Étude par cytométrie de flux de la phagocytose de conidies aspergillaires par des leucocytes humains (influence de l'infection à cytomégalovirus)

Aspergillus fumigatus est un champignon filamenteux, responsable d'une infection gravissime chez les immunodéprimés : l'aspergillose invasive. Parmi les différentes lignes de défenses utilisées par l'organisme pour éliminer le champignon, l'immunité cellulaire non spécifique occupe une place importante.Notre objectif était d'étudier, sur un modèle in vitro et à partir d'échantillon de sang total, certaines étapes essentielles de la phagocytose des conidies d'A. fumigatus : l'adhérence et le " burst oxydatif ".La première étape de ce travail consistait à développer une technique utilisant la cytométrie en flux pour quantifier le " burst oxydatif " des leucocytes humains après stimulation par des conidies d'A. fumigatus.Dans une deuxième étape, nous avons voulu déterminer l'influence d'une infection à cytomégalovirus sur l'adhérence des conidies aspergillaires à la surface des macrophages et sur l'expression de deux récepteurs b2 intégrines CR3 et CR4, susceptibles d'intervenir dans ce phénomène d'adhérence. De part les résultats obtenus, cette étude représente une nouvelle approche de la cytométrie en microbiologie et surtout en mycologie.GRENOBLE1-BU Médecine pharm. (385162101) / SudocSudocFranceF

PscF is a major component of the Pseudomonas aeruginosa type III secretion needle.

International audiencePseudomonas aeruginosa, a Gram-negative opportunistic pathogen, translocates exoenzymes (Exo) directly into the eukaryotic cell cytoplasm. This is accomplished by a type III secretion/translocation machinery. Here, we show that the P. aeruginosa type III secretory needle structure is composed essentially of PscF, a protein required for secretion and P. aeruginosa cytotoxicity. Partially purified needles, detached from the bacterial surface, are 60-80 nm in length and 7 nm in width, resembling needles from Yersinia spp.. YscF of Yersinia enterocolitica was able to functionally complement the pscF deletion, but required 11 P. aeruginosa-specific amino acids at the N-terminus for its function

The V Antigen of Pseudomonas aeruginosa Is Required for Assembly of the Functional PopB/PopD Translocation Pore in Host Cell Membranes

Pseudomonas aeruginosa efficiently intoxicates eukaryotic cells through the activity of the type III secretion-translocation system (TTSS). Gene deletions within the translocation operon pcrGVH-popBD abolish pore-forming activity of P. aeruginosa strains with macrophages and TTSS-dependent hemolysis. Here we investigated the requirements for PcrV, PopB, and PopD in pore formation by analyzing specific mutants using red blood cells (RBCs) and fibroblasts expressing green fluorescent protein fused to actin. Simultaneous secretion of three proteins, PopB, PopD, and PcrV, was required to achieve wild-type hemolysis and effector translocation. Deletion of pcrV in a cytotoxic strain did not affect secretion of PopB and PopD but abolished hemolytic activity and translocation of effectors into fibroblasts. Notably, the PcrV-deficient mutant was not capable of inserting PopD into host cell membranes, whereas PopB and PopD, but not PcrV, were readily found within membranes of wild-type-infected RBCs. Immunoprecipitation experiments performed by using a liposome model of pore assembly revealed a direct interaction between PopD and PopB but not between PopD and PcrV. Consequently, PcrV is necessary for the functional assembly of the PopB/D translocon complex but does not interact directly with pore-forming Pop proteins

The PscE-PscF-PscG complex controls type III secretion needle biogenesis in Pseudomonas aeruginosa.

International audienceType III secretion (T3S) systems play key roles in pathogenicity of many Gram-negative bacteria and are employed to inject toxins directly into the cytoplasm of target cells. They are composed of over 20 different proteins that associate into a basal structure that traverses both inner and outer bacterial membranes and a hollow, needle-like structure through which toxins travel. The PscF protein is the main component of the Pseudomonas aeruginosa T3S needle. Here we demonstrate that PscF, when purified on its own, is able to form needle-like fibers of 8 nm in width and >1 microm in length. In addition, we demonstrate for the first time that the T3S needle subunit requires two cytoplasmic partners, PscE and PscG, in P. aeruginosa, which trap PscF in a ternary, 1:1:1 complex, thus blocking it in a monomeric state. Knock-out mutants deficient in PscE and PscG are non-cytotoxic, lack PscF, and are unable to export PscF encoded extrachromosomally. Temperature-scanning circular dichroism measurements show that the PscE-PscF-PscG complex is thermally stable and displays a cooperative unfolding/refolding pattern. Thus, PscE and PscG prevent PscF from polymerizing prematurely in the P. aeruginosa cytoplasm and keep it in a secretion prone conformation, strategies which may be shared by other pathogens that employ the T3S system for infection