Histoplasma capsulatum is one of the best-studied dimorphic fungal pathogens, and most biological work has focused on specific characteristics that enable the yeast form to be a successful intracellular parasite of macrophages. New molecular genetic tools have allowed us to evaluate the expression and prove the importance of two yeast phase-specific factors: CBP, a secreted protein that is essential for virulence, and alpha-(1,3)-glucan, a virulence-associated cell wall polysaccharide. However, the mechanisms that explain their role in fungal pathogenicity have remained a complete mystery until recently.

We have now used NMR to solve the structure of CBP, revealing that this protein is a protease-resistant homodimer and a member of the saposin family of lipid- and membrane-binding proteins. It is likely that CBP is involved in lipid binding, lipid metabolism, and/or membrane remodeling in the phagolysosomal compartment in which Histoplasma resides. We have taken two approaches to study alpha-(1,3)-glucan: the first is a forward genetics strategy, using Agrobacterium-mediated insertional mutagenesis, to identify genes implicated in the regulation, synthesis, and processing of alpha-(1,3)-glucan. The second approach uses reverse genetics, combining fungal gene disruption with mammalian RNA-interference, to study the genes involved in production of and response to alpha-(1,3)-glucan. This work has revealed that alpha-(1,3)-glucan on the surface of Histoplasma yeasts masks recognition of the underlying beta-glucan by dectin-1, a macrophage pattern-recognition receptor that is critical in the innate immune response to fungi.

Frédérique Moyrand¹, Thierry Fontaine², Guilhem Janbon¹

1Unité de Mycologie Moléculaire, Institut Pasteur, Paris, France, ²Unité des Aspergillus, Institut Pasteur, Paris, France

The interactions between a pathogen and the infected host are the key to the pathogenesis of many infections. These involve different types of surfaces molecules and can be proteins, lipids or polysaccharides. However, most studies focus on proteins because they are easier to eliminate or modify through gene mutation. Polysaccharides are much more complicated to modify and their genetics, at least in eukaryotes, is far from being completely understood. However, it is obvious that, as in bacteria, their structures have a major effect on their function and on the virulence of the micro-organisms. The cell surface of the pathogenic basidiomycete yeast Cryptococcus neoformans is mainly composed of polysaccharides. The C. neoformans capsule represents a fascinating structure and we study the pathways that control its biosynthesis. It primarily comprised of two polysaccharides: glucuronoxylomannan (GXM, 88% of the capsule mass) and galactoxylomannan (GalXM, 7% of the capsule mass). Whereas most studies have focused on GXM, the genetics of the synthesis of galactomannan remained completely unknown. We have identified two paralogous genes, UGE1 and UGE2 encoding UDP-glucose/UDP-galactose epimerases. We demonstrated that UGE1 is necessary for GalXM biosynthesis and virulence whereas UGE2 is necessary for C. neoformans to grow on galactose at 30°C. Surprisingly, at 37°C, a uge2∆ mutant strain grow on galactose and this grow is dependent on the presence of UGE1. We analyzed how the temperature and the sugar source influence the function of the function of this enzyme in the biology of C. neoformans.

David Moyes¹, Celia Murciano², Julian Naglik¹

1King's College London, London, United Kingdom, ²University of Valencia, Valencia, Spain

The mucosal epithelium has immense importance in host defence and surveillance, as it is the cell layer that initially encounters most microorganisms. Immune responsiveness begins with pathogen recognition, usually through surface pattern recognition receptors, such as toll-like receptors. Candida albicans is the most common fungal pathogen of humans and recognition of this pathogen activates signalling pathways, which ultimately leads to an appropriate immune response through the induction of a specific set of cytokines and chemokines. Purpose: To determine how oral and vaginal epithelial cells respond to C. albicans and which signalling pathways are activated.

Methods: The innate immune response of oral (TR146) and vaginal (A431) epithelial monolayers and organotypic reconstituted human epithelium (RHE) to different Candida species and C. albicans yeast and hyphal forms was assayed. TLR gene expression was determined using real-time RT-PCR, and cytokine and chemokine protein levels measured by luminex. Epithelial cell damage was evaluated by the release of lactate dehydrogenase (LDH). Activation of NF-кB and MAP kinase signalling pathways was assessed by Western blotting.

Conclusion: C. albicans induces strong proinflammatory cytokine responses in epithelial cells predominantly through the NF-кB and MAP kinase signalling pathways.

Pathogenic fungi have to acquire nutrients from the host during invasive growth. However, different body sites may provide different nutrient compositions and the pathogens have to adapt rapidly to the changing environmental conditions. Additionally, inhaled conidia of filamentous fungi, e.g. Aspergillus fumigatus, might require different prerequisites for germination than growing hyphae that are able to secrete enzymes into the host tissue for making nutrients available. Even more, yeast strains, such as Candida albicans, differ in their physiology from filamentous fungi and may use alternative metabolic pathways for host invasion and nutrition.

Several studies have investigated the role of various metabolic pathways in pathogenicity of fungi. Interestingly, distinct differences in the role of single pathways in pathogenesis of fungal species have been observed and will be presented. One example derives from the role of the isocitrate lyase (icl), a key enzyme of the anaplerotic glyoxylate cycle. All human pathogenic fungi studied so far showed an increased expression of the icl gene after phagocytosis by macrophages but only Candida albicans icl deletion mutants were severely attenuated in virulence. Cryptococcus neoformans and Aspergillus fumigatus mutants retained full virulence, implicating an essential role of lipid metabolism only for C. albicans. In contrast, we showed that the methylcitrate cycle of A. fumigatus is essential during pathogenesis for the removal of toxic propionyl-CoA, which derives from protein degradation during invasive growth. However, enzymes of this cycle are not present in the genome of C. albicans. Furthermore, disruption of the lysine biosynthetic pathway led to avirulent A. fumigatus strains, whereas a pathogenic S. cerevisiae strain displayed no altered phenotypes when compared to the wild type.

