Type VI secretion in Pseudomonas species: bacterial competition and biocontrol

Lead Research Organisation: Imperial College London
Department Name: Life Sciences

Abstract

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.

Technical Summary

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.

Planned Impact

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).

Publications

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Karampatzakis A (2017) Probing the internal micromechanical properties of biofilms by Brillouin imaging. in NPJ biofilms and microbiomes

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Allsopp LP (2017) RsmA and AmrZ orchestrate the assembly of all three type VI secretion systems in . in Proceedings of the National Academy of Sciences of the United States of America

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Filloux A (2018) Multiple Structures Disclose the Secretins' Secrets. in Journal of bacteriology

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Bernal P (2018) Type VI secretion systems in plant-associated bacteria. in Environmental microbiology

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Cain AK (2019) Complete Genome Sequence of Pseudomonas aeruginosa Reference Strain PAK. in Microbiology resource announcements

 
Description Pseudomonas putida is able to protect plants from infection by plant pathogens, such as Xanthomonas or Agrobacterium.
The protection is dependent on an active T6SS which directly injects toxins into the bacterial pathogen and kills it. We have now identified a novel T6SS component in Pseudomonas putida, which we called TagX and which is not found in other T6SS classes.
We have now found that tagX is a key component of the T6SS and interacts with the baseplate component TssA. In this respect irt appears that TagX is a functional homologue of TagJ from P. aeruginosa nd is able to localize with -in the ring formed by TssA. This feature is thus an alternative to the long versions of the TssA family in which wcase that is the TssA C terminus which folds back into the lumen of the ring.
Exploitation Route Our findings have been published in ISME J in January 2017 and in a review published in Environmental Microbiology in 2017
Our new findings on TagX is a groundbreaking observations that we intend to publish in EMBO J.
Sectors Agriculture, Food and Drink,Environment

 
Description H2020-MSCA-IF-2014. Marie Curie Fellowship to Laurent Dortet - 01/02/2016 to 31/01/2018
Amount € 195,454 (EUR)
Organisation Marie Sklodowska-Curie Actions 
Sector Academic/University
Country Global
Start 02/2016 
End 01/2018
 
Description H2020-MSCA-IF-2014. Marie Curie Fellowship to Patricia Bernal - 01/02/2016 to 31/01/2018
Amount € 195,454 (EUR)
Organisation Marie Sklodowska-Curie Actions 
Sector Academic/University
Country Global
Start 02/2016 
End 01/2018
 
Description MRC PhD Stiudentship to Sophie Howard
Amount £79,500 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 10/2016 
End 10/2019
 
Description Talentia Fellowship to Patricia Bernal - Spanish Council
Amount € 166,489 (EUR)
Organisation Spanish National Research Council (CSIC) 
Sector Public
Country European Union (EU)
Start 03/2015 
End 08/2016
 
Description Biochemistry of the Pseudomonas T6SS - Despoina Mavridou 
Organisation Imperial College London
Department Department of Life Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution We have engineeered the constrcuts to overecpress tagX-tssA and tagJ-tssA from P. putida and P. aeruginosa, respectively
Collaborator Contribution Despoina Mavridou has performed biochemical analysis of the TagX-TssA and TagX-TssA complexes by co-purification approaches and electron microscopy analysis.
Impact expect high impact publication in EMBO J
Start Year 2018
 
Description CSIC-Granada-Spain-2014-Marian Llamas 
Organisation Spanish National Research Council (CSIC)
Country European Union (EU) 
Sector Public 
PI Contribution Collaboration stemming from the attribution of an EMBO short term fellowship to Patricia Bernal. Patricia stayed at Imperial College London from October to December 2014. Patricia Bernal has now secured a Talent Hub fellowship and a Marie Curie fellowship so that she could stay at Imperial until 2018. This work has also been the basis for the BBSRC award number: BB/N002539/1 which will commence on March 1 2016
Collaborator Contribution Study of the T6SS in Pseudomonas Putida
Impact EMBO Short term fellowship to Patricia Bernal Talent Hub fellowship to Patricia Bernal Marie Curie fellowship to Patricia Bernal
Start Year 2014
 
