Transcriptomics; whole-genome transcriptional profiles; Pneumocystis jirovecii
Luraschi Amanda, Richard Sophie, Hauser Philippe Marcel (2019), Reply to Nevez and Le Gal, “Caspofungin and Pneumocystis Pneumonia: It Is Time To Go Ahead”, in Antimicrobial Agents and Chemotherapy
, 63(10), 01320.
Luraschi A., Richard S., Almeida J. M. G. C. F., Pagni M., Cushion M. T., Hauser P. M. (2019), Expression and Immunostaining Analyses Suggest that Pneumocystis Primary Homothallism Involves Trophic Cells Displaying Both Plus and Minus Pheromone Receptors, in mBio
, 10(4), e01145.
Luraschi A., Richard S., Hauser P. M. (2018), Site-Directed Mutagenesis of the 1,3-β-Glucan Synthase Catalytic Subunit of Pneumocystis jirovecii and Susceptibility Assays Suggest Its Sensitivity to Caspofungin, in Antimicrobial Agents and Chemotherapy
, 62(12), 01159.
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), 00221.
Luraschi Amanda, Cissé Ousmane H., Pagni Marco, Hauser Philippe M. (2017), Identification and Functional Ascertainment of the Pneumocystis jirovecii Potential Drug Targets Gsc1 and Kre6 Involved in Glucan Synthesis, in Journal of Eukaryotic Microbiology
, 64(4), 481-490.
Background: Pneumocystis jirovecii is a fungus which 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 slowed down the understanding of its biology and pathogenicity. We recently succeeded to sequence the P. jirovecii genome from the microbiome present in a single bronchoalveolar lavage (BAL) specimen of a single patient with Pneumocystis pneumonia (PcP). The availability of this genome allows new analyses to be performed, such as transcriptomics. The reservoir of P. jirovecii includes patients without recognized PcP who are colonized with a low fungal load. The fungus is probably in a different physiological state (slow growth) during colonization than during active PcP (rapid growth). Consequently, the fungal genome is probably expressed differentially, virulence factors being over-expressed during PcP. Comparative whole-genome transcriptional analyses can be used to identify these virulence factors. A recognized virulence factor of P. jirovecii is the gene family encoding isoforms of the major surface glycoprotein (MSG) involved in antigenic variation and adhesion to target host lung cells. Only one MSG isoform localized downstream of a conserved expression site present at a single copy per genome is believed to be expressed at a time in a given cell. We hypothesize that the diversity of the expressed MSGs within P. jirovecii populations is smaller during colonization than during PcP because of an increased pressure by the human immune system. Another crucial feature of P. jirovecii is its probable obligate sexual reproduction within human lungs. We recently identified the mating type (MAT) genes responsible for sexual differentiation as well as other sex-related genes in the genome of P. jirovecii. The structure of the MAT locus and the identity of the genes present suggest self-fertility through primary homothallism. Transcriptional analyses of these genes within human lungs are required to further characterize this sexual reproduction. Specific aims: - to identify the virulence factors of P. jirovecii (Part 1).- to study the expression and diversity of P. jirovecii MSGs (Part 2).- to characterize the occurrence and mechanisms of the sexual reproduction of P. jirovecii (Part 3).Experimental design :Part 1.: Identification of the virulence factors of P. jirovecii P. jirovecii transcriptomes will be determined using illumina sequencing of the enriched cDNAs and the differential expression of the whole genome between the two conditions will be analyzed. Potential virulence factors over-expressed during PcP will be investigated. Part 2.: Study of the expression and diversity of P. jirovecii MSGsThe differential expression of the MSGs between colonization and PcP will be analyzed in the whole-genome transcriptional profiles obtained in part 1 using the expression site as surrogate marker. The diversity of the expressed MSGs will be determined by the analysis of the MSGs linked downstream to the expression site within the transcriptional raw reads. Part 3.: Characterization of the sexual reproduction of P. jirovecii 3.1. Characterization of the expression of MAT and sex-related genes within human lungsThe differential expression of the P. jirovecii MAT and other sex-related genes between colonization and PcP will be analyzed in the whole-genome transcriptional profiles obtained in part 1. These analyses will allow characterizing the system of P. jirovecii sexual reproduction, as well as assessing its occurrence during colonization and PcP. 3.2. Detection of receptors to mating pheromones on the surface of trophic formsThe receptors to pheromones of mating types Minus and Plus will be stained on the surface of P. jirovecii trophic cells using fluorescent antibodies. Controls will consist in staining Saccharomyces cerevisiae cells expressing the P. jirovecii receptors. These experiments will allow further assessing P. jirovecii sexuality, and determining if each cell expresses both mating types or only one.Importance and impact:In vivo transcriptomics during human infections are particularly relevant because, as shown for other fungal pathogens, animal and in vitro models do not reproduce fully the natural infections. The identification of the virulence factors of P. jirovecii and the characterization of its sexual reproduction may help fighting the disease by the identification of potential new targets for therapeutic intervention. The results may lead to new knowledge associated with the fungal opportunistic life style which may have broad applications to other fungi pathogenic to humans.