De novo sequencing of the genome of the human pathogenic fungus Pneumocystis jirovecii and study of Pneumocystis pneumonia lung microbiome

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. 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 new techniques of random DNA amplification and high throughput sequencing will 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.

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. It will serve two main 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.

High 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. Possible associations between specific compositions of the microbiome and various parameters will be investigated.

Most knowledge about the genus Pneumocystis was obtained from P. carinii, the rat model of infection. 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. Finally, the results will add to the basic knowledge of the microbiome of human lungs infected or not with P. jirovecii, about which few is known.