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Expression and functional analyses of the major surface antigens of the human pathogenic fungus Pneumocystis jirovecii

English title Expression and functional analyses of the major surface antigens of the human pathogenic fungus Pneumocystis jirovecii
Applicant Hauser Philippe
Number 192802
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 01.05.2020 - 30.04.2024
Approved amount 800'000.00
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All Disciplines (2)

Discipline
Experimental Microbiology
Medical Microbiology

Keywords (7)

CCS deep long reads sequencing; protein heterologous expression; Surface antigen; surface antigenic variation; Pneumocystis jirovecii; adhesion; transcriptomics

Lay Summary (French)

Lead
Pneumocystis jirovecii est un champignon qui provoque une pneumonie mortelle chez les patients immunodéprimés en raison du sida, d'une transplantation d'organe ou d'un cancer. Nous avons récemment réussi à assembler les sous-télomères de cet agent pathogène et à identifier six familles de gènes hypervariables qui codent les principales glycoprotéines de surface (MSG). Ces protéines constituent un facteur potentiel de virulence et/ou de colonisation car elles génèrent une variation antigénique de surface permettant vraisemblablement d'échapper au système immunitaire humain, tout en assurant l'adhésion aux cellules hôtes. Dans le présent projet, nous étudierons l'expression de ces protéines aux niveaux de l'ADN et de l'ARN, ainsi que leurs fonctions.
Lay summary
Dans une première partie, nous caractériserons les répertoires exprimés et non exprimés de la famille I des MSG de P. jirovecii chez les patients utilisant le séquençage circulaire de consensus à haut débit. Nous comparerons la composition des répertoires entre des patients de différentes localisations géographiques et avec diverses conditions sous-jacentes. 

Dans une deuxième partie, nous détecterons le MSG de la famille I exposéà la surface des sous-populations de P. jirovecii in vivo en utilisant des anticorps polyclonaux. Ces expériences permettront de tester l'hypothèse de l'existence de sous-populations cellulaires exprimant chacune une isoforme spécifique de MSG-I.

Dans une troisième partie, nous étudierons l'expression des MSG de P. jirovecii chez les patients au niveau de l'ARN en utilisant un enrichissement des séquences de P. jirovecii par hybridation avec des oligonucléotides couvrant l'ORFeome. Cela permettra de comprendre le mode et le niveau d'expression des six familles de MSGs.

Dans une quatrième partie, nous allons étudier la fonction adhésive des MSG de P. jirovecii par expression hétérogène dans Saccharomyces cerevisiae. Ces cellules seront testées dans des tests d'adhérence sur cellules entières. Ces expériences permettront de détermined à quelles molécules adhèrent les MSG des différentes familles. 

Importance et impact :
Les résultats permettront de comprendre l'interaction entre le champignon et les cellules hôtes, et éventuellement d'identifier de nouvelles cibles thérapeutiques. Le caractère unique du système étudié pourrait également permettre de mieux comprendre l'immunité humaine contre les pathogènes fongiques.

Direct link to Lay Summary Last update: 15.05.2020

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Associated projects

Number Title Start Funding scheme
146135 Characterization of the telomeres and comparative genomics of the human pathogenic fungus Pneumocystis jirovecii 01.05.2013 Project funding
182162 Construction of yeast cells covered with native glycoproteins of the human pathogenic fungus Pneumocystis jirovecii 01.07.2018 Scientific Exchanges
165825 Whole-genome and sex-related transcriptomics of the pathogenic fungus Pneumocystis jirovecii within human lungs 01.05.2016 Project funding

