Microaerophilic Conditions Increase Viability and Affect Responses of <i>Pneumocystis carinii</i> to Drugs In Vitro

Joffrion, Tiffany M.; Collins, Margaret S.; Cushion, Melanie T.

doi:10.1111/j.1550-7408.2006.00196.x

Cited by 5 publications

(3 citation statements)

References 11 publications

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“…The evidence at hand that argues against fermentation by P. carinii includes the lack of a homolog to pyruvate dehydrogenase in the unigene set or the genomic database and its inability to survive under anaerobic conditions. Recent studies have shown that P. carinii rapidly loses viability in an anaerobic atmosphere and seems to require some oxygen, although it is able to survive in an atmosphere of reduced oxygen levels [68]. On the other hand, its genome contains 3 genes encoding putative alcohol dehydrogenases.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2007

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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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Section: Discussionmentioning

confidence: 99%

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

et al. 2007

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“…There is some evidence that this might occur. Our laboratory explored the effects of 3 different gas mixtures on the ATP content of P. carinii in an RPMI 1640-based medium ( Joffrion et al, 2006 ). A significant increase in ATP content was observed for fungi grown under microaerophilic conditions (10–15% O2; 7–15% CO2) when compared to standard conditions of 5% CO2.…”

Section: What Elements Are Missing From All These Cultivation Efforts?mentioning

confidence: 99%

The Persistent Challenge of Pneumocystis Growth Outside the Mammalian Lung: Past and Future Approaches

et al. 2021

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The pathogenic fungi in the genus, Pneumocystis, have eluded attempts to continuously grow them in an ex vivo cultivation system. New data from transcriptomic and genomic sequencing studies have identified a myriad of absent metabolic pathways, helping to define their host obligate nature. These nutrients, factors, and co-factors are acquired from their mammalian host and provide clues to further supplementation of existing media formulations. Likewise, a new appreciation of the pivotal role for the sexual cycle in the survival and dissemination of the infection suggests that Pneumocystis species are obligated to undergo mating and sexual reproduction in their life cycle with a questionable role for an asexual cycle. The lack of ascus formation in any previous cultivation attempts may explain the failure to identify a sustainable system. Many characteristics of these ascomycetes suggest a biotrophic existence within the lungs of the mammalian hosts. In the present review, previous attempts at growing these fungi ex vivo are summarized. The significance of their life cycle is considered, and a list of potential supplements based on the genomic and transcriptomic studies is presented. State of the art technologies such as metabolomics, organoids, lung-on-a chip, and air lift cultures are discussed as potential growth systems.

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“…1), as well as recent studies showing the ability of P. carinii to thrive under reduced oxygen conditions in vitro (26) led us to posit that Pneumocystis spp. follow a pathway to biofilm formation within the host lung, similar to that of other fungal pathogens.…”

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Biofilm Formation by Pneumocystis spp

2009

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Pneumocystis spp. can cause a lethal pneumonia in hosts with debilitated immune systems. The manner in which these fungal infections spread throughout the lung, the life cycles of the organisms, and their strategies used for survival within the mammalian host are largely unknown, due in part to the lack of a continuous cultivation method. Biofilm formation is one strategy used by microbes for protection against environmental assaults, for communication and differentiation, and as foci for dissemination. We posited that the attachment and growth of Pneumocystis within the lung alveoli is akin to biofilm formation. An in vitro system comprised of insert wells suspended in multiwell plates containing supplemented RPMI 1640 medium supported biofilm formation by P. carinii (from rat) and P. murina (from mouse).Dramatic morphological changes accompanied the transition to a biofilm. Cyst and trophic forms became highly refractile and produced branching formations that anastomosed into large macroscopic clusters that spread across the insert. Confocal microscopy revealed stacking of viable organisms enmeshed in concanavalin A-staining extracellular matrix. Biofilms matured over a 3-week time period and could be passaged. These passaged organisms were able to cause infection in immunosuppressed rodents. Biofilm formation was inhibited by farnesol, a quorum-sensing molecule in Candida spp., suggesting that a similar communication system may be operational in the Pneumocystis biofilms. Intense staining with a monoclonal antibody to the major surface glycoproteins and an increase in (1,3)-␤-Dglucan content suggest that these components contributed to the refractile properties. Identification of this biofilm process provides a tractable in vitro system that should fundamentally advance the study of Pneumocystis.Organisms in the fungal genus Pneumocystis cause an oftenlethal pneumonia in mammals with a compromised immune status. To date, there have been five formally described species, although all mammalian species examined to date appear to host at least one Pneumocystis species. Those that have been described are P. jirovecii from humans (20), P. carinii (53) and P. wakefieldiae (10, 11) from rats, P. murina (30) from mice, and P. oryctolagi (16) from rabbits.Limited progress has been made in understanding the life cycle, transmission, and natural history of any Pneumocystis species, due in large part to the absence of a continuous in vitro culture system. Although the incidence of frank pneumonia caused by these organisms has decreased in developed countries such as the United States and European countries, mounting evidence points to new niches being exploited by these fungi. The presence of P. jirovecii in patients with underlying chronic diseases such as chronic obstructive pulmonary disease has been suggested to be a comorbidity factor (42-44). A series of recent case reports have identified P. jirovecii as a significant cause of infection in patients being treated with tumor necrosis factor alpha inhibitors for rheum...

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Microaerophilic Conditions Increase Viability and Affect Responses of Pneumocystis carinii to Drugs In Vitro

Cited by 5 publications

References 11 publications

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

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

The Persistent Challenge of Pneumocystis Growth Outside the Mammalian Lung: Past and Future Approaches

Biofilm Formation by Pneumocystis spp

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