Klebsiella pneumoniae type VI secretion system: a weapon for innate immunity warfare

Lead Research Organisation: Queen's University of Belfast
Department Name: Centre for Experimental Medicine

Abstract

The global problem of antimicrobial resistance (AMR) is fast becoming one of the major scientific and health issues of modern times. No surprisingly, AMR is included in the recently release UK government "National Risk Register of Civil Emergencies" that may directly affect the UK over the next 5 years. More than 80,000 deaths are estimated if there is a widespread outbreak of a resistant microbe. The development of new antibiotics is slow and difficult work but bacterial resistance is decreasing our arsenal of existing drugs. A post-antibiotic era - in which common infections and minor injuries can kill - far from being an apocalyptic fantasy, is a very real possibility for the 21st Century. The recent O'Neill review on AMR sets out the global threat by highliting that "drug-resistant infections already kill hundreds of thousands a year globally, and by 2050 that figure could be more than 10 million". Of particular concern is the mounting prevalence of infections caused by multidrug resistant Gram-negative bacteria, in particular Klebsiella pneumoniae. This pathogen has been singled out as an "urgent threat to human health" by the UK Government, the U.S. Centers for Disease Control and Prevention, and the World Health Organization due to extremely drug resistant strains. However, there is scant evidence on K. pneumoniae pathogenesis at the molecular and cellular level. Therefore, it is both urgent and necessary to better understand its pathophysiology to be able to design new strategies to treat Klebsiella infections.
Previous studies from the laboratory support the notion that Klebsiella subverts the activation of host defence mechanisms to survive in the lung. While we have progressed on understanding the cellular pathways manipulated by the pathogen to block inflammation, there is a major gap on decoding the anti-immune factors employed by Klebsiella. By applying a multidisciplinar approach encompassing cellular and molecular microbiology, innate immunity, structural bioinformatics; and exploiting Saccharomyces cerevisiae to express heterologous proteins, we will embark on harnessing basic knowledge about how Klebsiella pneumoniae type VI secretion system (T6SS)-delivered effectors block the activation of cell intrinsic immunity. T6SS is a recently discovered nanomachinery that bacteria use to deliver proteins to a recipient cell (either a competitor bacteria and/or an eukaryotic cell). We will characterize Klebsiella T6SS at the molecular level. We will dissect how T66S-delivered proteins antagonize the activation of the signalling pathway controlling the majority of host defense responses upon infection. And, finally, we will illuminate a hithertho unknown Klebsiella virulence strategy based on targeting mitochondrial dynamics.
Harnessing the host-pathogen interface opens the avenue for new antimicrobial therapeutics. Interference with pathogen virulence and/or signalling pathways hijacked by pathogens for their own benefit is an especially compelling approach, as it is thought to apply less selective pressure for the development of resistance than traditional strategies, which are aimed at killing pathogens or preventing their growth. It is therefore believed that such targets - if found and validated during the research - will meet big interest at pharmaceutical companies.

Technical Summary

Subversion of host defence systems is considered important for pathogen survival during the early stages of infection. This proposal will investigate one of the most remarkable anti-host strategies: quenching host signalling pathways by translocation of bacterial proteins into the cell. By combining cellular and molecular microbiology, innate immunity, structural bioinformatics; and exploiting Saccharomyces cerevisiae to express heterologous proteins, we will embark on harnessing basic knowledge about how Klebsiella pneumoniae type VI secretion system (T6SS)-delivered effectors block the activation of cell intrinsic immunity. K. pneumoniae has been recently singled out as an "urgent threat to human health" by the UK Government, the U.S. Centers for Disease Control and Prevention, and the World Health Organization due to extremely drug resistant strains. However, there is scant evidence on K. pneumoniae pathogenesis at the molecular and cellular levels. Therefore, it is both urgent and necessary to better understand its pathophysiology to be able to design new strategies to treat Klebsiella infections. We will pursue the following question at the forefront of research in infection biology: (i) decoding Klebsiella factors involved in re-programming cell functions; (ii) uncovering the action of Klebsiella T6SS effectors to counteract cell intrinsic immunity; and (iii) illuminating new virulence strategies of Klebsiella (targeting mitochondrial dynamics and cytoskeleton). This innovative project rises to the challenges of improving our understanding of host immunity, enhancing antimicrobial stewardship and combatting multi-resistant infections. A better understanding of the host-pathogen interface, offers the potential to develop therapies based on targeting host factors manipulated by the pathogen for its own benefit (host directed therapeutics) and the Klebsiella factors needed for (anti-virulence therapeutics).