Description Collaboration Professor Cayo Ramos (Malaga University) 
Organisation University of Malaga
Country Spain 
Sector Academic/University 
PI Contribution Isabel Aragon visited our laboratory on an EMBO short term fellowship to investigate the role of c-di-GMP in virulence asscoiated with Pseudomonas savastanoi and the similarities with Pseudomonas aeruginosa
Collaborator Contribution The Ramos laboratory provided all strains and reagents required to perform the study on P. savastanoi
Impact We published a paper in Environmental Microbiology describing the role in virulence of the diguanylate cyclase DgcP in Pseudomonas savastanoi and Pseudomonas aeruginosa
Start Year 2012
 
Description Statistical analysis of the spatio-temporal distribution of the T6SS in bacteria 
Organisation Imperial College London
Country United Kingdom 
Sector Academic/University 
PI Contribution Engineering bacterial strains with fluorescently labelled T6SS components and recording videos
Collaborator Contribution Mathematical and statistical analysis of the T6SS foci distribution in bacterial cells and bacterial populations.
Impact Hiring a Wellcome Trust PhD student, Scott Ward, who is hosted by Niall Adams in the Department of Mathematics
Start Year 2017
 
Description A talk at NTU in Singapore by Prof Alain Filloux 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Talk by the PI Alain Filloux to PI, Postdoctoral fellows and PhD students from the SCELSE Institute at the NTU in Singapore.
Role of c--di-GMP in biofilm formation and immune evasion and hos it relates to the central regulatory network RsmA/SadC
Year(s) Of Engagement Activity 2016
 
Description A weapon for bacterial warfare. Talk Patricia Bernal at the V International Symposium SRUK 2017, London, United Kingdom, July 7-9, 2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact Presentation at the postgraduate community in London
Year(s) Of Engagement Activity 2017
 
Description Academia Sinica (IPMB), Taipei, Taiwan 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact talk in the context of networking with our collaborator Erh-Min Lai
Year(s) Of Engagement Activity 2015
 
Description Co-cordination of the Imperial Festiva 2018 - Patricia Bernal 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Patricia has coordinated the section "Bug Zones" for the Imperial Festival 2018. This covered initiation to microbiology and microbial pathogens for school children. there were several boots installed on the ground floor of Flowers Building (South Kensington campus) and this involved also several other members of the laboratory (Panayiota Pissaridou, Laura Nolan, Kira Glatzel, Sophie Howard, Luke Allsopp). Patricia was also in the jury for the "Science Toy Award" 2018 during the festival.
Year(s) Of Engagement Activity 2018
 
Description European Course on Microbial Evolution and Molecular Epidemiology, ENS Lyon 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Undergraduate students
Results and Impact Course for for French Microbiologists at the Ecole Normale Supérieure de Lyon
Year(s) Of Engagement Activity 2015
 
Description Imperial Festival - Super Bugs Zone - 27-29 April 2018 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Open Day odf the CMBI at the annula Impetrial Festival. Several stands at the Supoer Bugs Zone describing multiple aspects of bacterial pathogens.
Year(s) Of Engagement Activity 2018
 
Description Imperial Festival Outreach - Super Bugs Zone - Educating chirldren to the world of Microbiology 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Several members of the Filloux's group, Laura Nolan (PDRA), Patricia Bernal (PDRA), Lukke Allsopp (PDRA), Thomas Wood (PhD student), Panayiota Pissaridou (PhD student), Sophie Howard (PhD student) and Sara Planamente (PDRA), designed a stand in May 2017 for the Imperial festival. These were describing various aspects of Microbiology from recognizing a bacterium to detailed molecular mechanisms of an antibacterial weapon, the T6SS.
Year(s) Of Engagement Activity 2017
 
