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Assembly and annotation of Pneumocystis jirovecii from the human lung microbiome.

ABSTRACT: Pneumocystis jirovecii is a fungus that causes Pneumocystis pneumonia in immunosuppressed patients and has been closely associated with AIDS since the beginning of the AIDS epidemic. Because in vitro cultivation of P. jirovecii is not possible, progress has been hindered in our understanding of its life cycle, mode of transmission, metabolic function, and genome. Limited amounts of P. jirovecii can be obtained from infected patients, but the occurrence of bacteria, other fungi, and human cells in clinical samples presents new challenges for whole-genome sequencing and downstream bioinformatic analysis. In a recent article, Cissé et al. used cell immunoprecipitation enrichment together with whole-genome amplification to generate sufficient quantities of DNA for Roche 454 and Illumina sequencing [O. H. Cissé, M. Pagni, and P. M. Hauser, mBio 4(1):e00428-12, 2012, doi:10.1128/mBio.00428-12]. In addition, a bioinformatic pipeline was devised to sort and assemble lung microbiome reads, thereby generating an 8.1-Mb P. jirovecii genome comprised of 356 contigs with an N50 (median length of all contigs) of 41.6 kb. Knowledge of this genome will open new avenues of research, including the identification of nutritional requirements for in vitro cultivation as well as the identification of new and novel drug and vaccine targets.

SUBMITTER: Cushion MT

PROVIDER: S-EPMC3634608 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

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Assembly and annotation of Pneumocystis jirovecii from the human lung microbiome.

Cushion Melanie T MT Keely Scott P SP

mBio 20130416 2

Pneumocystis jirovecii is a fungus that causes Pneumocystis pneumonia in immunosuppressed patients and has been closely associated with AIDS since the beginning of the AIDS epidemic. Because in vitro cultivation of P. jirovecii is not possible, progress has been hindered in our understanding of its life cycle, mode of transmission, metabolic function, and genome. Limited amounts of P. jirovecii can be obtained from infected patients, but the occurrence of bacteria, other fungi, and human cells i ...[more]

PMID: 23592264

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Project description:Microbial pathogens commonly escape the human immune system by varying surface proteins. We investigated the mechanisms used for that purpose by Pneumocystis jirovecii This uncultivable fungus is an obligate pulmonary pathogen that in immunocompromised individuals causes pneumonia, a major life-threatening infection. Long-read PacBio sequencing was used to assemble a core of subtelomeres of a single P. jirovecii strain from a bronchoalveolar lavage fluid specimen from a single patient. A total of 113 genes encoding surface proteins were identified, including 28 pseudogenes. These genes formed a subtelomeric gene superfamily, which included five families encoding adhesive glycosylphosphatidylinositol (GPI)-anchored glycoproteins and one family encoding excreted glycoproteins. Numerical analyses suggested that diversification of the glycoproteins relies on mosaic genes created by ectopic recombination and occurs only within each family. DNA motifs suggested that all genes are expressed independently, except those of the family encoding the most abundant surface glycoproteins, which are subject to mutually exclusive expression. PCR analyses showed that exchange of the expressed gene of the latter family occurs frequently, possibly favored by the location of the genes proximal to the telomere because this allows concomitant telomere exchange. Our observations suggest that (i) the P. jirovecii cell surface is made of a complex mixture of different surface proteins, with a majority of a single isoform of the most abundant glycoprotein, (ii) genetic mosaicism within each family ensures variation of the glycoproteins, and (iii) the strategy of the fungus consists of the continuous production of new subpopulations composed of cells that are antigenically different.IMPORTANCEPneumocystis jirovecii is a fungus causing severe pneumonia in immunocompromised individuals. It is the second most frequent life-threatening invasive fungal infection. We have studied the mechanisms of antigenic variation used by this pathogen to escape the human immune system, a strategy commonly used by pathogenic microorganisms. Using a new DNA sequencing technology generating long reads, we could characterize the highly repetitive gene families encoding the proteins that are present on the cellular surface of this pest. These gene families are localized in the regions close to the ends of all chromosomes, the subtelomeres. Such chromosomal localization was found to favor genetic recombinations between members of each gene family and to allow diversification of these proteins continuously over time. This pathogen seems to use a strategy of antigenic variation consisting of the continuous production of new subpopulations composed of cells that are antigenically different. Such a strategy is unique among human pathogens.

Project description:BACKGROUND:Thoracic ultrasound is helpful to evaluate lung pathology in patients with acute dyspnea. Several studies have demonstrated the efficacy of point-of-care ultrasound in patients with extrapulmonary TB and HIV co-infection. This retrospective, open-label case-control study explores the role of lung ultrasound in the diagnosis of Pneumocystis jirovecii pneumonia (PJP) in HIV-positive patients. In particular, it highlights the potential role of specific sonographic features that may be unique to this population. METHODS:The record of all HIV-positive patients admitted from 1.1.2013 to 31.6.2017 to the Department of Infectious Diseases and Tropical Medicine of san Bortolo Hospital, Vicenza, Italy, with a discharge diagnosis of acute lung injury (ALI) and who received point-of-care ultrasound of the chest for clinical purposes was included in the analysis. The patients were scanned according with the evidence-based recommendation. RESULTS:Of 273 HIV-positive patients whose records were reviewed, 81 (29.6%) were diagnosed with ALI. Complete documentation was available for 24 patients, of which 14 (58.3%) had microbiologically confirmed PJP (PJP+) and 10 (41.7%) had other conditions (PJP-). B-lines, subpleural consolidations, and cystic changes were significantly more frequent in patients with PJP (14/14 vs. 6/10, p = 0.0198; 14/14 vs. 4/10, p = 0.0016; 8/14 vs. 0/10, p = 0.0019, respectively). In particular, B-lines and subpleural consolidations were present in all PJP+ patients in our cohort giving a sensitivity of 100%, but their specificity was low (45 and 60%, respectively). On the contrary, the presence of consolidations with cystic changes had a very high specificity for PJP (100%), but low sensitivity (57%). Pleural effusions and consolidations with linear air bronchograms were not observed in PJP+ patients. CONCLUSIONS:B-lines, subpleural consolidations, and cystic changes are suggestive of PJP. Lung consolidation with air bronchograms and pleural effusion should prompt suspicion of other etiologies. These findings have the potential to be useful in the daily management of HIV-positive patients in resource-limited settings where other diagnostic tools are rarely available.

Dataset Information

Assembly and annotation of Pneumocystis jirovecii from the human lung microbiome.

Publications

Assembly and annotation of Pneumocystis jirovecii from the human lung microbiome.

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