Investigation of the Type VI secretion system-associated toxins of Burkholderia thailandensis

Informations projet

BURK-6

N° de convention de subvention: 657766

DOI

10.3030/657766

Projet clôturé

Date de signature de la CE 1 Avril 2015

Date de début 1 Septembre 2015

Date de fin 31 Août 2018

Financé au titre de

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Coût total

€ 276 107,40

Contribution de l’UE

€ 276 107,40

Coordonné par

LONDON SCHOOL OF HYGIENE AND TROPICAL MEDICINE ROYAL CHARTER
United Kingdom

Periodic Reporting for period 2 - BURK-6 (Investigation of the Type VI secretion system-associated toxins of Burkholderia thailandensis)

Période du rapport: 2017-09-01 au 2018-08-31

Résumé du contexte et des objectifs généraux du projet

Problem addressed:
Burkholderia pseudomallei causes melioidosis, a neglected tropical infectious disease, which treatment is complicated by long phases of latency and a high antibiotic resistance. Bacterial secretion systems, which act as small syringes, play a central role in infectious diseases by enabling pathogenic bacteria to deliver toxins and virulence factors into infected target cells.
Why is this important for society:
To date, there is no vaccine against Burkholderia pseudomallei, which is also renown for its high resistance to antibiotic treatment.
Uncovering the toxins delivered by Bacterial secretion systems is essential for the understanding of bacterial pathogenesis and the design of new antimicrobial therapies.
Overall objectives:
Using high-throughput and cross-disciplinary approaches in a surrogate model, this project aims at identifying and characterising novel toxins delivered by Burkholderia pseudomallei, an emerging life-threatening pathogen for which vaccines are unavailable.

Travail effectué depuis le début du projet jusqu’à la fin de la période considérée dans le rapport et principaux résultats atteints jusqu’à présent

This research proposal aimed at identifying new virulence effectors in the bacterial pathogen B. thailandensis, a surrogate model of Burkholderia pseudomallei. In light of new information, raised after the proposal submission in September 2014, this research project has expanded its focus to a novel T3SS effector (namely EV1), using the same rationale described in the proposal.
The MSCA research fellow, Abderrahman Hachani (AH), has been granted a Global MSCA Fellowship (36 months), hosted by the London School of Hygiene and Tropical Medicine (LSHTM-ITD-PMB) and is conducted in two research institute: a 24 months outgoing phase at the Department of Microbiology and Immunology at the Pete Doherty Institute, University of Melbourne, Australia and a 12-month return phase at the LSHTM in the UK. The first 24 months of this fellowship focussing on 2 work packages (WP), for which he trained in technologies essential to study the role of EV1.
WP1 was dedicated to characterising the infection steps of B. thailandensis (Bt) to pinpoint and identify the potential cellular pathways putatively affected by EV1 during infection. AH has been trained in Confocal Microscopy, Transmission Electron Microscopy, High-throughput microscopy, High-throughput proteomics, and Genetic screens using the Yeast Two Hybrid system in order to identify the EV1 host targets. The genetic and biochemical screens have uncovered a eukaryotic partner to EV1 (EV1P) which has been characterised during WP2.
In absence of available EV1P knock-out models, AH received training in eukaryotic genome-editing using Clustered Regularly Interspaced Short Palindromic Repeat- Cas9 (CRISPR-CAS9) technology to successfully generate the desired mutation in a cellular model. Using the parameters identified in WP1, AH identified and characterised the biological roles of EV1 and EV1P during Bt infection.
The results obtained in WP1 and WP2 have enabled the discovery of a new cellular biology pathway used by the host cell to control the damage caused by motile intracellular bacterial pathogens.

During the final period,
the results obtained during the first period about EV1 have been presented to several experts in the fields of Cellular Biology and Cellular Microbiology (at University of Geneva, Switzerland) , and at Imperial College London and at the London School of Hygiene and Tropical Medicine (London-UK) and have been received favourably and this reviewing process has enabled the grant holder to include several control experiments necessary for the submission of the EV1 manuscript.

During the final period, the grant holder (AH) has focused his effort to delineate the role of two genes, homologous genes, A1 and A2, which were identified as necessary for the virulence of Burkholderia Pseudomallei. AH has used his newly acquired expertise in imaging and host-pathogen interaction to demonstrate the differential role exhibited by the proteins A1 and A2 in virulence and resistance to antibiotics, respectively of Burkholderia species.
The current results are now ready for publication along with the data on EV1 and EVP1 and will be submitted to PNAS and Cell host and microbe respectively.

Progrès au-delà de l’état des connaissances et impact potentiel prévu (y compris l’impact socio-économique et les conséquences sociétales plus larges du projet jusqu’à présent)

The evolution of the Burk-6 project has provided novel avenues of research beyond its primary focus in Basic Molecular Microbiology. Indeed, the applicant has discovered a novel role for an essential eukaryotic protein for which multiple functions were attributed in mammalian physiology and were unrelated to infectious diseases. This discovery has led to the development of new perspectives in the control of the cytoskeleton in eukaryotic cells beyond the current knowledge.
Results expected at the end of the current project are:
1) Dissemination of the new discoveries through 2 publications in high impact journals:
a) Manuscript 1 is currently prepared for the journal “Cell Host and Microbe” and is describing the role of two eukaryotic factors in the control of intracellular bacterial pathogens.
b) Manuscript 2 is currently prepared for the journal “PNAS” and is describing the impact of 2 homologous lipoproteins in the virulence and antibiotic resistance of Burkholderia species.

2) Dissemination of the new discoveries through scientific conferences: Gordon Conference in bacterial cell surface 2020

3) The potential impacts of the results are:
a) The creation of new research avenues linking metabolism and cytoskeleton homeostasis using intracellular bacterial and viral pathogens by the applicant. These overarching themes will progress European knowledge in basic science and will positively impact our current knowledge in medicine and drug design.
b) The identification of new therapeutic avenues targeting both virulence and antibiotic resistance in emerging pathogens
3) Actions were taken in 2019 for funding this new venture with the NHMRC/EU 2020 between Australia and the European union
The wider Socio-Economic impacts are:
By spending 24 months at the PDI in Melbourne, the applicant has promoted and expanded the impact of EU scientific research in a third country (see dissemination and exploitation or results in point 6). By creating an extended collaborative network with Australian world experts in cell biology, microbiology, proteomics and genomics, the applicant has establish himself as a recognised future leader in Microbiology.Additionally AH has initiated a collaborative project, with the Genomic department and the Immunology department at UoM on the recognition of persistent intracellular bacterial pathogens and for which a seed funding to UoM has been submitted in March 2018. Furthermore, the applicant skills in communication and expertise in T6SS and Pseudomonas infection, have been recognised by the Partner institution in Melbourne where: 1) he has been chosen by the Microbiology teaching committee to deliver as invited lecturer to 3rd year undergraduates in Microbiology 3 consecutive yearly lectures on T6SS, which have been received with >90% positive feedback by the students, 2) The applicant has been elected by the Theme leaders at the PDI (Genomics, Host-Pathogen Interactions, Virology and Genomics) to convene the Host-Pathogen Interaction theme Seminar Series (spanning 10 seminars per year at the PDI, 3) the applicant has been elected by the communication committee at the PDI as representative of host-pathogen interaction theme.

HeLa cell infected with Burkholderia thailandensis (green) using an actin tail (red)