Abstract

Background:Pneumocystis jirovecii is a fungus that causes life-threatening pneumonia in patients who are immuno-compromised because of AIDS, organ transplantation, or cancer. The absence of in vitro culture method for this pathogen complicates the study of its biology and pathogenicity. Using single molecule real-time sequencing, we recently succeeded in assembling its subtelomeres from a single clinical specimen and identifying six hypervariable gene families that encode major surface glycoproteins (MSG). These proteins constitute a potential virulence factor because they generate surface antigenic variation allowing presumably escape from the human immune system, while ensuring adhesion to host cells. MSGs of family I (MSG-I) are thought to be the most abundant at the cell surface, and only one isoform would be expressed at a time in each P. jirovecii cell. This mutually exclusive expression would rely on the expression of a single isoform under the control of a transcription promoter that is present at a single copy per genome, whereas the other genes of the family I have no promoter. However, at the population level, expression of several different isoforms is observed. Each gene of the other five MSG families possesses its own promoter, the mode and level of expression remaining uncharacterized. Recombinations between the genes of each MSG family would generate gene mosaicism that contributes to antigenic variation. MSGs-I are known be involved in adhesion to type I alveolar epithelial cell, whereas the function of the MSGs of the other families has not been investigated yet.Working hypothesis: P. jirovecii has a unique system of antigenic variation: it segregates continuously cells expressing each a specific MSG-I isoform, as well as in minority a mixture of mosaic isoforms of the other MSG families. Consequently, each P. jirovecii population is made of subpopulations that are antigenically different.Specific aims:Part 1: Characterization of the expressed and non-expressed repertoires of P. jirovecii MSG-I in patients. The full-length MSG-I genes linked, or not linked, to the single copy promoter will be amplified from patients’ samples using generic primers. The PCR products will be deep sequenced using high throughput circular consensus sequencing that generates long and highly accurate reads. We will compare the composition of the expressed and non-expressed repertoires of MSG-I between patients from different geographical locations and with various underlying conditions. The results will help understanding the mechanisms of mutually exclusive expression, and thus the strategy of antigenic variation of the fungus. These repertoires will also allow investigating possible hotspots of recombination using bioinformatics, as well as selecting peptides in order to raise antibodies for part 2 of the project.Part 2: Detection of the MSG-I displayed on the surface of P. jirovecii subpopulations in vivo. We will raise polyclonal antibodies against the most abundantly expressed MSG-I isoforms identified within given patients in part 1. These antibodies will be used to detect these isoforms on the surface of P. jirovecii cells using co-staining in immunochemistry and recombinant yeasts as control. These experiments will allow testing the hypothesis of the existence of cell subpopulations expressing each a specific MSG-I isoform.Part 3: Study of the expression of P. jirovecii MSGs in patients by RNAseq. We will use complementary DNAs (cDNA) from patients’ samples enriched in P. jirovecii cDNA by hybridization to oligonucleotides covering the ORFeome, including MSGs. We will attribute illumina cDNA reads to a single MSG family using generalized profiles in silico. The number of isoforms of each MSG family expressed will be determined using a dedicated bioinformatics procedure, as well as by PCR followed by sequencing. This will provide insights into the mode and level of expression of all six MSG families.Part 4: Study of the adhesive function of P. jirovecii MSGs4.1. Heterologous expression of full-length MSGs and whole-cell adherence assays. Specific MSGs of each family will be expressed in Saccharomyces cerevisiae and displayed at the cell surface. We will use a wild type strain as well as a strain mimicking the absence of hyper-mannan glycosylation in P. jirovecii. These cells will be used in whole-cell adherence assays to relevant human proteins and receptors adsorbed on magnetic beads using cell counting as readout. We will also study adherence in absence of glycosylation. These experiments will assess if the MSGs of the different families are adhesins, as well as to which molecules they adhere.4.2. Heterologous expression of MSG domains and adherence assays. Thirteen domains composing the MSGs of the six families will be expressed fused to a carrier protein in Escherichia coli as well as in S. cerevisiae strains with or without hyper-mannan glycosylation. After purification, we will analyze each domain in the adherence assays set up in part 4.1. using SDS-PAGE as readout. These experiments will allow investigating the specific adhesive properties of these domains.4.3. Heterologous expression of full-length MSGs and identification of human ligands. Specific MSGs of each family will be expressed without its GPI anchor signal in the highly efficient heterologous expression system of Pichia pastoris, and purified from the growth medium. We will also use expression in S. cerevisiae without hyper-mannan glycosylation. Each purified MSG will be mixed with human lung epithelial cells in vitro. We will identify the human protein ligands interacting with each MSG by co-immunoprecipitation using an antibody directed against the MSG, followed by peptides sequencing using tandem mass spectrophotometry. These experiments will allow identifying new host receptors for P. jirovecii potentially present on epithelial cells.Importance and impact:The in vivo studies of human infections that we propose (parts 1 to 3) are particularly relevant because animal and in vitro models do not reproduce fully the natural infections. The proposed analyses of the P. jirovecii system of antigenic variation and adhesion will help understanding the interaction between the fungus and host cells, possibly identifying new drug targets. The uniqueness of the system studied may also lead to a better understanding the human immunity against fungal pathogens.
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