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Remodelling of mycobacterial peptidoglycan during cell division and in tuberculosis disease

English title Remodelling of mycobacterial peptidoglycan during cell division and in tuberculosis disease
Applicant McKinney John
Number 170912
Funding scheme South Africa
Research institution Global Health Institute EPFL SV-DO
Institution of higher education EPF Lausanne - EPFL
Main discipline Medical Microbiology
Start/End 01.10.2017 - 30.09.2022
Approved amount 335'964.00
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All Disciplines (2)

Discipline
Medical Microbiology
Experimental Microbiology

Keywords (8)

cell division; remodelling; single cell imaging; fluorescent probes; host immunity; mycobacterium tuberculosis; peptidoglycan; cell wall

Lay Summary (French)

Lead
Avec 8 millions de nouveaux cas et 1,5 million de décès par an, la tuberculose reste une source importante de souffrance pour l’humanité. En outre, environ 2 milliards d’individus dans le monde sont porteurs de la maladie sous sa forme latente, ce qui constitue une énorme source de nouveaux cas potentiels. Compte tenu de l’importance de la prolifération bactérienne dans le développement de la maladie sous sa forme active, notre connaissance de la croissance cellulaire, du remodelage de la paroi cellulaire et de la division cellulaire dans les mycobactéries est insuffisante
Lay summary

Contenu et objectifs du travail de recherché

La prolifération bactérienne implique une série complexe et soigneusement coordonnée d’événements tels que la réplication chromosomique, la biogénèse du matériau de la paroi cellulaire, l’extension de la cellule, la cytokinese suivie de la séparation des nouvelles cellules filles. Cette proposition vise à combler nos lacunes dans le processus de division des cellules mycobactériennes, en construisant des souches mutantes dépourvues d’enzymes spécifiques impliquées dans ces processus et en conduisant une analyse phénotypique de ces mutants (en utilisant la génétique, la protéomique et l’immunologie), ainsi qu’en les analysant au niveau de la cellule unique par vidéo microscopie (time-lapse). Plus précisément, nous souhaitons étudier deux groupes d’enzymes mycobactériennes (i) amidases impliquées dans le transformation du peptidoglycane et impliqués dans la division cellulaire, la séparation cellulaire, la résistance aux antibiotiques et le recyclage du peptidoglycane et (ii) les facteurs favorisant la réanimation, qui ont été impliqués dans la modulation de la réactivation des bactéries non réplicantes. Dans l’ensemble, le programme de recherche proposé couvre la recherche biomédicale fondamentale et la science translationnelle dans le but de sonder la biologie du remodelage de la surface cellulaire dans le bacille tuberculeux.

Contexte scientifique et social du projet de recherche

 Au cours des 40 dernières années, il n’y a eu qu’un nouveau médicament approuvé pour le traitement de la tuberculose. Compte tenu du rôle important du remodelage de la paroi cellulaire et de la division cellulaire pour l’établissement de l’infection, nous espérons que l’étude de ces processus révélera de nouvelles vulnérabilités qui pourront être exploitées pour tuer l’organisme.

