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, 2 (5), e423

Transcriptome of Pneumocystis Carinii During Fulminate Infection: Carbohydrate Metabolism and the Concept of a Compatible Parasite

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Transcriptome of Pneumocystis Carinii During Fulminate Infection: Carbohydrate Metabolism and the Concept of a Compatible Parasite

Melanie T Cushion et al. PLoS One.

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<or=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 ( approximately 80%), molecular binding and catalytic activities (approximately 70%), and were primarily derived from cell and organellar compartments (approximately 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.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. EST Analysis and Unigene Process.
The raw sequence reads of the Expressed Sequence Tags were first purged of poor quality sequence (arrows to right of starting point). The resulting 3,896 reads were deposited to NHLBI GenBank and further analyzed for similarities to genes and gene products in the UNI-PROT_TREMBL databases using BLASTn and BLASTx and putative function with BLAST2GO and KASS. The raw reads were then processed using an iterative scheme to form the unigene set starting with a primary screen to purge sequences originating from the cloning vector, rat host and bacteria, followed by a trimming of the sequence ends to reduce poor quality sequence, using an in house program (qTrim). After trimming, the sequences were again purged of contaminants using BLASTn and BLASTx, then assembled using the CAP3 assembly program. After another round of contaminant removal, 1,632 cDNA clones representing putative unique genes were selected for full sequencing. These sequences were then subjected to the same qTrim program, then assembled by CAP3. The primer design program, Primer3 was used to design primers to close gaps in those clone sequence that did not represent full sequences. After another round of assembly, the cDNA sequences were compared to one another for sequence identity using BLASTn, to identify any redundant gene sequences. This resulted in a set of 1042 unique sequences (both contigs and singletons) of which 994 had significant similarities (E≤10−6) to genes within the UNI-PROT_TREMBL and 48 did not have significant similarities to existing genes (E≥10−6). The unigene set was then analyzed for putative functions by BLAST2GO and KAAS.
Figure 2
Figure 2. Binning of P. carinii EST gene homologs by general organism groups.
After trimming the raw EST reads for poor quality sequence (qTrim), the ESTs were analyzed for similarities to gene products of other organisms using BLASTx. Significance was set at E≤10−6.
Figure 3
Figure 3. Binning of putative fungal gene homologs of P. carinii ESTs.
EST sequences with significant similarities to fungal genes with BLASTx scores of E≤10−6 were binned according to fungal species.
Figure 4
Figure 4. Distribution of P. carinii ESTs with significant similarities to known Pneumocystis genes.
EST sequences with significant similarities to known P. carinii genes with BLASTx scores of E≤10−6
Figure 5
Figure 5. BLAST2GO categories of ESTs.
ESTs were analyzed using the BLAST2GO software. Shown are level 2 categories for Biological Processes (A); Molecular Function (B); and Cellular Components (C).
Figure 6
Figure 6. Binning of putative fungal gene homologs of P. carinii Unigenes.
The 918 unigene sequences with significant similarities fungal genes (BLASTx of E≤10−6 ) were binned according to fungal organism of origin.

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