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  • LabArchives

    Generic Work thumbnail: LabArchives
    Type:
    Generic Work
    Description/Abstract:
    LabArchives Test
    Creator/Author:
    Grant, Tiffany
    Submitter:
    Tiffany Grant
    Date Uploaded:
    03/15/2017
    Date Modified:
    03/15/2017
    License:
    All rights reserved

  • Functional Characterization and Localization of Pneumocystis lanosterol synthase

    Article thumbnail: Functional Characterization and Localization of Pneumocystis lanosterol synthase
    Type:
    Article
    Description/Abstract:
    Organisms in the genus Pneumocystis are ubiquitous, opportunistic pathogenic fungi capable of causing a lethal pneumonia in immunocompromised mammalian hosts. Pneumocystis spp. are unique members of the fungal kingdom due to the absence of ergosterol in their cellular membranes. Although these organisms were thought to obtain cholesterol by scavenging, transcriptional analyses indicate that Pneumocystis carinii encodes gene homologs involved in sterol biosynthesis. To better understand the sterol pathway in these uncultivable fungi, yeast deletion strains were used to interrogate the function and localization of P. carinii lanosterol synthase (ERG7). The expression of PcErg7p in an ERG7-null mutant of the yeast Saccharomyces cerevisiae did not alter its growth rate and produced a functional lanosterol synthase, as evidenced by the presence of lanosterol detected by gas chromatographic analysis in levels comparable to that produced by the yeast enzyme. Western blotting and fluorescence microscopy revealed that, like the S. cerevisiae Erg7p, the PcErg7p localized to lipid particles in yeast. Using fluorescence microscopy, we show for the first time the presence of apparent lipid particles in P. carinii and the localization of PcErg7p to lipid particles in P. carinii. The detection of lipid particles in P. carinii and their association with PcErg7p therein provide strong evidence that the enzyme serves a similar function in P. carinii. Moreover, the yeast heterologous system should be a useful tool for further analysis of the P. carinii sterol pathway.
    Creator/Author:
    Grant, Tiffany
    Submitter:
    Tiffany Grant
    Date Uploaded:
    04/14/2016
    Date Modified:
    04/14/2016
    License:
    All rights reserved

  • Sterol biosynthesis and sterol uptake in the funal pathogen Pneumocsytis carinii

    Article thumbnail: Sterol biosynthesis and sterol uptake in the funal pathogen Pneumocsytis carinii
    Type:
    Article
    Description/Abstract:
    Members of the fungal genus Pneumocystis colonize healthy mammalian hosts without causing apparent disease, but colonization in immunocompromised hosts may result in a potentially fatal pneumonia known as Pneumocystis pneumonia. Although Pneumocystis are fungi, this genus has characteristics that make it atypical among other fungi. Pneumocystis do not appear to synthesize the major fungal sterol, ergosterol, and biochemical analyses have shown that they utilize cholesterol rather than ergosterol as the bulk sterol. Pneumocystis carinii appears to scavenge exogenous sterols, including cholesterol, from its mammalian host. As a result, it has long been held that their ability to scavenge cholesterol from their hosts, and their inability to undergo sterol biosynthesis, makes them resistant to antifungal drugs that target ergosterol or ergosterol biosynthesis. However, genome scans and in vitro assays indicate the presence of sterol biosynthetic genes within the P. carinii genome, and targeted inhibition of these enzymes resulted in reduced viability of P. carinii, suggesting that these enzymes are functional within the organism. Heterologous expression of P. carinii sterol genes, along with biochemical analyses of the lipid content of P. carinii cellular membranes, have provided an insight into sterol biosynthesis and the sterol-scavenging mechanisms used by these fungi.
    Creator/Author:
    Grant, Tiffany
    Submitter:
    Tiffany Grant
    Date Uploaded:
    04/14/2016
    Date Modified:
    04/14/2016
    License:
    All rights reserved

  • Sterol biosynthesis and sterol uptake in the fungal pathogen Pneumocsytis carinii

    Document thumbnail: Sterol biosynthesis and sterol uptake in the fungal pathogen Pneumocsytis carinii
    Type:
    Document
    Description/Abstract:
    Fungi in the genus Pneumocystis are the cause of a potentially life threatening pneumonia, Pneumocystis pneumonia (PCP). The understanding of the lifecycle, metabolism, and drug development has been hindered due to a lack of a long term in vitro culture system. Unlike most other fungi, members of the genus Pneumocystis do not appear to synthesize the major fungal sterol, ergosterol. However, genome scans and in vitro assays suggest the presence of functional genes involved in a sterol pathway. One of the goals of this work was to characterize the P. carinii sterol enzyme, lanosterol synthase (Erg7p), an essential enzyme of the sterol pathway. The activity of P. carinii Erg7p was assessed by heterologous expression of P. carinii Erg7p in a Saccharomyces cerevisiae Erg7p null mutant. Growth rates and lanosterol production were similar between S. cerevisiae expressing the P. carinii enzyme and S. cerevisiae expressing its own Erg7p under the same conditions, indicating that not only does P. carinii produce a functional Erg7p, but also that the enzyme functionally complements the S. cerevisiae enzyme. Western blotting and fluorescent localization studies revealed that P. carinii Erg7p localizes to lipid particles in S. cerevisiae as does S. cerevisiae Erg7p. A novel finding of these studies, was that P. carinii contains lipid particles, and that P. carinii Erg7p localizes to lipid particles in P. carinii. These studies indicate that P. carinii Erg7p functions similar to the S. cerevisiae enzyme, and may perform a similar function in P. carinii. Biochemical analyses of sterols within the membranes of P. carinii have shown that it utilizes cholesterol rather than ergosterol as its bulk sterol. However, P. carinii does not appear to synthesize cholesterol from a de novo pathway, but rather scavenges exogenous sterols from its mammalian host. S. cerevisiae is induced to undergo sterol scavenging under anaerobic conditions. Consequently, another goal of this work was to provide information on the effect of O2 on sterol biosynthesis and sterol scavenging by P. carinii. ATP measurements revealed that the viability of P. carinii is severely decreased when maintained under hypoxic conditions, and this decrease correlated with an increase in drug susceptibility. We show that uptake of exogenous cholesterol by P. carinii occurred under normal O2 tensions, indicating that sterol scavenging is not limited to anaerobic conditions. Microarray analysis indicated that hypoxic maintenance of P. carinii resulted in decreased transcription of several genes involved in sterol and lipid biosynthesis suggesting that while hypoxic conditions down-regulated genes involved in sterol biosynthesis, down-regulation of sterol biosynthesis is not a requirement for sterol scavenging in P. carinii. The ability of P. carinii to scavenge exogenous sterols under normal O2 tensions at which the sterol pathway is unaffected provides evidence that sterol scavenging may be the primary means that P. carinii utilizes to obtain its sterols.
    Creator/Author:
    Grant, Tiffany
    Submitter:
    Tiffany Grant
    Date Uploaded:
    04/14/2016
    Date Modified:
    08/09/2016
    License:
    All rights reserved