Direct link to Lay Summary Last update: 11.09.2017

Responsible applicant and co-applicants

Employees

Project partner

Publications

Publication
Cell surface biosynthesis and remodelling pathways in mycobacteria reveal new drug targets
Shaku Moagi, Ealand Christopher, Kana Bavesh (2020), Cell surface biosynthesis and remodelling pathways in mycobacteria reveal new drug targets, in Frontiers in Cellular and Infection Microbiology , 10, 603382.
Characterization of putative DD-carboxypeptidase-encoding genes in Mycobacterium smegmatis
Ealand Christopher S., Asmal Rukaya, Mashigo Lethabo, Campbell Lisa, Kana Bavesh D. (2019), Characterization of putative DD-carboxypeptidase-encoding genes in Mycobacterium smegmatis, in Scientific Reports, 9(1), 5194-5194.
L,D-Transpeptidase Specific Probe Reveals Spatial Activity of Peptidoglycan Cross-Linking
Pidgeon Sean, ApostolosAlexis, NelsonJulia, ShakuMoagi, RimalBinayak, IslamM Nurul, CrickDean, KimSung Joon, PavelkaMartin, KanaBavesh, PiresMarcos (2019), L,D-Transpeptidase Specific Probe Reveals Spatial Activity of Peptidoglycan Cross-Linking, in ACS Chemical Biology, 14(10), 2185-2196.
Remembering the Host in Tuberculosis Drug Development
Frank Daniel J, Horne David J, Dutta Noton K, Shaku Moagi Tube, Madensein Rajhmun, Hawn Thomas R, Steyn Adrie J C, Karakousis Petros C, Kana Bavesh Davandra, Meintjes Graeme, Laughon Barbara, Tanvir Zaid (2019), Remembering the Host in Tuberculosis Drug Development, in The Journal of Infectious Diseases, 219(10), 1518-1524.
Resuscitation-Promoting Factors Are Required for Mycobacterium smegmatis Biofilm Formation
Ealand Christopher, Rimal Binayak, Chang James, Mashigo Lethabo, Chengalroyen Melissa, Mapela Lusanda, Beukes Germar, Machowski Edith, Kim Sung Joon, Kana Bavesh (2018), Resuscitation-Promoting Factors Are Required for Mycobacterium smegmatis Biofilm Formation, in Applied and Environmental Microbiology, 84(17), e00687-18.

Collaboration

Group / person Country
Types of collaboration
Prof. Carolyn Bertozzi (PhD) United States of America (North America)
- in-depth/constructive exchanges on approaches, methods or results
- Publication

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
BACTERIAL MORPHOGENESIS, SURVIVAL AND VIRULENCE: REGULATION IN 4D Poster A phenotyping toolkit for studying mycobacterial physiology at the single-cell level 24.11.2019 Cape Town, South Africa Kana Bavesh;
PdB-Spier Symposium Talk given at a conference Single-cell approaches towards understanding bacterial persistence to antibioticsbiotics 23.11.2019 Cape Town, South Africa Kana Bavesh;
Keystone Symposium – “Tuberculosis: Mechanisms, pathogenesis and treatment” Poster Elucidating the role of the peptidoglycan hydrolase Ami1 during mycobacterial cell division 01.11.2019 Banff, Canada Von Schultz Sofia;
EMBO workshop - "Bacterial cell division: Closing the gap” Poster Elucidating the role of the peptidoglycan hydrolase Ami1 during mycobacterial cell division 09.06.2019 Lund, Sweden Von Schultz Sofia;
Bill and Melinda Gates Foundation Grand Challenges Annual Meeting Talk given at a conference Single-cell approaches for probing phenotypic heterogeneity in Mycobacterium tuberculosis during infection 15.10.2018 Berlin, Germany McKinney John;
Global Health Institute Retreat Poster Elucidating the role of peptidoglycan remodelling enzymes in mycobacterial cell division 18.06.2018 Zermatt, Switzerland Von Schultz Sofia;
EMBO Workshop: Bacterial Persistence and Antimicrobial Therapy Talk given at a conference Ampicillin persistence in uropathogenic E. coli is independent of growth rate at the single-cell level 10.06.2018 Ascona, Switzerland McKinney John;
Microbiology in the new Millennium: from molecules to communities Talk given at a conference Single-cell approaches to probing phenotypic heterogeneity in Mycobacterium tuberculosis 27.10.2017 Bose Institute, Kolkata, India McKinney John;
IMTechCon: An industry-academia meet Talk given at a conference Microengineering for Microbiology 04.10.2017 IMTECH, Chandigarh, India McKinney John;


Self-organised

Title Date Place
BACTERIAL MORPHOGENESIS, SURVIVAL AND VIRULENCE: REGULATION IN 4D 24.11.2019 Cape Town, South Africa

Associated projects

Number Title Start Funding scheme
149082 Single cell analysis of peptidoglycan remodelling and resuscitation in mycobacteria: Implications for TB disease 01.11.2013 South Africa