Description Invited talks for Luke Allsopp at the London School of Hygiene and University of Kent on the T6SS 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact Invited talks recognizing Luke Allsopp has a valued PDRA from the Filloux lab and highlighting his research on T6SS
Year(s) Of Engagement Activity 2017
 
Description Nanyang Technological University (NTU), Singapore Center for Environmental Life Sciences Engineering (SCELSE). 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact talk in the context of Networking with future collaborators in Singapore on biofilm, c-di-GMP signalling and T6SS
Year(s) Of Engagement Activity 2016
 
Description National Chung-Hsing University, Taiwan 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Networking with our collaborator Erh-Min Lai in Taiwan
Year(s) Of Engagement Activity 2016,2017
 
Description Patricia Bernal presentation at the 6th Congress of European Microbiologists, Maastricht, The Netherlands, 2015 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Bernal P., Civantos, C., Filloux, A. and Llamas M.A. Type VI secretion in the soil bacterium Pseudomonas putida: role in bacterial competition. Poster presented at the 6th Congress of European Microbiologists, Maastricht, The Netherlands, June 7-11, 2015.
Year(s) Of Engagement Activity 2015
 
Description Poster at the YMS conference in Dundee by Patricia Bernal 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Patricia Bernal presented a poster on the work with Pseudomonas putida as a plant warden against phytopathogens.
Bernal P., Llamas M.A. and Filloux, A. Type VI secretion system: a biocontrol weapon exploited by Pseudomonas putida to outcompete phytopathogens. Poster presented at the Young Microbiologists Symposium 2016, Dundee, United Kingdom, June 29-30, 2016.
Year(s) Of Engagement Activity 2016
URL http://www.lifesci.dundee.ac.uk/other/yms/
 
Description Poster presentation by Thomas Wood at the FEMS 2017 meeting on the T6SS effector VgrG2b 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact PhD student from the Filloyx's group presenting work on the T6SS
Year(s) Of Engagement Activity 2017
 
Description Poster presented by Patricia Bernal on Pseudomonas putida T6SS-International Symposium SRUK 2016, London 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Poster presented by patricia Bernal on T6SS-dependent biocontrol by Pseudomonas putida
Bernal P., Llamas M.A. and Filloux, A. The Pseudomonas putida Type VI secretion system is used as a biocontrol weapon against phytopathogens. Poster presented at the IV International Symposium SRUK 2016, London, United Kingdom, July 9-10, 2016
Year(s) Of Engagement Activity 2016
 
Description Poster presented by Sara Planamente, Panyiota Pissaridou and Sarah Wettstadt at the Pseudomonas conference in Liverpool - The T6SS machinery and its effectors 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Presentation of our work on the T6SS
Year(s) Of Engagement Activity 2017
 
Description Posters from Luke Allsopp, Thomas Wood and Sarah Wettsadt at the 5th Molecular Microbiology and 4th Midlands Molecular Microbiology Meeting in Birmingham 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact PDRA and PhD students from the Filloux lab, Luke Allsopp, Thomas Wood and Sarah Wettsadt, presented talks and poster on the T6SS
Year(s) Of Engagement Activity 2017
 
Description School Visit WIX (Clapham London) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Luke Allsoop and Patricia Bernal visited the scool WIX in London to initiate year two kids to Microbiology (Two classes)
Year(s) Of Engagement Activity 2018
 
Description Talk Alain Filloux Academia Sinica in Taipei 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact From January 15th, 2017 to January 23rd 2017, Alain Filloux visited Erh-Min Lai laboratory and to further discuss the collaboration and give a talk.
Year(s) Of Engagement Activity 2017
 
Description The Pseudomonas putida T6SS is a plant warden against phytopathogens. Talk Patricia Bernal at the I Department of Life Sciences Postdoc Symposium, London, UK, March 30th, 2017 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact Invited talk to communicate research within the postgraduate community in London
Year(s) Of Engagement Activity 2017