Data will be presented, which define the differences between fungal pathogens and possible explanations will be given to elucidate the role of specific pathways in pathogenesis of different species.

Sebastian Pilsyk, Andrzej Paszewski

A unique tight sulfate permease mutant sB1^pr was used as a recipient strain for cloning and characterization of genes which can complement its phenotype (Piłsyk et al.). The "button" phenotype of the mutant is the joint effect of two very specific mutations in the sB promoter and in the nonconserved region of the pigP gene encoding subunit of GPI-N-acetylglucosaminyltransferase (GPI-GnT). A human ortholog of the pigP gene was previously shown to be involved in Down Syndrome (Shibuya et al.).

The pigP gene codes for a protein which is responsible for GPI anchoring and is involved in vesicular transport in the endoplasmic reticulum. An impairment of this function may disturb the sorting of membrane proteins, including sulfate permease. Conditional disruption of the pigP gene in the wild-type strain leads to the "button" phenotype on solid media, similar to that of the sB1^pr mutant. However, in contrast to sB1^pr, the growth defect of the disruptant is not cured by methionine. The disruptant grows fairly well in liquid minimal medium. It appears, therefore, that the PigP protein is essential for growth only on solid media whereas it is dispensible in liquid medium.

Furthermore, the pigP strain shows a different pattern of excreted proteins than the wild type. A 34kDa alkaline serine protease (ALP) is abundantly excreted by the mutant. This protease is the major allergen produced by a number of pathogenic fungi.

Piłsyk S, Natorff R, Sieńko M, Paszewski A. (2007) Fungal Genet Biol. 44, 715-25, Shibuya K, Kudoh J, Minoshima S, Kawasaki K, Asakawa S, Shimizu N. (2000) 21q22.2. Biochem Biophys Res Commun. 271, 693-8.

Franziska Lessing, Olaf Kniemeyer, Axel A. Brakhage

With the increasing number of immunocompromised individuals Aspergillus fumigatus has become one of the most important opportunistic fungal pathogens. During infection A. fumigatus is confronted with a number of defence mechanisms in the host, in particular neutrophiles and macrophages, which kill conidia by producing reactive oxygen intermediates (ROI).

We identified a homolog of the AP1 like transcription factor Yap1 from yeast in A. fumigatus, which we designated Afyap1. In yeast, Yap1p was found to be a global regulator for oxidative stress response and required for the protection of the cell against H₂O₂ and other ROI. Similarly, deletion of Afyap1 led to an A. fumigatus mutant strain which showed drastically increased sensitivity against ROI. Nuclear localisation of an Afyap1-eGFP fusion in A. fumigatus was dependent on the presence of H₂O₂ and diamide. To identify new targets of Afyap1, we compared the proteome pattern of H₂O₂ treated and non-treated wild-type mycelia and of an Afyap1 deletion strain by 2D-gel analysis. Moreover, despite the importance of Afyap1 for defence against ROS, the Afyap1 deletion mutant was not reduced in pathogenicity in a low dose murine infection model for invasive aspergillosis. These data indicate that at least in the mouse infection model, ROI do not play a significant role in killing of A. fumigatus by immune effector cells.

E Harrison, A Pasqualotto, S Howard, M Anderson, D Denning, P Bowyer

Aspergillomas caused by Aspergillus fumigatus are a rare but life threatening form of fungal disease. Currently we do not understand the basis of aspergilloma formation. Aspergillomas form as a large fungal mass in the lung. It is supposed that they form in pre-existing cavites in the lung and develop over a number of years until they are noticed when patients present with fever coughing or haemoptysis. Aspergillomas are generally treated with antifungal agents but due to their large size are prone to become resistant and may, in certain cases, need to be removed by surgery. Aspergillomas carry a 40% 5 years survival, and it not clear whether antifungal therapy is helpful. We have obtained ethical approval to take surgically resected aspergillomas and analyse them in our laboratory.

We have obtained evidence that the fungus arises from a single inoculum and appears to evolve during growth in the chest. Recent work using comparative genome hybridisation-microarray based analysis shows that the fungus adapts to the constrained and hostile lung/drug environment by large scale loss and duplication of between 30 and 150 genes as well as by mutations to azole resistance in the cyp51A gene. This displays a previously unsuspected and unprecendented ability of the fungus to evolve rapidly over a short period. This ability may have important implications in fungal pathogenicity generally.

Christian B. Fleck, Matthias Brock

Phospholipases are assumed to weaken the membrane stability of mammalian cells by releasing fatty acids from the phospholipid layer. It has been shown that phospholipase activity contributes to virulence of different Candida species. However, although phospholipase activity was also detected in supernatants of Aspergillus fumigatus isolates, neither one of the responsible proteins has been purified nor has their impact on virulence been studied.

Since the genome of A. fumigatus contains several genes coding for secreted phospholipases, a deletion strategy for all coding genes is not recommended. Therefore, we used phospholipid-containing media to screen the supernatants for putative phospholipases by 2-D-gel electrophoresis. Interestingly, although more than 100 protein spots were analysed, none of them coded for one of the annotated phospholipases. In contrast, two extracellular lipases were found, which may contribute to the phospholipase activities detected in former studies. Since both proteins were absent from medium of glucose grown cells, both lipases are specifically induced in the presence of, at least, phospholipids. Currently, we are overproducing the enzymes in A. fumigatus to study their substrate specificity and to use the purified proteins to investigate them for their ability to cause membrane destruction of different mammalian cell lines. Additionally, gene deletions will reveal, whether these lipases contribute to virulence of A. fumigatus.