Project description:Pneumocystis carinii is an ascomycete phylogenetically related to Schizosaccharomyces pombe. Little is known about gene regulation in P. carinii. The removal of introns from pre-mRNA requires spliceosomal recognition of the intron-exon boundary. In S. pombe and higher eukaryotes, this boundary and a branch site within the intron are conserved. We recently demonstrated that P. carinii cdc2 cDNA can complement S. pombe containing conditional mutations of cdc2, an essential gene involved in cell cycle regulation. We next tested whether P. carinii genomic cdc2 (with six introns) could also complement S. pombe cdc2 mutants and found genomic sequences incapable of this activity. Reverse transcriptase PCR confirmed the inability of the S. pombe cdc2 mutants to splice the P. carinii genomic cdc2. Analysis of 83 introns from 19 P. carinii protein-encoding genes demonstrated that the sequence GTWWDW functions as a donor consensus in P. carinii, whereas YAG serves as an acceptor consensus. These sequences are similar in S. pombe; however, a branch site sequence was not found in the P. carinii genes studied.
Project description:Pneumocystis pneumonia remains an important complication of immune suppression. The cell wall of Pneumocystis has been demonstrated to potently stimulate host inflammatory responses, with most studies focusing on β-glucan components of the Pneumocystis cell wall. In the current study, we have elaborated the potential role of chitins and chitinases in Pneumocystis pneumonia. We demonstrated differential host mammalian chitinase expression during Pneumocystis pneumonia. We further characterized a chitin synthase gene in Pneumocystis carinii termed Pcchs5, a gene with considerable homolog to the fungal chitin biosynthesis protein Chs5. We also observed the impact of chitinase digestion on Pneumocystis-induced host inflammatory responses by measuring TNFα release and mammalian chitinase expression by cultured lung epithelial and macrophage cells stimulated with Pneumocystis cell wall isolates in the presence and absence of exogenous chitinase digestion. These findings provide evidence supporting a chitin biosynthetic pathway in Pneumocystis organisms and that chitinases modulate inflammatory responses in lung cells. We further demonstrate lung expression of chitinase molecules during Pneumocystis pneumonia.
Project description:In the fungus Pneumocystis carinii, at least three gene families (PRT1, MSR, and MSG) have the potential to generate high-frequency antigenic variation, which is likely to be a strategy by which this parasitic fungus is able to prolong its survival in the rat lung. Members of these gene families are clustered at chromosome termini, a location that fosters recombination, which has been implicated in selective expression of MSG genes. To gain insight into the architecture, evolution, and regulation of these gene clusters, six telomeric segments of the genome were sequenced. Each of the segments began with one or more unique genes, after which were members of different gene families, arranged in a head-to-tail array. The three-gene repeat PRT1-MSR-MSG was common, suggesting that duplications of these repeats have contributed to expansion of all three families. However, members of a gene family in an array were no more similar to one another than to members in other arrays, indicating rapid divergence after duplication. The intergenic spacers were more conserved than the genes and contained sequence motifs also present in subtelomeres, which in other species have been implicated in gene expression and recombination. Long mononucleotide tracts were present in some MSR genes. These unstable sequences can be expected to suffer frequent frameshift mutations, providing P. carinii with another mechanism to generate antigen variation.
Project description:Mice immunized with recombinant mouse Pneumocystis carinii antigen A12-thioredoxin fusion protein developed an antibody response that recognized P. carinii antigens, as determined by Western blotting and immunofluorescence analysis. Compared to mice immunized with thioredoxin alone, mice immunized with A12-thioredoxin had significantly reduced lung P. carinii burdens after CD4+ T-cell depletion and challenge with P. carinii.
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.