Pneumocystis jirovecii; Pneumocystis carinii; genome sequencing; high throughput; Saccharomyces cerevisiae; Schizzosaccharomyces pombe; heterologous expression
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Summary of research planBackground : Pneumocystis jirovecii is a fungus which causes severe pneumonia specifically in immunocompromised humans. The lack of a culture method in vitro has hampered progress in the understanding of its biology. Numerous studies showed that P. jirovecii resistance to the most efficacious drugs is emerging. Pneumocystis carinii, the species that specifically infects the rat, can be isolated from lungs of experimentally infected animals; a draft assembly of its genome is available. On the other hand, sequencing P. jirovecii genome is complicated by the fact that its DNA can be isolated only from bronchoalveolar lavage specimens or lungs of patients with Pneumocystis pneumonia which implies small samples and contamination with other DNAs, including from the host. The microbiome present in lungs of patients with Pneumocystis pneumonia has not been investigated so far.Working hypothesis : Random DNA amplification followed by high throughput sequencing allow to sequence P. jirovecii genome from bronchoalveolar lavage specimens of patients with Pneumocystis pneumonia, and to study the lung microbiome associated with Pneumocystis pneumonia.Specific aims :Part 1 : De novo sequencing and analysis of P. jirovecii genomeThe genome of P. jirovecii will be sequenced using high throughput sequencing of a bronchoalveolar lavage specimen of a patient containing a high proportion of P. jirovecii DNA. After identification of the reads attributable to P. jirovecii by in silico comparison with relevant genomes, P. jirovecii genome will be assembled, annotated, and release in the public domain. The genome sequence will serve three purposes :1.The P. jirovecii orthologs of the potential drugs targets proposed in P. carinii will be isolated by homology, and amplified by PCR from clinical specimens. The function of these genes will be assessed by complementation of deletion mutants of Saccharomyces cerevisiae. 2.The P. jirovecii genome will be compared to those of close relatives of the Archiascomycetes class which can be grown in vitro in order to identify possible missing metabolic pathways responsible for the absence of growth of the Pneumocystis species in vitro. This part will also involve sequencing the genome of Taphrina deformans, a plant pathogen fungus.3.The completed P. jirovecii genome will allow to search for the presence of this pathogen in silico by scanning the environmental metagenomic sequences released in the public domain.Part 2 : Study of Pneumocystis pneumonia lung microbiomeHigh throughput sequencing of DNA from bronchoalveolar lavage specimens will be used to characterize the microbiome present in the lungs of several patients with Pneumocystis pneumonia by comparison to control patients. This study may help understand factors that may predispose to the disease and may reveal previously undetected micoorganisms or virus which could be associated with P. jirovecii. Possible associations between specific compositions of the microbiome and various parameters will be investigated. Specific PCRs in the laboratory and review of patients’ charts will be involved.Expected value of the proposed project : Most knowledge about the genus Pneumocystis was obtained from P. carinii because the rat model of infection provides many fungal cells and allows to test the efficacy of potential new drugs. The sequence of P. jirovecii genome will allow to get insights into the biology of the pathogen that actually infects humans, and to better characterize the actual targets against which new drugs should be developed. This is important because drug resistance is emerging in P. jirovecii. The analysis of P. jirovecii genome may also reveal why it could not be grown in vitro or suggest supplements that may allow growth in vitro, providing a key tool for research. The results may lead to new knowledge associated with the fungal opportunistic life style which may have broad applications to other pathogenic fungi. Finally, the results will add to the basic knowledge of S. pombe and T. deformans, as well as of the microbiome of human lungs infected or not with P. jirovecii, about which few is known.