Pathogenic factors

Functional genome annotation

• Development of molecular tools for functional genomics
  
  In collaboration with Dr. Christopher Thornton from the University of Exeter (UK) and Dr. Thomas Guillemette from the FungiSem team at the IRHS in Angers, a protocol for obtaining protoplasts has been developed over the last few months, as well as a method for transformation and gene invalidation using the hygromycin B resistance gene.
  
  Nevertheless, functional complementation, such as the preparation of multiple mutants, will require the search for additional selection markers to complement the existing panel. In parallel, expression systems will be developed to assess the impact of gene overexpression.

• Melanin synthesis pathway
  
  In addition to protecting fungal cells against UV rays or elevated temperatures, melanin is also known to ensure wall rigidity, bind metals and store water and ions to prevent desiccation. Melanin also plays an important role in pathogenicity, protecting fungi from antimicrobial peptides and antifungals, and masking wall polysaccahrides to prevent their recognition by PRPs. Inhibition of melanin synthesis is associated with loss of pathogenicity in Sporothrix schenkii and A. fumigatus. In the latter, dihydroxynaphthalene (DHN)-melanin also plays an indirect role in pathogenicity, by participating in the assembly of various spore wall components and the surface expression of molecules involved in adhesion and hydrophobins.
  
  In addition, invalidation of the ALB1 gene encoding polyketide synthase results in a loss of virulence and increased sensitivity to host defense systems, notably oxygen radicals, while contradictory results have been reported for invalidation of genes encoding enzymes involved in the later stages of this biosynthetic pathway.
  
  takes place essentially, if not exclusively, via the dihydroxynaphthalene (DHN-melanin) pathway, a complex metabolic pathway that involves six clustered genes in A. fumigatus. Bioinformatics analysis of the S. apiospermum genome has identified the corresponding cluster. The various genes will therefore be inactivated and the phenotypic characterization of the mutants will be undertaken.

Melanin synthesis pathway in A. fumigatus and organisation of the genes involved.

• Oxidative stress enzymes
  
  In this field, we have purified and characterised in S. boydii two of the enzymes involved in the degradation of oxygen radicals produced by phagocytic cells, a cytosolic superoxide dismutase and a monofunctional catalase, and sequenced the corresponding genes. In addition, recombinant proteins were produced, the value of which in the serodiagnosis of scedosporiosis was demonstrated.
  
  The characterisation of oxidative stress enzymes is being pursued as part of Cindy Staerck's thesis. A bioinformatics analysis revealed the presence in the S. apiospermum genome of 7 genes encoding superoxide dismutases, 3 encoding catalases, 7 peroxidases, 6 thioredoxins/glutaredoxins and 5 nitrate and nitrite reductases. The expression of these genes is assessed under oxidative stress conditions and in S. apiospermum phagocytic cell/germ tube co-culture systems. The best candidates will be invalidated, and the impact of these mutations on pathogenicity will be clarified.

• Cyclic AMP signalling pathway
  
  In eukaryotes, the cyclic AMP (cAMP) signalling cascade is found in both humans and pathogenic fungi. An external stimulus, usually relayed by a G protein-coupled receptor, activates adenylate cyclase, which in turn activates protein kinase A to synthesise cAMP. Activation of adenylate cyclase by carbonic anhydrase via the synthesis of bicarbonate ions appears to be more specific to fungi.
  
  Preliminary work shows that exposure of S. apiospermum cultures to an atmosphere enriched in carbon dioxide, such as the addition of bicarbonate or an adenylate cyclase agonist to the culture medium, stimulates hyphal growth and increases the rate of spore germination. These results confirm that, as in other pathogenic fungal species, the partial pressure of CO2 (ppCO2) regulates morphogenesis in S. apiospermum, probably via the cAMP pathway.
  We are therefore currently studying the signalling cascade activated by the increase in ppCO2 in S. apiospermum, in order to characterise the molecular mechanisms regulating morphogenesis in this fungus via this signalling cascade and to determine its possible involvement in the low intrinsic sensitivity of this fungus to antifungal agents.
  
  Bioinformatics analysis was used to identify the various genes in the S. apiospermum genome the various genes potentially involved in the response to CO2, encoding carbonic anhydrase, phosphodiesterase which enables the recycling of cAMP and thus ensures transient activation of the pathway, the catalytic and regulatory subunits of protein kinase A, the transcription factor Efg1 which targets protein kinase A, and an orthologue of the transcription factor Rca1 which regulates the expression of carbonic anhydrase in C. albicans. These genes of interest will be inactivated by deletion and the phenotype of the resulting mutants will be determined.

cyclic AMP signalling pathway

• Histidine kinases and associated cell signalling pathways
  
  Histidine-aspartate kinase signalling pathways are used by bacteria, plants and fungi to perceive a wide range of environmental signals and propose an appropriate response. While the successive phosphorylation patterns involved in these pathways are fairly simple in prokaryotes, they are generally more complex in plants and pathogenic fungi, where they are referred to as multiple phosphorylation relays.
  Recent work has focused on the involvement of histidine-aspartate kinase systems in morphogenesis and virulence in various fungal pathogens. It is now accepted that the primary players in these pathways, namely histidine kinases (HK), are virulence factors in C. albicans, Cryptococcus neoformans and dimorphic fungi.

An initial bioinformatics analysis of the S. apiospermum genome shows that this fungus has six distinct genes potentially encoding HKs. The function of these genes is currently being investigated as part of Anaïs Hérivaux's thesis.

Theoretical diagram showing the canonical organisation of histidine aspartate kinase systems in fungi.

Comparative genomics approaches

Scedosporium apiospermum is now considered to be a complex of five morphologically indistinguishable species, only four of which are known to be involved in cystic fibrosis.

The S. apiospermum complex includes a fifth species called Scedosporium dehoogii, which is abundant in the environment and equally virulent in an experimental model of immunocompromised mice, but curiously rare in human pathology.

In this study, carried out in collaboration with the EcoFun (Dr Bruno Le Cam) and BioInfo (Dr Jean-Pierre Renou) teams at the IRHS (Angers), the genome of the pathogenic species of the S. apiospermum complex will be compared with that of S. dehoogii. To overcome the polymorphism that exists in all living species, the genome of five strains of each species will be sequenced and a comparative genomics bioinformatics analysis will be carried out within the complex. This research project is expected to identify genes potentially involved in the pathogenicity of S. apiospermum.