Abstract

SummaryBacterial proliferation involves a complex series of events with a principle component being cell division which requires careful coordination of chromosomal replication, biogenesis of cell wall precursors, extension of the cell, cytokinesis and final separation of nascent daughter cells. The inherent complexity of the entire process, involving a large number of enzymes, mostly absent in humans, exposes numerous points of vulnerability in bacterial metabolism that can be targeted for drug development. Consistent with this notion, the bacterial cell wall, especially the peptidoglycan (PG) component, has been the target of numerous clinically successful antimicrobial agents. PG, a sugar-peptide macromolecule, is a critical component of bacterial cells that contributes to maintenance of cell shape, size, cytoplasmic turgor, and numerous other essential processes. Although PG biology has been extensively studied in other organisms, the metabolic processes related to biosynthesis, crosslinking, and remodelling of PG in mycobacterial pathogens remains largely unexplored. The research proposed within the context of this Swiss-South Africa Joint Research Program aims to address this knowledge gap through the study of several families of PG remodelling enzymes in Mycobacterium tuberculosis, the causative agent of tuberculosis (TB). The enzymes chosen for this study include amidases, endopeptidases and lytic transglycosylases, all of which show great promise as drug targets. The experimental plan involves the generation of deletion/depletion mutants, defective for these enzymes, followed by a multipronged approach to characterize the behaviour of the resulting strains using various microbiological approaches including single-cell, time-lapse microscopy to monitor and measure real-time cell elongation and division defects that are expected to be associated with PG remodelling deficiencies. If division is dysregulated in mutant cells, the cell division and elongation machinery will be visualized in these strains, using fluorescent reporters, to provide mechanistic insight into the functional network of PG remodelling enzymes. Additional reporter strains will also be constructed that allow for monitoring initiation of chromosome replication, followed by partitioning during elongation and cytokinesis. To assess temporal and spatial incorporation of new PG subunits, the incorporation of novel chemical bioorthogonal probes into the cell wall of mutant cells will be monitored using single-cell time-lapse microscopy. In addition, the structural and compositional changes in the PG of mutant strains will also be analysed by LCMS to determine how loss of certain PG remodelling enzymes affects biosynthesis, crosslinking and turnover of cell wall structure. Recognition of PG by the host immune system forms a critical component of the innate response to bacterial infection. However, this aspect has been relatively unexplored in TB infection and requires urgent attention. The mutants that we will generate provide an ideal framework to further interrogate the immune response to PG breakdown products during TB disease. Remodelling of PG during bacterial growth in macrophages is expected to release muropeptides that are potent agonists of the NOD1/2 innate immune effectors. To further assess this, primary murine bone-marrow derived macrophages, J774.1 macrophage-like cells, and THP1-macrophages will be infected with mutants defective for PG remodelling enzymes, followed by an assessment of cytokine and chemokine production. In addition, purified PG from mutant strains will also be used to stimulate macrophages, followed by quantification of cytokine production. When combined with the above-mentioned ultra-structural analysis of the PG, these experiments will allow for a granular description of how mycobacterial-derived PG breakdown products modulate host immunity. To further explore the role of PG remodelling amidases during infection, we will assess the virulence of M. tuberculosis amidase-defective mutants in the murine model of TB infection. As a third component of the proposed research program, the role of PG remodelling lytic transglycosylases in modulating the growth of bacteria in sputum derived from TB diseased patients will be studied. Resuscitation promoting factors (Rpfs), a group of secreted lytic transglycosylases, implicated in enhancing bacterial culturability have been of particular interest due to their ability to enhance the recovery of non-replicating bacteria. It has been demonstrated that a large proportion of sputum-derived bacteria required Rpf-supplementation for growth and the presence of such organisms has also been confirmed in the murine model of TB infection. To further assess this, the growth of sputum-derived organisms from South African patients with active TB disease, prior to the initiation of treatment, in the presence and absence of Rpfs will be monitored using single-cell time-lapse microscopy. The growth rates of individual bacteria, before and after Rpf-supplementation will be monitored to determine the proportion of differentially culturable bacteria in sputum. The susceptibility of these bacteria to rifampicin or isoniazid treatment will also be monitored at a single-cell level to determine if Rpf-dependent organisms display inherent tolerance to antibiotics. It is expected that these experiments will provide notable insight into the phenomenon of bacterial persistence in TB disease and the consequent requirement for protracted treatment. Collectively, the proposed research program spans fundamental biomedical research and translational science with the aim of probing the biology of cell surface remodelling in the tubercle bacillus. The work will be conducted in the context of a cohesive partnership between South African, Swiss and appending US collaborators all of whom have highly specialized expertise in areas of mycobacterial metabolism, microscopy, clinical microbiology and immunology. Furthermore, numerous PhD students from South Africa will be trained whilst conducting research within this framework of study.
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