Project

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Identification and characterization of new factors involved in cell wall homeostasis in Gram-negative bacteria

Applicant Fumeaux Coralie
Number 185713
Funding scheme PRIMA
Research institution Institut de Microbiologie - CHUV Faculté de Biologie et Médecine Université de Lausanne
Institution of higher education University of Lausanne - LA
Main discipline Experimental Microbiology
Start/End 01.02.2020 - 31.01.2025
Approved amount 1'363'601.00
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All Disciplines (3)

Discipline
Experimental Microbiology
Medical Microbiology
Molecular Biology

Keywords (11)

antibiotics; Klebsiella pneumoniae; Tn-seq; Pseudomonas aeruginosa; screen; resistance; Acinetobacter baumannii; FACS; peptidoglycan; beta-lactams; proteomics

Lay Summary (French)

Lead
Etude de l’enveloppe chez les bactéries Gram négatif et les mécanismes de résistance aux antibiotiques
Lay summary

Avec l’émergence de plus en plus fréquente de bactéries pathogènes résistantes aux antibiotiques, l’agriculture, la santé publique et animale se trouvent menacées. Un besoin urgent de découvrir de nouvelles cibles pour le développement de molécules antimicrobiennes existe donc. 

De nombreux antibiotiques ciblent la formation du peptidoglycane (paroi bactérienne) qui entoure et donne sa rigidité et sa forme aux cellules. En inhibant la synthèse de cette paroi, la croissance et la division se voient stoppées, causant la mort des bactéries. Malheureusement, nous ne connaissons que très peu les mécanismes moléculaires et les modes de régulation de l’assemblage et les modifications que subit cette paroi. Afin de comprendre de façon plus détaillée comment le peptidoglycane est remanié durant le cycle cellulaire, le laboratoire marie des approches génétiques et biochimiques, avec l’espoir d’identifier de nouvelles cibles potentielles pour le design ou le screening de nouveaux antibactériens.

Le laboratoire a décidé de concentrer ses recherches sur trois espèces de bacilles Gram négatif : Pseudomonas aeruginosa, Klebsiella pneumoniae et les espèces d’Acinetobacter. Ces trois espèces bactériennes font partie de la liste établie par l’Organisation Mondiale de la Santé (OMS) des agents pathogènes critiques pour lesquels le développement de nouveaux antibiotiques est absolument requis.

Direct link to Lay Summary Last update: 27.07.2020

Responsible applicant and co-applicants

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Abstract

The peptidoglycan (PG) or cell wall, is a unique and essential structural element in the cell envelope of most bacteria. Due to its essential role in maintaining bacterial cell shape and resisting internal osmotic pressure, weakening the cell wall is catastrophic for the integrity of the cell. Agents that disrupt cell wall production, such as beta-lactams, are potent antibiotics that are extremely useful in medical practice. However, since the introduction of the first antibiotics, the emergence of resistance to almost all clinically used antibiotics and the development of multidrug resistant strains have become a serious concern. Innovative approaches are therefore necessary to discover new bacterial vulnerabilities. In this proposal, I describe various genetic and biochemical approaches to explore how cell wall homeostasis is maintained in the Gram-negative bacteria Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii, all three members of the World Health Organization (WHO) high-priority pathogen list. Many Gram-negative bacteria, including the opportunistic pathogen P. aeruginosa, are resistant to beta-lactam antibiotics due to a chromosomally encoded beta-lactamase called AmpC. The expression of the ampC gene is tightly regulated and its induction is linked to cell wall stress. I therefore reasoned that a fusion of the ampC promoter to a reporter gene allowing for a colorimetric readout on agar plates (e.g. lacZ) would serve as a useful reporter for the identification of new factors involved in maintaining cell wall homeostasis. My pilot study using this reporter (PampC::lacZ), successfully identified three new factors with poorly understood functions involved in cell wall assembly and drug resistance. In one published example, a hit from my screen identified a missing enzyme involved in the cell wall recycling pathway that is required for the resistance of P. aeruginosa to the antibiotic fosfomycin. The first objective of the proposed project is to investigate the function of the last factor identified in the pilot screen. My preliminary data indicate it is likely to be playing novel and unexpected roles in cell envelope homeostasis and antibiotic resistance. Given the success of my pilot study, I will also develop a high-throughput version of my genetic reporter screen employing fluorescence-activated cell sorting (FACS) and expand the scope of the project to include studies of cell wall biogenesis in two additional drug resistant Gram-negative bacteria, A. baumannii and K. pneumoniae. A comparative analysis of cell wall homeostasis in the three organisms will help identify the most conserved and important mechanisms as key targets for antibiotic development.These complementary approaches will uncover new mechanisms involved in PG biogenesis and drug resistance. The results will reveal new targets and lead to a better understanding of how to disrupt these processes for the development of antibiotics effective against these problematic pathogens. The high-throughput screen will also unearth many additional leads to follow-up, seeding my lab with exciting new projects to pursue for the years to come.
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