Mycobacterial Pathogenesis and Novel Therapeutic Targets

THE PROJECTS

Mycobacterium tuberculosis, the etiologic agent of tuberculosis, represents a major cause of death worldwide due to a unique infectious agent. Since the mid-80s, there has been a prominent progression of the disease, substantiated by the spread of the HIV pandemic and the emergence of multidrug-resistant M. tuberculosis strains. In addition, atypical mycobacteria, including Mycobacterium abscessus, represent an emerging health problem in industrialized countries, and are notorious for being highly resistant to most antibiotic treatments. Thus, there is an urgent need to develop new therapies to combat these infections. One key aspect characterizing pathogenic mycobacteria resides in their capacity to persist within the phagocytic cells for several years/decades, which is strongly associated with the presence of a very unusual cell envelope. These cell wall components play a key role in driving host-pathogen interactions necessary for the establishment and persistence of the infection and represent valid targets for several antitubercular drugs.

In this context, we explore the mycobacterial cell envelope to decipher its role in the physiopathological events characterizing the infection and to identify new pharmacological targets. Our work focuses on major cell wall (glycol)lipidic components with respect to their biosynthesis, regulation, and contribution/role in virulence in pathogenic mycobacteria.

Our research program aims to investigate virulence factors in Mycobacterium tuberculosis as well as in non-tuberculous mycobacteria, such as M. abscessus, by studying genes involved in biosynthesis, catabolism and transport of mycobacterial cell wall components. Among these components are mycolic acids (long-chain fatty acids) and a vast array of lipids and glycolipids sharing exotic structures and participating in the immunopathology of the infection. Besides determining the metabolic pathways and the mechanisms controlling expression of these molecules, we are also interested in elucidating the structures of the (glycol)lipid components and their biological functions to better define their role in the physiopathology of the infection and to discover new targets of pharmacological interest. The molecular mechanisms responsible for virulence and physiopathology of atypical mycobacteria remaining elusive, we also develop alternative models of infection to apprehend and describe new mechanisms of immune evasion and persistence in the infected host.

Our studies can be summarized by 3 mains interconnected goals.

Aim 1: Microbiology of tuberculous and non-tuberculous mycobacteria.

We focus essentially on the biosynthetic and catabolic pathways of lipidic and glycolipidic cell wall associated components.

Genetic, biochemical and crystallographic studies are employed to discover and characterize enzymes involved in the biosynthesis, transport and remodeling of the mycobacterial cell wall. The objective here consists of understanding the role of components of the envelope in the adaptation of pathogenic mycobacteria to their environment and to establish a successful infection process within the host as well as to identify and validate new targets of therapeutic interest to fight against mycobacterial infections. This aim includes also the elucidation of structures and biological functions of glycolipids in atypical mycobacteria (M. marinum and M. abscessus complex) and to determine their contribution in the the pro-inflammatory response and granuloma formation.

Schematic structure of the mycobacterial envelope. Besides a plasmic membrane, the cell wall consists of a complex network of proteins, lipoglycans and (glyco)lipids, which participate directly in the interaction with the host cells. Because of their uniqueness in Mycobacterium, the enzymes involved in the biosynthesis of the cell wall components represent attractive targets for future drug development.

Aim 2: Mode of action of antimycobacterial drugs.

We study the mechanisms of activation and action of molecules inhibiting the biosynthesis of cell wall components, with a special emphasis on mycolic acids.

This aim consists essentially of determining the molecular targets and mode of action of several antimycobacterial drugs, of synthesizing structural analogues that are more efficient and less toxic than the parental molecules and to evaluate the anti-mycobacterial properties of these compounds in vitro and in vivo in a zebrafish model of infection (see Aim 3).

Another key aspect of this axis consists to describe the mechanisms responsible for the intrinsic resistance of atypical mycobacteria to most antitubercular drugs and to discover new active molecules against these species. This implies the screening of chemical libraries, the selection of resistant strains to the selected active inhibitors and identification/characterization of the therapeutic targets through the combination of genetic/biochemical and crystallographic techniques.

Three-dimensional structure of Ag85C bound to a potent inhibitor cyclophostin

Aim 3: Alternative models of infection to study mycobacterial virulence.

We develop amoeba and zebrafish (Danio rerio) models to identify new virulence genes and to image in real time the chronology of the infection.

The amoeba model is particularly adapted to the screening of transposon libraries to select for instance attenuated mutants of M. marinum or M. abscessus and to evaluate and compare their capacity of intracellular growth and survival. The zebrafish is also used to evaluate and compare virulence of mutants and, due to its optical transparency, this model is particularly suited to image the infection process at a spatiotemporal level. In this context, it has been successfully used to propose mycobacterial cords as a new mechanism of immune evasion by preventing mycobacteria to be phagocytosed by macrophages and neutrophils. In addition, embryos can be used to visualize, in real time, the pharmacological activity of molecules/drugs or to study the role of cell wall components in the induction of an inflammatory granulomatous response during infection with M. marinum and M. abscessus.

Zebrafish embryo infected with the rough variant of M. abscessus expressing mCherry (red). Microinjection was performed in the caudal vein in the mpx::GFP transgenic line harbouring green fluorescent neutrophils. The image shows the presence of a massive mycobacterial cord (red) surrounded by neuthrophils (green) in the brain.

POST-DOC, PhD and undergraduate students

We are looking for strong candidates (microbiologists, biochemists, structural biologists) as well as researchers or technicians/engineers with positions at CNRS, INSERM or University who are interested by our interdisciplinary approaches, combining basic and translational research in mycobacteriology.

Team leader

Laurent KREMER

DR1 INSERM HDR
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At a glance

Model organisms

Mycobacterium
Danio rerio (zebrafish)

Biological process studied
The mycobacterial cell wall: its role in the pathophysiology of the infection, in the development of new therapeutic molecules and in resistance to antibiotics.

Techniques used
Microbiology
Genetics
Animal experimentation (zebrafish)
Cellular biology
Biochemistry
Structural Biology

Medical applications
Antimycobacterial drug discovery

Founding

          

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