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Characterization of the telomeres and comparative genomics of the human pathogenic fungus Pneumocystis jirovecii

English title Characterization of the telomeres and comparative genomics of the human pathogenic fungus Pneumocystis jirovecii
Applicant Hauser Philippe
Number 146135
Funding scheme Project funding
Research institution Institut de Microbiologie - CHUV Faculté de Biologie et Médecine Université de Lausanne
Institution of higher education University of Lausanne - LA
Main discipline Experimental Microbiology
Start/End 01.05.2013 - 30.04.2016
Approved amount 343'960.00
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All Disciplines (2)

Discipline
Experimental Microbiology
Medical Microbiology

Keywords (6)

Pneumocystis murina; Schizzosaccharomyces pombe; Pneumocystis carinii; Saccharomyces cerevisiae; high throughput; Pneumocystis jirovecii

Lay Summary (French)

Lead
Les patients immunocompromis sont à risques pour une infection par Les patients immunocompromis sont à risques pour une infection par Pneumocystis jirovecii, un champignon qui causent des pneumonies sévères. Nous avons assemblé le génome de P. jirovecii grâce au séquençage à haut débit du microbiome présent dans un échantillon clinique d'un seul patient. Les séquences d'ADN courtes obtenues avec la méthode de séquençage utilisée n'ont toutefois pas permis d'assembler les télomères.
Lay summary
Hypothèse de travail : la nouvelle méthode de séquençage en temps réel de molécules uniques permet de caractériser la structure des télomères de P. jirovecii, ainsi que de générer des génomes d’espèces de Pneumocystis d’une qualité adéquate pour les comparaison.Objectifs :Partie 1 : Caractérisation de la structure des télomères de P. jirovecii. L’ADN génomique sera obtenus à partir d’un seul lavage broncho-alvéolaire d’un seul patient contenant une grande quantité de P. jirovecii. La structure sera caractérisée grâce aux longues séquences obtenues grâce à la méthode de séquençage en temps réel de molécules uniques. . Ces familles de gènes sont responsables pour un système de variation antigénique et sont d'une importance capitale pour ce parasite.Partie 2 : Comparaison de génomes de différentes espèces de Pneumocystis. Les génomes de P. murina and P. carinii, espèces infectant respectivement les souris et les rats, seront séquencés et annotés grâce au séquençage à haut débit. La synténie et les éventuels réarrangements seront établis. La comparaison des génomes d’espèces cousines appartenant au même groupe taxonomique (Archiascomycetes) permettra de comprendre la spécificité d’hôte et la réticence des différentes espèces du genre Pneumocystis à croître au laboratoire. Valeur attendue du projet : La caractérisation des télomères est potentiellement cruciale pour combattre la maladie. De nouvelles cibles pour médicaments ou développer un vaccin pourraient être identifiées cat la variation antigénique est un élément clé dans la pathogenèse de ceparasite. La comparaison des espèces de Pneumocystis pourrait permettre de comprendre les bases moléculaires de la spécificité d’hôte. La comparaison avec des cousins plus éloigné pourrait permettre d’identifier des suppléments permettant la croissance de ces champignons au laboratoire, ce qui serait un outil important pour la recherche. Les résultats pourraient avoir des applications pour d’autres champignons pathogènes.

 

Direct link to Lay Summary Last update: 08.04.2013

Responsible applicant and co-applicants

Employees

Publications

Publication
Functional and Expression Analyses of the Pneumocystis MAT Genes Suggest Obligate Sexuality through Primary Homothallism within Host Lungs
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.
Mechanisms of Surface Antigenic Variation in the Human Pathogenic Fungus Pneumocystis jirovecii
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.
Comparative genomics suggests primary homothallism of Pneumocystis species.
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.
Functional characterization of the Pneumocystis jirovecii potential drug targets dhfs and abz2 involved in folate biosynthesis.
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.
Comparative genomics suggests that the human pathogenic fungus Pneumocystis jirovecii acquired obligate biotrophy through gene loss.
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.
Genomic insights into the fungal pathogens of the genus pneumocystis: obligate biotrophs of humans and other mammals.
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.
Molecular epidemiology of Pneumocystis outbreaks
Hauser M. Philippe, Kovacs Joseph (2014), Molecular epidemiology of Pneumocystis outbreaks, in Kurzai Oliver (ed.), Springer, Berlin, 191-203.

Collaboration

Group / person Country
Types of collaboration
A. Fonseca, J. Almeida, Universita nova di Lisboa Portugal (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
K. Mühlethaler, Institut für Infektionskrankheiten, Universität Bern Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
M. Pagni, Swiss Bioinformatics Institute, Vital-it Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
14th International Workshop on Opportunistic Protists Talk given at a conference Further evidence of primary homothallism in Pneumocystis species 09.08.2017 Cincinnati, United States of America Hauser Philippe; Luraschi Amanda;
14th International Workshop on Opportunistic Protists Talk given at a conference Mechanisms of antigenic variation in the human pathogenic fungus Pneumocystis jirovecii 09.08.2017 Cincinnati, United States of America Hauser Philippe; Luraschi Amanda;
73rd Annual Assembly of Swiss Society of Microbiology Poster Functional characterization of the Pneumocystis jirovecii potential drug targets dhfs and abz2 involved in folate biosynthesis 28.05.2015 Lugano, Switzerland Luraschi Amanda;
13th International Workshop on Opportunistic Protists Talk given at a conference Comparative Genomics Suggests That the Human Pathogenic Fungus Pneumocystis jirovecii Acquired Obligate Biotrophy through Gene Loss 13.11.2014 Sevilla, Spain Hauser Philippe;
13th International Workshop on Opportunistic Protists Talk given at a conference Comparative genomics suggests primary homothallism of Pneumocystis spp. 13.11.2014 Sevilla, Spain Hauser Philippe;


Awards

Title Year
Master thesis, Faculty of Biology and Medicine award 2015

Associated projects

Number Title Start Funding scheme
192802 Expression and functional analyses of the major surface antigens of the human pathogenic fungus Pneumocystis jirovecii 01.05.2020 Project funding
124998 De novo sequencing of the genome of the human pathogenic fungus Pneumocystis jirovecii and study of Pneumocystis pneumonia lung microbiome 01.10.2009 Project funding
165825 Whole-genome and sex-related transcriptomics of the pathogenic fungus Pneumocystis jirovecii within human lungs 01.05.2016 Project funding

Abstract

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.
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