Project description:Pneumocystis is a relevant genetic system to study centromere formation in relation with host adaptation. How centromeres are formed and maintained in strongly host adapted fungal pathogens is poorly investigated. Centromeres are genomic regions that coordinate accurate chromosomal segregation during mitosis and meiosis. Yet, despite their essential function, centromeres evolve rapidly across eukaryotes. CENP-A, a variant of histone H3 is the epigenetic marker that define centromeres in most eukaryotes. Centromeres are often the sites of chromosomal breaks which contribute to genome shuffling and promote speciation by inhibiting gene flow. Genome shuffling allows genome reconfiguration suitable for survival in new environment such as pathogen adaptation to new hosts. Here, we study the evolution of centromeres in closely related species of mammalian specific pathogens of the fungal phylum of Ascomycota. Long term culture of Pneumocystis species is currently untenable. Using heterologous complementation, we show that Pneumocystis CENP-A ortholog is functionally equivalent to fission yeast Cnp1. Using a short-term in vitro culture, infected animal models and ChIP-seq, we identified centromeres in three Pneumocystis species that diverged ~100 Mya ago. Each species has 17 unique short regional centromeres (< 10kb) in 17 monocentric chromosomes. The centromeres are flanked by heterochromatin. They span active genes, lack conserved DNA sequence motifs, and repeats.These features suggest an epigenetic specification of centromere function.
Project description:This study was done to show the utility of precision-cut lung slices (PCLS) in supporting the survival of Pneumocystis murina in vitro.
Project description:The major surface glycoprotein (Msg) is the most abundant surface protein of Pneumocystis species. Given that Msg is present on both the cyst and trophic form of Pneumocystis, and dendritic cells play a critical role in initiating host immune responses, we undertook studies to examine activation of bone marrow-derived myeloid dendritic cells by Msg purified from P. murina. Incubation of dendritic cells with Msg did not lead to increased expression of CD40, CD80, CD86, or MHCII, or increased secretion of any of 10 cytokines. Microarray analysis identified very few differentially expressed genes. In contrast, LPS activated dendritic cells by all of these assays. However, Msg did bind to mouse mannose macrophage receptor and human DC-SIGN, two C-type lectins expressed by dendritic cells that are important in recognition of pathogen-associated high mannose glycoproteins. Deglycosylation of Msg demonstrated that this binding was dependent on glycosylation. These studies suggest that Pneumocystis has developed a mechanism to avoid activation of dendritic cells, potentially by the previously identified loss of genes that are responsible for the high level of protein mannosylation found in other fungi.
Project description:Pneumocystis is a relevant genetic system to study centromere formation in relation with host adaptation. How centromeres are formed and maintained in strongly host adapted fungal pathogens is poorly investigated. Centromeres are genomic regions that coordinate accurate chromosomal segregation during mitosis and meiosis. Yet, despite their essential function, centromeres evolve rapidly across eukaryotes. CENP-A, a variant of histone H3 is the epigenetic marker that define centromeres in most eukaryotes. Centromeres are often the sites of chromosomal breaks which contribute to genome shuffling and promote speciation by inhibiting gene flow. Genome shuffling allows genome reconfiguration suitable for survival in new environment such as pathogen adaptation to new hosts. Here, we study the evolution of centromeres in closely related species of mammalian specific pathogens of the fungal phylum of Ascomycota. Long term culture of Pneumocystis species is currently untenable. Using heterologous complementation, we show that Pneumocystis CENP-A ortholog is functionally equivalent to fission yeast Cnp1. Using a short-term in vitro culture, infected animal models and ChIP-seq, we identified centromeres in three Pneumocystis species that diverged ~100 Mya ago. Each species has 17 unique short regional centromeres (< 10kb) in 17 monocentric chromosomes. The centromeres are flanked by heterochromatin. They span active genes, lack conserved DNA sequence motifs, and repeats.These features suggest an epigenetic specification of centromere function.