> Pathogénie microbienne moléculaire - INSERM U389
• Summary
• Objectives
• Genetics
• Cell Biology
• Inflammation
• Immunology
• Vaccines
INTRODUCTION: ESTABLISHING PARADIGMS OF BACTERIAL INVASION BY FUSING MOLECULAR GENETICS AND CELL BIOLOGY, AND MAKING STRATEGIC DECISIONS FOR VACCINE DEVELOPMENT (1979-1989).
Invasive bacteria, which have to recapitulate a complex program of interactions with eukaryotic cells, are good candidates to decipher the molecular basis of complex steps encompassing entry into cells, intracellular survival and growth, cell killing, etc…Shigella has been selected, more than twenty years ago, as a model of rupture, invasion and inflammatory destruction of the intestinal epithelium.
In 1979-1980, work done by Philippe Sansonetti at Institut Pasteur in Paris and at the Walter Reed Army Institute of Research (Washington DC) in Sam Formal’s group demonstrated that the invasive phenotype of this microrganism was encoded by a large virulence plasmid ( , , ). Based on this evidence, and by a combination of plasmid transfer and interrupted conjugation of chromosomal fragments with various Shigella Hfr donor strains, a fully invasive Shigella was reconstructed, step by step, from a totally avirulent Escherichia coli K12 recipient strain ). This work which is generally considered as pioneering in the genomics of bacterial pathogens has set the stage for further studies of Shigella pathogenesis and for the development of vaccine strategies.

un In 1985, Listeria monocytogenes was introduced in order to develop a Gram positive model of brain infection, concurrent with the Gram negative model of intestinal infection by Shigella. In parallel to the inactivation and cloning of the plasmid genes of Shigella involved in epithelial cell invasion ( ) and the discovery of virR, the master chromosomal regulator of invasion genes according to temperature ( ), the first approach of transposon mutagenesis in Listeria monocytogenes was carried out, demonstrating the crucial role of listeriolysin O (LLO) in the pathogenesis of the disease ( ), and more specifically, in the capacity to grow inside epithelial cells ( ) and to cause protective immunity in mice ( ).
These studies stimulated to examine in more detail the biology of infected cells, and to develop increasingly sophisticated cell assays and cell imaging technologies, including confocal microscopy and video microscopy, naturally leading to merge cell biology and molecular genetics in a discipline that would be later identified as “Cellular Microbiology”.
Applying these techniques of cell biology, these pathogens were shown to remodel the host cell actin cytoskeleton for their entry into non-phagocytic cells, particularly Shigella ( ). Additionally, Shigella and Listeria were shown to lyse their phagocytic vacuole and to gain access to the cytoplasm ( , ). Shigella was also shown for the first time to be able to move intracellularly and pass from cell to cell in a process dependent on actin polymerisation. A bacterial protein from the outer membrane, IcsA, was responsible for this actin-dependent movement within the host cell (Sansonetti, FASEB Conference on Molecular Mechanisms of Infection, Copper Mountain, 1988, ). This was quickly followed by similar demonstration in Listeria monocytogenes ( ). These studies were among those which stimulated both the microbiology and the cell biology communities to join forces in order to identify and analyse bacterial effectors, cellular receptors and signalling cascades that lead to the remodelling of the mammalian cell cytoskeleton.

Leading from these basic molecular studies summarised in a review ( ), as soon as from 1987, it was realised that icsA, which encoded for efficient cell to cell spread in an epithelial layer, could represent a “core” mutation to attenuate the pathogenicity of Shigella, thus providing the basis for the development of a live, rationally-attenuated, oral vaccine. Vaccine candidates against the two major species/serotypes causing dysentery in the developing world: Shigella flexneri 2a, and Shigella dysenteriae 1, were constructed. In addition to icsA, mutations in the major iron chelating systems of these bacteria (Aerobactin: iuc-iut, in S.flexneri and Enterochelin: ent-fep, in S.dysenteriae 1) were introduced, knowing that chelation of Fe3+ was a significant factor for Shigella growth in the intestinal tissue ( ). StxA, the gene encoding the catalytic subunit of Shiga toxin, a potent cytotoxin that is exclusively produced by S.dysenteriae 1, was also deleted. In order to assay for attenuation, immunogenicity and protective capacity of these vaccine candidates, animal models of infection were developed and standardised, including the macaque monkey. The disease in macaques closely mimics shigellosis observed in humans. Application of this model, allowed to validate the vaccination strategy by demonstrating attenuation and protective capacity of the vaccine candidates ( ). I also allowed to anticipate that studying the development of infectious processes at the level of the tissue would become increasingly important.
It helped to realize that the next step to Cellular Microbiology would be Tissue Microbiology and from that period on, experimental approaches have combined in vitro and in vivo studies. This strong incentive to “go in vivo” was initiated by two discoveries in the macaque monkey model: (i) Shiga toxin is essentially a cytotoxin with vascular tropism that destroys the capillary system of the intestinal mucosa thereby adding a component of ischemic colitis to invasive dysentery, thereby explaining the severity of infections by S.dysenteriae 1 ( ); and (ii) the follicle-associated epithelium (FAE) that covers the mucosal lymphoid follicles (i.e. the inductive sites of mucosal immunity) represents the primary site of initial entry of Shigella into the intestinal mucosa ( ).