Pneumocystis murina; Schizzosaccharomyces pombe; Pneumocystis carinii; Saccharomyces cerevisiae; high throughput; Pneumocystis jirovecii
Richard S., Almeida J. M. G. C. F., Cissé O. H., Luraschi A., Nielsen O., Pagni M., Hauser P. M. (2018), Functional and Expression Analyses of the Pneumocystis MAT Genes Suggest Obligate Sexuality through Primary Homothallism within Host Lungs, in mBio
, 9(1), e02201-e02211.
Schmid-Siegert Emanuel, Richard Sophie, Luraschi Amanda, Mühlethaler Konrad, Pagni Marco, Hauser Philippe M. (2017), Mechanisms of Surface Antigenic Variation in the Human Pathogenic Fungus Pneumocystis jirovecii, in mBio
, 8(6), e01470-e01487.
Almeida João M G C F, Cissé Ousmane H, Fonseca Álvaro, Pagni Marco, Hauser Philippe M (2015), Comparative genomics suggests primary homothallism of Pneumocystis species., in mBio
, 6(1), 1-8.
Luraschi A, Cissé O H, Monod M, Pagni M, Hauser P M (2015), Functional characterization of the Pneumocystis jirovecii potential drug targets dhfs and abz2 involved in folate biosynthesis., in Antimicrobial agents and chemotherapy
, 59(5), 2560-6.
Cissé Ousmane H, Pagni Marco, Hauser Philippe M (2014), Comparative genomics suggests that the human pathogenic fungus Pneumocystis jirovecii acquired obligate biotrophy through gene loss., in Genome biology and evolution
, 6(8), 1938-48.
Hauser Philippe M (2014), Genomic insights into the fungal pathogens of the genus pneumocystis: obligate biotrophs of humans and other mammals., in PLoS pathogens
, 10(11), 1004425-1004425.
Hauser M. Philippe, Kovacs Joseph (2014), Molecular epidemiology of Pneumocystis outbreaks, in Kurzai Oliver (ed.), Springer, Berlin, 191-203.
Background: Immunocompromised patients with AIDS, organ transplantation and cancer are at risk of infection by Pneumocystis jirovecii, a fungus which causes severe pneumonia. However, research is hindered by the absence of in vitro culture method and thus the genome sequence of this pathogen was lacking. During the previous grant, we succeeded in assembling the P. jirovecii genome from the high throughput metagenome sequencing of a single bronchoalveolar lavage fluid specimen of a single patient with Pneumocystis pneumonia, using a specifically developed Bioinformatics procedure. The P. jirovecii genome obtained is fragmented in 358 contigs totaling 8.1Mb. Its availability allows new analyses to be performed, such as RNA sequencing and comparative genomics. The small reads obtained with the sequencing technologies used to sequence P. jirovecii genome did not allow assembling telomeres because of the repetitive nature of both their sequences and the subtelomeric gene families encoding surface proteins. These gene families are believed to encode a system of surface antigen variation and are thus of great importance in the biology of this colonizing parasite, so that characterization of telomeres structure, expression, and function remains a crucial challenge. Sequencing P. murina and P. carinii genomes, the species infecting respectively mice and rats, and their comparison with P. jirovecii genome would allow improving exploitation of these animal models of the human infection. Comparative genomics with Archiascomycetes relatives should help understanding the host specificity and reluctance to grow in vitro of Pneumocystis spp, but gap closure of P. jirovecii genome, as well as sequencing other Pneumocystis spp are prerequisites.Working hypothesis: Single molecule real-time sequencing and RNA sequencing allow characterizing the structure and expression of P. jirovecii telomeres, as well as generating genomes of Pneumocystis spp suitable for comparative genomics. Specific aims:Part 1: Characterization of the structure of P. jirovecii telomeres and expression of the subtelomeric gene familiesGenomic DNA and total RNA will be obtained from a single bronchoalveolar lavage specimen of a single patient containing a high load of a single P. jirovecii genotype. The selection of the suitable sample will involve genotyping to exclude co-infection with several genotypes and load quantification. Telomeres will be purified using a newly developed method of DNA-DNA hybridization capture. Their structure will be characterized using long DNA reads obtained with single molecule real-time sequencing. The expression of the subtelomeric surface antigen and other gene families will be investigated using deep cDNA sequencing (RNAseq) on the same sample.Part 2: Comparative genomics of Pneumocystis spp. and Archiascomycetes relativesIn the frame of an international collaboration, we will sequence and annotate P. murina and P. carinii genomes using high throughput sequencing and RNAseq. This will be facilitated by the availability of P. jirovecii genome and our Bioinformatics expertise acquired during the previous grant. Synteny and potential genome rearrangements between the three Pneumocystis genomes will be established. The genome of Taphrina deformans that we recently completed, as well as of other Archiascomycetes relatives recently released in the public domain, will be compared to those of Pneumocystis spp.Expected value of the proposed project: Characterization of P. jirovecii telomeres is crucial for understanding the organization and the expression of the subtelomeric gene families, and thus potentially for fighting the disease. It may lead to the identification of potential new targets for therapeutic intervention and vaccine development because these gene families are believed to be responsible for surface antigen variation, a key feature in the pathogenesis of this important human pathogen. Determination of P. carinii and P. murina genome sequences is crucial as their infections are the most used animal models of the human infection. Comparative genomics of Pneumocystis spp will help understanding the host-specificity of P. jirovecii, also an important feature of this pathogen. Comparison of Pneumocystis spp genomes with those of Archiascomycetes relatives may reveal why P. jirovecii could not be grown in vitro and 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.