Intracellular oxidative response of human monocytes and granulocytes to different strains of Aspergillus fumigatus.

Aspergillus fumigatus is one of the most prevalent airborne fungal pathogens, causing severe and often fatal infections. Its fungal virulence factors have not been clearly identified. Reactive oxygen species produced by phagocytic cells are potent fungicides for A. fumigatus. The aim of this study was to examine the influence of conidia pigmentation, fungal development stage and genotype strain on human leucocytes oxidative response. Various A. fumigatus strains were used and the oxidative response was analysed by flow cytometry. A significant difference was observed between live- and killed-conidia. A pigmentless strain gave an important intracellular oxidative response compared with pigmented strains. But no difference was observed between strains isolated from patients with invasive aspergillosis (IA) and bronchial colonisation. The modification of healthy phagocytes' oxidative response caused by A. fumigatus components is not sufficient to explain the virulence of fungus and to predict an evolution of patients with IA.

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

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

Type 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.

Human cytomegalovirus downregulates complement receptors (CR3, CR4) and decreases phagocytosis by macrophages.

Human cytomegalovirus (HCMV) infection is associated with an increased susceptibility to opportunistic infections. Although the subversion of adaptive immune responses has been extensively studied, the consequences of HCMV infection on natural immune responses are not well documented. A striking selective downmodulation of CD11b/CD18 (CR3) or CD11c/CD18 (CR4) was found upon HCMV infection, on two models, the monocytic THP-1 cell line and monocyte- derived macrophages. HCMV-infected macrophages have an altered adhesion/phagocytic capacity to Candida albicans, a pathogen responsible for some opportunistic infections in immunocompromised patients. These results suggest a new mechanism implicated in the augmentation of opportunistic infections in HCMV patients.

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.