Planned Impact

Who will benefit from this research? Academics will be the main short to medium term beneficiary. This state-of-the-art project represents a significant step forward on our understanding of Klebsiella infection biology and will illuminate general principles of microbial pathogenesis. The research will enhance the career development of Dr Palacios (named Researcher). Dr. Palacios was responsible for a significant amount of the background research. Industry: our investigations could result in the transfer of knowledge from academia to industry to develop new therapies and even products of commercial interest. Effectors and T6SS delivery system could become targets to develop new antimicrobials (anti-virulence therapeutics). Our study of Klebsiella effectors could potentially lead to the use of these proteins to re-programme cellular functions. The enormous potential of bacterial effectors to manipulate eukaryotic systems places these proteins at the center to develop cellular engineering science. Effectors offer great potential as biotechnological tools. It is also possible to use effectors and delivery systems for immunomodulation, vaccine development or gene therapy purposes.This knowledge transfer would increase wealth and foster economic competitiveness of UK-based industry. On top of this, this knowledge transfer could result in new therapies hence being benefitial for the UK health system. Public bodies: The project and the information generated will benefit UK based government bodies such as the Chief Medical Officer, the Dept of Health, and the Dept for Environment, Food and Rural Affaires as the data will provide an evidence-base for policy developments. This proposal is clearly aligned with the strategic action "supporting the development of new antimicrobials and alternative treatments" outline in the UK antimicrobial resistance strategy 2013-2018. General public: Antimcirobial resistance is one of the pressing health issues. It is advisable to increase public awareness about the potential threats and to provide the UK national regulatory bodies, with a top-class knowledge platform to maintain the unique position of UK as an area of research excellence on infection biology. This project offers also a unique opportunity to enhance the attractiveness of science for young people.

How will they benefit from this research?: Knowledge of value to the academic sector will be communicated by publication in peer-reviewed journals, oral and poster presentations at conferences and via invited lectures. Exchange of staff and students will promote knowledge transfer between collaborative groups. Staff working on the project will receive training on complementary skills (group management, know-how transfer, and entrepreneurship) which together with the cutting-edge research training received will give them all options for either an excellent career in academia, industry, or to develop a business plan for their own start-up enterprise. Knowledge transfer to industry on new targets to treat infections and biotechnological tools might have economic potential since royalty payments can reach numbers in the magnitude of several millions or tens of millions. The grant will have impact on the wider public sector by continuing our program of scientific communication. The laboratory hosts undergraduates to engage them in the fundamentals of scientific research. A video clip on infection biology will serve to disseminate our research work. Social media will be targeted via Twitter (@josebengoechea). Dr. Palacios will be involved in the public communication activities of this project.The planned symposium will disseminate our results to key stakeholders (scientists, clinitians and senior officials). There will be an outreach presentation covering the importance of microbiology for human health. This session will be opened to the general public.
 
