Members of the genus Pneumocystis are fungal pathogens that cause pneumonia in a wide variety of mammals with debilitated immune systems. Little is known about their basic biological functions, including life cycle, since no species can be cultured continuously outside the mammalian lung. To better understand the pathological process, about 4500 ESTS derived from sequencing of the poly(A) tail ends of P. carinii mRNAs during fulminate infection were annotated and functionally characterized as unassembled reads, and then clustered and reduced to a unigene set with 1042 members. Because of the presence of sequences from other microbial genomes and the rat host, the analysis and compression to a unigene set was necessarily an iterative process. BLASTx analysis of the unassembled reads (UR) vs. the Uni-Prot and TREMBL databases revealed 56% had similarities to existing polypeptides at E values of≤10−6, with the remainder lacking any significant homology. The most abundant transcripts in the UR were associated with stress responses, energy production, transcription and translation. Most (70%) of the UR had similarities to proteins from filamentous fungi (e.g., Aspergillus, Neurospora) and existing P. carinii gene products. In contrast, similarities to proteins of the yeast-like fungi, Schizosaccharomyces pombe and Saccharomyces cerevisiae, predominated in the unigene set. Gene Ontology analysis using BLAST2GO revealed P. carinii dedicated most of its transcripts to cellular and physiological processes (∼80%), molecular binding and catalytic activities (∼70%), and were primarily derived from cell and organellar compartments (∼80%). KEGG Pathway mapping showed the putative P. carinii genes represented most standard metabolic pathways and cellular processes, including the tricarboxylic acid cycle, glycolysis, amino acid biosynthesis, cell cycle and mitochondrial function. Several gene homologs associated with mating, meiosis, and sterol biosynthesis in fungi were identified. Genes encoding the major surface glycoprotein family (MSG), heat shock (HSP70), and proteases (PROT/KEX) were the most abundantly expressed of known P. carinii genes. The apparent presence of many metabolic pathways in P. carinii, sexual reproduction within the host, and lack of an invasive infection process in the immunologically intact host suggest members of the genus Pneumocystis may be adapted parasites and have a compatible relationship with their mammalian hosts. This study represents the first characterization of the expressed genes of a non-culturable fungal pathogen of mammals during the infective process.

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With the exception of a few genes, most of the mitochondrial (mt) genome of Pneumocystis carinii has not previously been sequenced. Shotgun sequences generated as a result of the Pneumocystis Genome Project (PGP) were assembled with the gap4 assembly program into a 23-kb contig. Annotation of the mt genome identified 4 open reading frames and 20 tRNAs in addition to 17 other genes: ATP synthase, subunits 6, 8, and 9; cytochrome c oxidase, subunits 1, 2, and 3; NADH dehydrogenase, subunits 1, 2, 3, 4, 4L, 5, and 6; apocytochrome b; RNase P RNA gene; and the mitochondrial large and small ribosomal RNA subunits. A 24-bp unit that repeated from one to five times was identified interior to the ends of the mt genome. Migration of the genome on CHEF gels was consistent with that of linear DNA and digestion with BAL31 showed a concomitant reduction in size of the genome, a characteristic of linear DNA. Together with the identification of terminal repeats similar to those found in other linear fungal mt genomes and the inability to join the ends by PCR, these data provide strong evidence that the mt genome of P. carinii is linear.

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