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synthase"],"date_uploaded_dtsi":"2016-04-14T00:00:00Z","date_modified_dtsi":"2016-04-14T00:00:00Z","isPartOf_ssim":["admin_set/default"],"doi_tesim":["doi:10.7945/C2HS33"],"college_tesim":["Other"],"department_tesim":["HSL Info \u0026 Edu Svcs"],"creator_tesim":["Grant, Tiffany"],"publisher_tesim":["Eukaryotic Cell"],"description_tesim":["Organisms in the genus Pneumocystis are ubiquitous, opportunistic pathogenic fungi capable of causing a\nlethal pneumonia in immunocompromised mammalian hosts. Pneumocystis spp. are unique members of the\nfungal kingdom due to the absence of ergosterol in their cellular membranes. Although these organisms were\nthought to obtain cholesterol by scavenging, transcriptional analyses indicate that Pneumocystis carinii encodes\ngene homologs involved in sterol biosynthesis. To better understand the sterol pathway in these uncultivable\nfungi, 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\nlanosterol 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."],"license_tesim":["http://rightsstatements.org/vocab/InC/1.0/"],"thumbnail_path_ss":"/downloads/ff3655656?file=thumbnail","suppressed_bsi":false,"actionable_workflow_roles_ssim":["admin_set/default-default-depositing"],"workflow_state_name_ssim":["deposited"],"member_ids_ssim":["ff3655656"],"file_set_ids_ssim":["ff3655656"],"visibility_ssi":"open","admin_set_tesim":["Default Admin Set"],"sort_title_ssi":"FUNCTIONAL CHARACTERIZATION AND LOCALIZATION OF PNEUMOCYSTIS LANOSTEROL SYNTHASE","human_readable_type_tesim":["Article"],"read_access_group_ssim":["public"],"edit_access_person_ssim":["joffritm@ucmail.uc.edu"],"nesting_collection__pathnames_ssim":["ff365564x"],"nesting_collection__deepest_nested_depth_isi":1,"_version_":1697076654875082752,"timestamp":"2021-04-15T03:46:58.727Z","score":0.00049999997},{"system_create_dtsi":"2017-10-27T01:06:00Z","system_modified_dtsi":"2019-06-06T15:07:06Z","has_model_ssim":["Article"],"id":"ff365562c","accessControl_ssim":["482cff88-ac68-4f54-b26c-dc4722dab1f6"],"hasRelatedMediaFragment_ssim":["ff365563n"],"hasRelatedImage_ssim":["ff365563n"],"depositor_ssim":["joffritm@ucmail.uc.edu"],"depositor_tesim":["joffritm@ucmail.uc.edu"],"title_tesim":["Sterol biosynthesis and sterol uptake in the funal pathogen Pneumocsytis carinii"],"date_uploaded_dtsi":"2016-04-14T00:00:00Z","date_modified_dtsi":"2016-04-14T00:00:00Z","isPartOf_ssim":["admin_set/default"],"doi_tesim":["doi:10.7945/C2NK51"],"college_tesim":["Other"],"department_tesim":["HSL Info \u0026 Edu Svcs"],"creator_tesim":["Grant, Tiffany"],"publisher_tesim":["FEMS Microbiology Letters"],"description_tesim":["Members of the fungal genus Pneumocystis colonize healthy mammalian hosts\nwithout causing apparent disease, but colonization in immunocompromised hosts\nmay result in a potentially fatal pneumonia known as Pneumocystis pneumonia.\nAlthough Pneumocystis are fungi, this genus has characteristics that make it atypical\namong other fungi. Pneumocystis do not appear to synthesize the major fungal sterol,\nergosterol, and biochemical analyses have shown that they utilize cholesterol rather\nthan 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,\nsuggesting that these enzymes are functional within the organism. Heterologous\nexpression of P. carinii sterol genes, along with biochemical analyses of the lipid\ncontent of P. carinii cellular membranes, have provided an insight into sterol\nbiosynthesis and the sterol-scavenging mechanisms used by these fungi."],"license_tesim":["http://rightsstatements.org/vocab/InC/1.0/"],"thumbnail_path_ss":"/downloads/ff365563n?file=thumbnail","suppressed_bsi":false,"actionable_workflow_roles_ssim":["admin_set/default-default-depositing"],"workflow_state_name_ssim":["deposited"],"member_ids_ssim":["ff365563n"],"file_set_ids_ssim":["ff365563n"],"visibility_ssi":"open","admin_set_tesim":["Default Admin Set"],"sort_title_ssi":"STEROL BIOSYNTHESIS AND STEROL UPTAKE IN THE FUNAL PATHOGEN PNEUMOCSYTIS CARINII","human_readable_type_tesim":["Article"],"read_access_group_ssim":["public"],"edit_access_person_ssim":["joffritm@ucmail.uc.edu"],"nesting_collection__pathnames_ssim":["ff365562c"],"nesting_collection__deepest_nested_depth_isi":1,"_version_":1697102252029771776,"timestamp":"2021-04-15T10:33:50.076Z","score":0.00049999997},{"system_create_dtsi":"2017-10-27T01:05:53Z","system_modified_dtsi":"2019-06-06T15:05:24Z","has_model_ssim":["Document"],"id":"ff365560t","accessControl_ssim":["b7d91108-5428-40bb-acc2-5c16a6e10e3d"],"hasRelatedMediaFragment_ssim":["ff3655613"],"hasRelatedImage_ssim":["ff3655613"],"depositor_ssim":["joffritm@ucmail.uc.edu"],"depositor_tesim":["joffritm@ucmail.uc.edu"],"title_tesim":["Sterol biosynthesis and sterol uptake in the fungal pathogen Pneumocsytis carinii"],"date_uploaded_dtsi":"2016-04-14T00:00:00Z","date_modified_dtsi":"2016-08-09T00:00:00Z","isPartOf_ssim":["admin_set/default"],"doi_tesim":["doi:10.7945/C2S889"],"college_tesim":["Other"],"department_tesim":["HSL Info \u0026 Edu Svcs"],"creator_tesim":["Grant, Tiffany"],"publisher_tesim":["University of Cincinnati"],"description_tesim":["\nFungi in the genus Pneumocystis are the cause of a potentially life threatening\npneumonia, 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.\n\nBiochemical 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\nexogenous sterols from its mammalian host. S. cerevisiae is induced to undergo sterol\nscavenging 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. 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