Description Of particular concern is the mounting prevalence of infections caused by multidrug resistant Gram-negative bacteria, in particular Klebsiella pneumoniae. This pathogen has
been singled out as an "urgent threat to human health" by the UK Government, the U.S. Centers for Disease Control and Prevention, and the World Health Organization due to extremely drug resistant strains. However, there is scant evidence on K. pneumoniae pathogenesis at the molecular and cellular level. Therefore, it is both urgent and necessary to better understand its pathophysiology to be able to design new strategies to treat Klebsiella infections. By applying a multidisciplinar approach encompassing cellular and molecular microbiology, innate immunity, structural bioinformatics; and exploiting Saccharomyces cerevisiae to express heterologous proteins, we have embarked on harnessing basic knowledge about how Klebsiella pneumoniae type VI secretion system (T6SS)-delivered effectors block the activation of cell intrinsic immunity. T6SS is a recently discovered nanomachinery that bacteria use to deliver proteins to a recipient cell (either a competitor bacteria and/or an eukaryotic cell). Our initial results have shown that Klebsiella delivers at least two proteins into eukarypotic cells via the T6SS to block NF-kB activation. Intriguing, these effectors activate a hitherto unknown signalling cascade responsible for cell immune homeostasis by sensing cytoskeleton dynamics. This signaling pathways plays a pivotal role in colitis hence making possible the manipulation of this pathway to develop new therapeutics. We have also discovered that Klebsiella employs the T6SS to compete with other bacteria. Our research has uncovered at least one effector, the immunity protein, and the mechanism of action of the effector.
Exploitation Route Upon publication of our findings, we anticipate a significant number of follow-up studies carried out by other groups since the effectors identified in this grant are also encoded by other bacterial pathogens (for humans and animals). Moreover, we envision that pharma and biotech companies will be interested in exploring the manipulation of signalling pathway controlling immune homeostasis we have already uncovered. Also, the potential exits for exploiting the effector proteins identify as biotechnological tools to manipulate cell biology.
Sectors Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology,Other

 
Description We have kept our programme of outreach on the global health issues of antibtioc resistant microorganisms. In the context of the Norther Ireland Science Festival (2017, 2018, 2019, 2020), we did introduce to the general public the concept of bacteria virulence and how bacteria can manipulate our cells by injecting proteins to control essential functions.
Sector Education
Impact Types Societal

 
Description DEL studentship
Amount £65,000 (GBP)
Organisation Government of Northern Ireland 
Sector Public
Country United Kingdom
Start 09/2016 
End 12/2020
 
Description DeF PhD studenship
Amount £70,000 (GBP)
Organisation Government of Northern Ireland 
Sector Public
Country United Kingdom
Start 09/2020 
End 03/2024
 
Title Toolkit to manipulate Klebsiella pneumoniae 
Description In the microbial pathogenesis field, it is essential to fulfill molecular Koch's postulate to conclusively confirm whether a factor expressed by a pathogen is indeed a virulence factor, being essential to construct mutants, and subsequently complement these mutants. Although there are tools available to manipulate K. pneumoniae, there is no reliable method in place to manipulate multidrug resistant K. pneumoniae strains. In this grant, we have developed such tools. Upon publication of the method and the tools (golden open access option), the plasmids will be deposited at Addgene hence being freely available to the scientific community. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact We anticipate that this breakthrough method will have a huge impact in the field since it will allow for the first time to manipulate clinical relevant multidrug resistant strains to better understand their antibiotic mechanisms of resistance and their virulence. 
 
Title Yeast to expresses T6SS effectors 
Description Yeast is used as a surrogate host to assess whether T6SS effectors may have a role interfering with mammalian functions. Yeast is a well established model investigate cell biology, and the availability of strains and others tools makes easy to obtain mechanistic information. 
Type Of Material Technology assay or reagent 
Year Produced 2020 
Provided To Others? Yes  
Impact The impact has not been realized yet. 
 
Description Collaboration Abo Akademy (Finland) 
Organisation Abo Akdemi University
Country Finland 
Sector Hospitals 
PI Contribution My team share reagents, knolwedge, and genomic data.
Collaborator Contribution Dr Tiina Salminen team carried out molecular modelling of Klebsiella proteins implicated in bacterial competition.
Impact This collaboration has resulted in an accepted publication in PlosPathogens. The collaboration is multidisciplinary bridging microbiology and biophysics.
Start Year 2018
 
Description Collaboration Abo University (Finland) 
Organisation Abo Akdemi University
Country Finland 
Sector Hospitals 
PI Contribution We have described a new K. pneumoniae type VI secretion effector which targets other bacteria. We have identified the anti-toxin, and the mechanism of action of the effector.
Collaborator Contribution The partner has modeled the effector and the anti-toxin to identify active sites, and inform structural analysis.
Impact This is a multidisciplinary collaboration bridging molecular microbiology and structural bioinformatics.
Start Year 2017
 
