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Background Aspergillus fumigatus is the most common agent of invasive aspergillosis, a feared complication in severely immunocompromised patients. Despite the recent commercialisation of new antifungal drugs, the prognosis for this infection remains uncertain. Thus, there is a real need to discover new targets for therapy. Particular attention has been paid to the biochemical composition and organisation of the fungal cell wall, because it mediates the host-fungus interplay. Conidia, which are responsible for infections, have melanin as one of the cell wall components. Melanin has been established as an important virulence factor, protecting the fungus against the host's immune defences. We suggested that it might also have an indirect role in virulence, because it is required for correct assembly of the cell wall layers of the conidia. Results We used three A. fumigatus isolates which grew as white or brown powdery colonies, to demonstrate the role of melanin. Firstly, sequencing the genes responsible for biosynthesis of melanin ( ALB1 , AYG1 , ARP1 , ARP2 , ABR1 and ABR2 ) showed point mutations (missense mutation, deletion or insertion) in the ALB1 gene for pigmentless isolates or in ARP2 for the brownish isolate. The isolates were then shown by scanning electron microscopy to produce numerous, typical conidial heads, except that the conidia were smooth-walled, as previously observed for laboratory mutants with mutations in the PKSP/ALB1 gene. Flow cytometry showed an increase in the fibronectin binding capacity of conidia from mutant isolates, together with a marked decrease in the binding of laminin to the conidial surface. A marked decrease in the electronegative charge of the conidia and cell surface hydrophobicity was also seen by microelectrophoresis and two-phase partitioning, respectively. Ultrastructural studies of mutant isolates detected considerable changes in the organisation of the conidial wall, with the loss of the outermost electron dense layer responsible for the ornamentations seen on the conidial surface in wild-type strains. Finally, analysis of the conidial surface of mutant isolates by atomic force microscopy demonstrated the absence of the outer cell wall rodlet layer which is composed of hydrophobins. Conclusion These results suggest that, in addition to a protective role against the host's immune defences, melanin is also a structural component of the conidial wall that is required for correct assembly of the cell wall layers and the expression at the conidial surface of adhesins and other virulence factors.

Although refrigerated storage slows the metabolism of volunteer donor RBCs, which is essential in transfusion medicine, cellular aging still occurs throughout this in vitro process. Storage-induced microerythrocytes (SMEs) are morphologically altered senescent RBCs that accumulate during storage and are cleared from circulation following transfusion. However, the molecular and cellular alterations that trigger clearance of this RBC subset remain to be identified. Using a staining protocol that sorts long-stored SMEs (i.e., CFSEhi) and morphologically normal RBCs (CFSElo), these in vitro aged cells were characterized. Metabolomics analysis identified depletion of energy, lipid-repair, and antioxidant metabolites in CFSEhi RBCs. By redox proteomics, irreversible protein oxidation primarily affected CFSEhi RBCs. By proteomics, 96 proteins, mostly in the proteostasis family, had relocated to CFSEhi RBC membranes. CFSEhi RBCs exhibited decreased proteasome activity and deformability; increased phosphatidylserine exposure, osmotic fragility, and endothelial cell adherence; and were cleared from the circulation during human spleen perfusion ex vivo. Conversely, molecular, cellular, and circulatory properties of long-stored CFSElo RBCs resembled those of short-stored RBCs. CFSEhi RBCs are morphologically and metabolically altered, have irreversibly oxidized and membrane-relocated proteins, and exhibit decreased proteasome activity. In vitro aging during storage selectively alters metabolism and proteostasis in these storage-induced senescent RBCs targeted for clearance.

The intracellular morphogenesis of flaviviruses has been well described, but flavivirus release from the host cell remains poorly documented. We took advantage of the optimized production of an attenuated chimeric yellow fever/dengue virus for vaccine purposes to study this phenomenon by microscopic approaches. Scanning electron microscopy (SEM) showed the release of numerous viral particles at the cell surface through a short-lived process. For transmission electron microscopy (TEM) studies of the intracellular ultrastructure of the small number of cells releasing viral particles at a given time, we developed a new correlative microscopy method: CSEMTEM (for correlative scanning electron microscopy - transmission electron microscopy). CSEMTEM analysis suggested that chimeric flavivirus particles were released as individual particles, in small exocytosis vesicles, via a regulated secretory pathway. Our morphological findings provide new insight into interactions between flaviviruses and cells and demonstrate that CSEMTEM is a useful new method, complementary to SEM observations of biological events by intracellular TEM investigations.

