Transcriptome of Pneumocystis carinii during Fulminate Infection: Carbohydrate Metabolism and the Concept of a Compatible Parasite

Abstract

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.

Functional Differentiation of tbf1 Orthologues in Fission and Budding Yeasts

“…A partial sequence corresponding to the Pctbf1 open reading frame (ORF) was identified in the unigene set of 1,042 expressed sequence tags (ESTs) isolated from a P. carinii cDNA library (the Pneumocystis Genome Project, http://pgp.cchmc.org) (14) by its homology to the Sptbf1 and ScTBF1 genes. Longer fragments of the Pctbf1 gene were obtained from the randomly amplified cDNA library by PCR (GenomiPhi DNA amplification kit; GE Healthcare, Otelfingen, Switzerland) with primers corresponding to adjacent regions of the genomic sequence in conjunction with the T3 primer located upstream of the multiple-cloning site in the Uni-ZAP XR vector.…”

“…Searches of the S. cerevisiae, S. pombe, and vertebrate proteomes for homologues of the 1,042 unique P. carinii ESTs (http://pgp.cchmc.org) (14) were performed under low-stringency conditions (E value of 1EϪ3) using the local alignment tool BLASTx. Several of the ESTs had homology with the C-terminal regions of ScTBF1 (YPL128C) and Sptbf1 (SPBC19G7.13), with maximum homology values of 1EϪ21 and 8EϪ23, respectively.…”

Pneumocystis Workshop: 10th Anniversary Summary

“…Prior to that, only about 40 gene accessions for any species of Pneumocystis were listed. A transcriptional profile (22) and gene inventory (http://pgp.cchmc.org) provided the community at large with sufficient genetic information to begin functional characterizations, predict metabolic pathways, and conduct phylogenetic analyses.…”

“…The transcriptome of P. carinii during fulminant infection was recently examined using microarray analysis (22). Custom slides were comprised of 70-mer signature sequences of the cDNA unigene set and of predicted fungal gene homologs identified in the assembled (119).…”

Functional Characterization of Pneumocystis carinii brl1 by Transspecies Complementation Analysis

“…The Pneumocystis Genome Project (http://pgp.cchmc.org) (12,15) has completed the sequencing of a nonredundant set of 1,042 expressed sequence tags (ESTs) from RNA isolated from a single rat infected with Pneumocystis carinii (3). About 34% of the genes with the highest homology to P. carinii ESTs are found in Schizosaccharomyces pombe, the closest relative to P. carinii among fungi with sequenced genomes (6), while approximately 15% are found in the more distantly related Saccharomyces cerevisiae (3). Our strategy has been to use bioinformatics in conjunction with functional complementation in S. cerevisiae or S. pombe to assess the function of identified P. carinii genes (5).…”