outcompetes Agrobacterium tumefaciens, another plant pathogen, in vitro when co-cultured on an agar plate. Remarkably, the T6SS-dependent warfare turns to the advantage of A. tumefaciens when both organisms are co-inoculated in planta. Although our research is basic research, molecular understanding of infection/colonization strategy employed by T6SS-proficient bacteria is instrumental to predict evolution of bacterial population and establishment of polymicrobial communitites such as for example in the gut. We established that the plant beneficial bacteria, Pseudomonas putida, and the plant pathogen, Pseudomonas syringae, both have a functional T6SS. Using engineered T6SS-proficient P. putida strains as agents to protect crops from the invasion of phytopathogens such as P. aeruginosa, P. syringae or A. tumefaciens can be invaluable to define biocontrol strategies. Bio-Iliberis, a biotech company that develops products based on microorganisms to favour agriculture and the environment, is a collaborator on our project. The company works routinely with several biocontrol agents including P. putida, testing the capacity to annihilate different phytopathogens by using in planta assays and have a long experience performing these experiments on crop plants. The involvement of BioIliberis guarantees long term applications of our research.
Our research is basic research, but will lead to biomedical applications, new antimicrobials, agricultural and societal impact. It will also have broader impact on understanding the fate of microbial population in nature and thus on ecology.
There will be an opportunity to organize a workshop on the site of Imperial College. The topic will be "Mechanisms of Interbacterial Cooperation and Competition", a follow up of the first of a series to be held in March 2015 at Washington DC. It will attract UK and European players in the field (Dundee University, Université de la Méditerannée; Basel University) together with groups from US (Harvard Medical School, University of Washington). Biotech and pharmaceutical companies (BioIliberis & Sanofi-Aventis), and editors (Nature) will be invited. Alain Filloux has expertise in conference organization, having been organizer of the Pseudomonas meeting (Marseille 2005) and chair of the GRC on Microbial adhesion (Newport 2009).