Context
Systemic fungal infections (fungemias) have emerged as important causes of morbidity and mortality in immunocompromised patients. In addition, hospital-related infections in patients not previously considered at risk have become a cause of major health concern.
Recent experiences of antifungal drug treatment failures combined with improvements in performance and standardization of antifungal susceptibility testing have drawn attention to the problem of antifungal resistance. Clinicians are particularly concerned by further drug-resistant fungi due to the increasing use of antifungal drugs, especially in settings such as hospitals where hospital-acquired infections are a growing problem.
There is a critical medical need for the development of antifungal agents. Since there are no fungal vaccines currently licensed the only clinical recourse is the use of therapeutics. Current antifungal treatments are limited by not being rapidly fungicidal, having limited spectrum, toxicity concerns, and emerging resistance.
Selected species
In our project, 3 reference genomes were used: Saccharomyces cerevisiae, Candida albicans and Aspergillus fumigatus to obtain antifungal targets against a large spectrum of microorganisms.
C. albicans is a causal agent of opportunistic oral and vaginal infections in humans. Under normal circumstances, C. albicans lives in 80% of the human population with no harmful effects, although overgrowth results in candidiasis. Candidiasis is often observed in immunocompromised individuals such as HIV-positive patient.
A. fumigatus is one of the most common Aspergillus species to cause disease in immunocompromised individuals. A. fumigatus is common in the natural environment and can also be found in the upper respiratory tracts of healthy individuals. Exposure to A. fumigatus in immunocompromised individuals can lead to aspergillosis, a pulmonary infection. Leukemia or bone marrow transplant patients are particularly touched.
S. cerevisiae can cause fungemia in immunocompromised individuals too. But in this project it has been used as a eucaryotic model organism.
Comparative Genomic
Comparative Genomic was used as a method of identification. 3 reference genomes were used: S. cerevisiae, C. albicans and A. fumigatus to obtain antifungal targets against a large spectrum of microorganism. A competition was made between Conservation between fungal genomes and Conservation between vertebrate genomes, in order to select the most conserved genes. Bioinformatics implementation of this strategy consisted in exhaustive optimal similarity searches of all putative ORFs.
Detailed sequence analysis
The antifungal drug must not be toxic for human that's why ideal candidates must be absent from the mammalian lineage. The implementation of this dialectic prioritization process requires the expertise in sequence/structure analysis and large-scale computing found in the IGS laboratory. The Increase of FCI (Conservation index between Fungi) vs decrease of VCI (Conservation index between vertebrates) conducted to the identification of 201 new targets.
A new selection was made with secondary criteria such as presence in C. albicans/S. cerevisiae essentiality in S. cerevisiae, known/unknown, structural homology and feasibility.
Production strategy
The structural genomic approach involved 4 different steps. We first did the high-throughput expression screening of C. albicans and S. cerevisiae proteins. The second step concerned the production and purification of the proteins of interest. At last, the crystallogenesis stage and the the structure determination were performed.
The functional annotation of potential ligand and the inhibitor identification were done with 3 approaches such as in silico annotation, enzymatic assay and structural annotation.
Status
A portfolio of available antifungal targets functionally annotated was obtained.
An example of antifungal target: CA1462
CA1462 is a thiamine pyrophosphokinase from C. albicans. To get further details about this protein, please click here (PDF file).