Gram positif | EPFL Graph Search

Les bactéries à Gram positif sont mises en évidence par une technique de coloration appelée coloration de Gram. Cette technique de coloration, qui permet de classifier les bactéries dans deux catégories générales, repose sur les caractéristiques membranaires et de paroi de la bactérie. Les bactéries à Gram positif apparaissent alors mauves au microscope. La coloration au Gram est un facteur déterminant dans la taxonomie (classification) bactérienne. Principe de coloration des Gram positif Après fixation des bactéries sur une lame (deux méthodes possibles, par fixation à l'éthanol à 90° ou par passage de la lame dans la flamme) :

la lame est plongée dans un premier colorant, le violet de gentiane (ou un produit proche, le cristal violet). Le violet de gentiane est un colorant puissant (mais toxique et cancérigène). Il traverse les parois et les membranes des bactéries et se fixe dans leur cytoplasme. Ainsi à cette étape toutes les cellules sont colorées en violet ;
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Analysis of a specific modular serine protease acting in the Drosophila Toll pathway

Valentin Sottas

In Drosophila, the Toll pathway plays an important role in the immune defense against Gram-positive bacteria and fungi. Molecular determinants coming from those pathogens are directly detected by pattern recognition receptors (PPRs) circulating in the hemolymph. It has been proposed that several serine protease cascades are activated by PRRs, leading to the activation of a cleaved form of the cytokine-like molecule Spätzle, the ligand of the Toll receptor. The results obtained during my master project demonstrate an essential role for ModSP, a modular serine protease acting in the activation of the Toll pathway upon Gram-positive and fungal infections. We demonstrate that ModSP integrates signals coming from GNBP3 and PGRP-SA recognition molecules and that ModSP sends this signal to Grass, a serine protease already known to activate SPE and thereby Spätzle. Further biochemical experiments show the interaction between ModSP and GNBP1 demonstrating an apical role of this serine protease in the proteolytic cascades leading to Toll pathway activation. We also find that ModSP is expressed in specific vesicles released from the fat bodies into the hemolymph. Also, preliminary studies suggest that ModSP does not participate in the melanization reaction, a secondary but important insect immune mechanism. Biochemical analysis done by some collaborators indicates that ModSP does not cleave Grass and that ModSP exhibits a high level of auto-proteolysis when this molecule is expressed. Our data reveal a conserved role of modular serine protease in the regulation of immune proteolytic cascade in insects

2009

Fluorescent D-Amino Acids for Super-resolution Microscopy of the Bacterial Cell Wall

Juliette Griffie, Suliana Manley, Jiangtao Qiao, Luc Reymond, Ophélie Rutschmann, Faustine Ambroisine Charline Ryckebusch, Willi Leopold Stepp, Chen Zhang

Fluorescent D-amino acids (FDAAs) have previ-ously been developed to enable in situ highlighting of locations of bacterial cell wall growth. Most bacterial cells lie at the edge of the diffraction limit of visible light; thus, resolving the precise details of peptidoglycan (PG) biosynthesis requires super-resolution micros-copy after probe incorporation. Single molecule localization microscopy (SMLM) has stringent requirements on the fluorophore photophysical properties and therefore has remained challenging in this context. Here, we report the synthesis and characterization of new FDAAs compatible with one-step labeling and SMLM imaging. We demonstrate the incorporation of our probes and their utility for visualizing PG at the nanoscale in Gram-negative, Gram-positive, and mycobacteria species. This improved FDAA toolkit will endow researchers with a nanoscale perspective on the spatial distribution of PG biosynthesis for a broad range of bacterial species.

AMER CHEMICAL SOC2022

Role and regulation of the melanization reaction in Drosophila immune response

Olivier Binggeli

In arthropods, the melanization reaction consists in the rapid synthesis of melanin at the site of infection and injury. A key enzyme in the melanization process is phenoloxidase (PO), which catalyzes the oxidation of phenols to quinones that subsequently polymerize into melanin. PO is synthesized under an inactive form called pro-PO (PPO) that is activated upon cleavage by a terminal protease of a serine protease cascade. The main objective of my PhD thesis is to understand the role and regulation of the melanization reaction in the Drosophila immune response. The Drosophila genome encodes three PPOs, two of which, PPO1 and PPO2, are produced by crystal cells, a larval-specific type of hemocyte. I generated flies carrying null mutations in PPO1 and PPO2 as well as PPO1, PPO2 double mutant flies. My results show that both PPO1 and PPO2 contribute to the PO activity in the hemolymph induced by septic injury. While PPO1 is involved in the early delivery of PO activity, PPO2 is stored in crystalline form in the crystal cells and can be deployed at a later phase of the immune response. My genetic analysis also reveals an important role of PPO1 and PPO2 in the resistance of flies to infection with Gram-positive bacteria and fungi. This study definitively establishes the importance of melanization in insect host defense. Serine proteases regulate many immune reactions, including blood clotting, the complement system in mammals, and melanization and Toll pathway activation in insects. A second objective of my thesis is to identify new serine proteases or serine protease inhibitors (serpins) participating in the Drosophila immune response. I performed an RNAi screen to identify genes encoding serine protease or serpins whose inactivation caused an immune phenotype. In parallel, I identified or generated mutations affecting some serine proteases and serpins and described their immune phenotype. This led to the identification of SPH9, a gene strongly induced by microbial infection, which encodes a serine protease homologue that negatively regulates the melanization reaction. Together, my results provide new insights on the melanization reaction in Drosophila.

EPFL2013

Role and regulation of the melanization reaction in Drosophila immune response

Olivier Binggeli

EPFL2013