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De Novo Fungal Lung Genome Sequence and Assembly Suggests P. jirovecii Is an Obligate Parasite

NEW YORK (GenomeWeb News) – A trio of Swiss researchers reported on their de novo sequence and assembly of the Pneumocystis jirovecii genome, a fungus that causes pneumonia in immune-compromised patients, such as people with AIDS, in mBio today. The group's analyses of the fungal genome suggested that P. jirovecii may be an obligate parasite.

As P. jirovecii cannot be cultured, the researchers also said that knowing its genome would be a boon for future study, especially to find ways to grow it in the lab or to uncover new drug targets to treat its infection.

"Since the organism cannot be grown in the lab for study, researchers have long made do with studying P. jirovecii's lab-friendly relatives, species that infect animals and plants, in order to explore the secrets of the human disease," said Philippe Hauser from the University of Lausanne in Switzerland and the senior author of the study in a statement. "It is obviously better to study [P. jirovecii's] genes rather [than] those of Pneumocystis species from animal models. The genome has both medical and evolutionary interests for the scientific community."

To tease out the P. jirovecii genome sequence, the researchers collected bronchoalveolar lavage fluid samples from patients with pneumonia caused by P. jirovecii. But since P. jirovecii cannot be cultured, they also had to turn to cell immunoprecipitation and random DNA amplification to achieve large enough samples of genomic DNA for sequencing. Two of the four samples were enriched in P. jirovecii cells using immunoprecipitation.

Genomic DNA was extracted from each of the bronchoalveolar lavage fluid samples, amplified, and sequenced to a low level by Roche 454 pyrosequencing. The resulting reads were then analyzed using a simplified classification pipeline to determine which samples had the highest levels of enrichment.

Additionally, Hauser and his colleagues noted that one sample appeared to contain P. carinii in addition to P. jirovecii, despite P. jirovecii being thought to be the only member of the Pneumocystis species to infect humans; P. carinii was known to infect rats.

For the rest of their analyses, the researchers focused on one highly enriched sample that came from a patient with chronic lymphocytic leukemia.

However, the reads the researchers accumulated from the bronchoalveolar lavage fluid sample represented the gamut of the human lung microbiome. To sort the P. jirovecii reads from reads from other organisms, the researchers "iteratively built stringent assembly of the reads to detect homology with the available genome of P. carinii, as well as with other fungal genomic data," they reported.

Additionally, the investigators deep-sequenced RNA from a bronchoalveolar lavage fluid sample from a separate patient with B-cell lymphoma to aid in annotating the P. jirovecii genome.

From this, Hauser and his colleagues reported an 8.1 megabase P. jirovecii genome assembly with 336 contigs and 3,878 coding sequences. By comparison, P. carinii has a 6.3 megabase genome with 4,541 coding regions.

Examination of the P. jirovecii genome revealed that it lacked a number of genes needed for important metabolic pathways, including amino acid synthesis pathways, suggesting that P. jirovecii is an obligate parasite. "The loss of these pathways is a hallmark of obligate parasites," Hauser and his colleagues write. "This strongly suggests that P. jirovecii scavenges these compounds from human lungs."

Additionally, the P. jirovecii genome contains a higher than usual amount of transporter genes that would aid in scavenging those missing, but necessary, compounds. P. jirovecii also lacks virulence factors and toxins common to other fungal pathogens.

"Together, these features are compatible with the view that P. jirovecii is an obligate parasite specialized in colonization of human lungs, which causes deadly disease only in immunocompromised individuals," the researchers said, adding that humans are likely its only host and reservoir.