To establish an infection, has to interact with eukaryotic cells. Invasion of non-phagocytic cells (i.e., epithelial, fibroblast and endothelial cells) involves either a trigger or a zipper mechanism mediated by the T3SS-1 or the invasin Rck, respectively. Another outer membrane protein, PagN, was also implicated in the invasion. However, other unknown invasion factors have been previously suggested. Our goal was to evaluate the invasion capability of a Typhimurium strain invalidated for the three known invasion factors. Non-phagocytic cell lines of several animal origins were tested in a gentamicin protection assay. In most cells, we observed a drastic decrease in the invasion rate between the wild-type and the triple mutant. However, in five cell lines, the triple mutant invaded cells at a similarly high level to the wild-type, suggesting the existence of unidentified invasion factors. For the wild-type and the triple mutant, scanning-electron microscopy, confocal imaging and use of biochemical inhibitors confirmed their cellular uptake and showed a zipper-like mechanism of internalization involving both clathrin- and non-clathrin-dependent pathways. Despite a functional T3SS-1, the wild-type bacteria seemed to use the same entry route as the mutant in our cell model. All together, these results demonstrate the existence of unknown invasion factors, which require further characterization.

Seed transmission constitutes a major component of the parasitic cycle for several fungal pathogens. However, very little is known concerning fungal or plant genetic factors that impact seed transmission and mechanisms underlying this key biological trait have yet to be clarified. Such lack of available data could be probably explained by the absence of suitable model pathosystem to study plant-fungus interactions during the plant reproductive phase. Here we report on setting up a new pathosystem that could facilitate the study of fungal seed transmission. Reproductive organs of Arabidopsis thaliana were inoculated with Alternaria brassicicola conidia. Parameters (floral vs fruit route, seed collection date, plant and silique developmental stages) that could influence the seed transmission efficiency were tested to define optimal seed infection conditions. Microscopic observations revealed that the fungus penetrates siliques through cellular junctions, replum and stomata, and into seed coats either directly or through cracks. The ability of the osmosensitive fungal mutant nik1[DELA]3 to transmit to A. thaliana seeds was analyzed. A significant decrease in seed transmission rate was observed compared to the wild-type parental strain, confirming that a functional osmoregulation pathway is required for efficient seed transmission of the fungus. Similarly, to test the role of flavonoids in seed coat protection against pathogens, a transparent testa Arabidopsis mutant (tt4-1) not producing any flavonoid was used as host plant. Unexpectedly, tt4-1 seeds were infected to a significantly lower extent than wild-type seeds, possibly due to over-accumulation of other antimicrobial metabolites. The Arabidopsis thaliana-Alternaria brassicicola pathosystem, that have been widely used to study plant-pathogen interactions during the vegetative phase, also proved to constitute a suitable model pathosystem for detailed analysis of plant-pathogen interactions during the reproductive phase. We demonstrated that it provides an excellent system for investigating the impact of different fungal or plant mutations on the seed transmission process and therefore paves the way towards future high-throughput screening of both Arabidopsis and fungal mutant.

Protocols allowing the in vitro culture of human hair follicles in a serum free-medium up to 9 days were developed 30 years ago. By using similar protocols, we achieved the prolonged maintenance in vitro of juvenile feather follicles (FF) microdissected from young chickens. Histology showed a preservation of the FF up to 7 days as well as feather morphology compatible with growth and/or differentiation. The integrity of the FF wall epithelium was confirmed by transmission electron microscopy at Day 5 and 7 of culture. A slight elongation of the feathers was detected up to 5 days for 75% of the examined feathers. By immunochemistry, we demonstrated the maintenance of expression and localization of two structural proteins: scaffoldin and fibronectin. Gene expression (assessed by qRT-PCR) of NCAM, LCAM, Wnt6, Notch1, and BMP4 was not altered. In contrast, Shh and HBS1 expression collapsed, DKK3 increased, and KRT14 transiently increased upon cultivation. This indicates that cultivation modifies the mRNA expression of a few genes, possibly due to reduced growth or cell differentiation in the feather, notably in the barb ridges. In conclusion, we have developed the first method that allows the culture and maintenance of chicken FF in vitro that preserves the structure and biology of the FF close to its in vivo state, despite transcriptional modifications of a few genes involved in feather development. This new culture model may serve to study feather interactions with pathogens or toxics and constitutes a way to reduce animal experimentation.

Monitoring virus assembly at the nanoscale in host cells remains a major challenge. Human immunodeficiency virus type 1 (HIV-1) components are addressed to the plasma membrane where they assemble to form spherical particles of 100 nm in diameter. Interestingly, HIV-1 Gag protein expression alone is sufficient to produce virus-like particles (VLPs) that resemble the immature virus. Here, we monitored VLP formation at the plasma membrane of host CD4+ T cells using a newly developed workflow allowing the analysis of long duration recordings of single-molecule Gag protein localisation and movement. Comparison of Gag assembling platforms in CD4+ T cells expressing wild type or assembly-defective Gag mutant proteins showed that VLP formation lasts roughly 15 minutes with an assembly time of 5 minutes. Trapping energy maps, built from membrane associated Gag protein movements, showed that one third of the assembling energy is due to direct Gag capsid-capsid interaction while the remaining two thirds require the nucleocapsid-RNA interactions. Finally, we show that the viral RNA genome does not increase the attraction of Gag at the membrane towards the assembling site but rather acts as a spatiotemporal coordinator of the membrane assembly process.