Description Collaboration Complutense University (Madrid, Spain) 
Organisation Complutense University of Madrid
Country Spain 
Sector Academic/University 
PI Contribution My team share with the collaborating partner reagents and tools to assess the biology of type Vi secretion effectors.
Collaborator Contribution The partner has carried out crucial experiments showing how type VI secretion effectors affect Saccharomyces cerevisiae. These findings have uncovered that yeast can be also used to study type VI secretion effectors.
Impact This is a multidisciplinary collaboration bridging microbiology and cell biology. This collaboration has crystallized in a manuscript to be submitted soon.
Start Year 2017
 
Description Collaboration University of Birmingham 
Organisation University of Birmingham
Department Institute of Microbiology and Infection
Country United Kingdom 
Sector Academic/University 
PI Contribution We did carry an intial bioinformatic analysis of the type VI secretion systems in four clinical relevant isolates. This analysis pave the way for a global analysis.
Collaborator Contribution The team led by Dr McNally has performed a bioinformatic analysis of the distribution of the type VI secretion clusters in the genus Klebsiella.
Impact This is a multidisciplinary collaboration bridging molecular microbiology and bioinformatics.
Start Year 2017
 
Description Collaboration West African Centre for Cell Biology of Infectious Pathogens 
Organisation University of Ghana
Department West Africa Centre for Cell Biology of Infectious Pathogens
Country Ghana 
Sector Academic/University 
PI Contribution We have undertaken an analysis of MDR infections in South Saharan countries. We brought our knowledge on molecular epidemiology and inferction biology of multidfrug resistant infections.
Collaborator Contribution WACCBIP contributes with his expertise on infections and immune responses, and their network of collaborators across sub Saharan countries.
Impact This is a multidisciplinary collaboration bridging molecular microbiology, molecular epidemiology, and immunology.
Start Year 2018
 
Description Media press release 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Press release highlighting our results demonstrating how Klebsiella is resistant to last line antibtiotic. This news was highlighted by BBSRC (URL included) and Queen's University Belfast. It has brought attention from Northern Ireland policy makers, and peers.
Year(s) Of Engagement Activity 2017
URL http://www.bbsrc.ac.uk/news/health/2017/170216-pr-queens-researchers-make-breakthrough-in-fight-agai...
 
Description Niorthern Ireland Science Festival 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact The laboratory organized a stand presenting how our RCUK-funded research is tackling the problem of antibiotic resistant infections. The purpose was to increase awareness of the health problem. More than 300 persons (including school kids) attended the event and our stand, the feedback indicates an increase awareness of the health problem and has translated into donations to our research team
Year(s) Of Engagement Activity 2019
URL https://www.nisciencefestival.com/
 
Description Northern Ireland Science Festival 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact The laboratory organized a stand presenting how our BBSRC-funded research is tackling the problem of antibiotic resistant infections. The purpose was to increase awareness of the health problem. More than 300 persons (including school kids) attended the event and our stand, the feedback indicates an increase awareness of the health problem and has translated into donations to our research team.
Year(s) Of Engagement Activity 2018
URL http://www.nisciencefestival.com/
 
Description Northern Ireland Science Festival 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact 100 visitors from all ages attended a range of demonstrations and interactive laboratory research activities on eye disease, diabetes/vascular disease, respiratory/infectious disease.
Year(s) Of Engagement Activity 2020
URL https://www.nisciencefestival.com/event.php?e=186
 
Description Northern Ireland science Festival 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact More than 500 visitors (families, teenagers,...) attended our stand on "Infections and antibiotic resistance" where they were informed of this health issue and how our BBSRC funded research is tackling this global health problem. Feedback indicates increase awareness.
Year(s) Of Engagement Activity 2017
URL http://www.nisciencefestival.com/event.php?e=461
 
Description School visit (South Eastern Regional College, SERC) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact 55 students attended for a school visit to the Institute. the visit riase awareness of research, and what the lab is doing to tackle antimicrobial resistance. Also students learnt about risk assessment, and lab management.
Year(s) Of Engagement Activity 2019