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A deeper understanding of the urease enzyme in the opportunistic yeast Cryptococcus neoformans
Thesis (PhD)--Stellenbosch University, 2020.
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| Format: | Thesis |
| Language: | en_ZA |
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Stellenbosch : Stellenbosch University
2020
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| _version_ | 1867613810647367680 |
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| access_status_str | Open Access |
| author | Toplis, Barbra-Ann |
| author2 | Botha, Alfred |
| author_browse | Botha, Alfred Toplis, Barbra-Ann |
| author_facet | Botha, Alfred Toplis, Barbra-Ann |
| author_sort | Toplis, Barbra-Ann |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (PhD)--Stellenbosch University, 2020. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/109450 |
| institution | Stellenbosch University (South Africa) |
| language | en_ZA |
| last_indexed | 2026-06-10T12:42:03.173Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2020 |
| publishDateRange | 2020 |
| publishDateSort | 2020 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/109450 A deeper understanding of the urease enzyme in the opportunistic yeast Cryptococcus neoformans Toplis, Barbra-Ann Botha, Alfred Stellenbosch University. Faculty of Science. Dept. of Microbiology. Cryptococcus neoformans Urease -- Inhibitors Enzymes -- Regulation Cryptococcus -- Effect of environment on Cryptococcus -- Physiology Urease enzyme -- Effect of temperature on UCTD Thesis (PhD)--Stellenbosch University, 2020. ENGLISH ABSTRACT: Urease enzyme production is a well-studied virulence mechanism of the opportunistic pathogen Cryptococcus neoformans. It is believed that the main function of urease is to provide these yeasts with a readily assimilable nitrogen source in the form of ammonia, via the hydrolysis of exogenous urea. The latter is encountered by cryptococci both within the human host, as well as in the natural environment. Although largely unexplored in C. neoformans, urease can also hydrolyse urea that originates endogenously from metabolic reactions within the yeast cell, such as those involved in polyamine biosynthesis. The overall aim of this study was therefore to determine whether cryptococcal urease is linked to metabolic pathways related to the yeast’s endogenous urea production. Thus, throughout the study, biochemical analyses were conducted on wild-type, urease-deficient and urease-reconstituted strains of C. neoformans H99. To investigate cryptococcal physiology at mammalian body temperature, all experiments were carried out at 37 °C in addition to 26 °C. The results revealed that urease plays a role in cryptococcal growth and compared to nutrient-rich conditions and a temperature of 26 °C, urease enzyme activity was highest under nutrient-limited conditions at 37 °C. The latter findings were obtained in the absence of exogenous urea, which together with the observation of elevated urea levels in crude extracts of urease-deficient cells, indicated that urea was produced endogenously by C. neoformans. In addition, it was found that arginase enzyme activity, a potential source of intracellular urea, was highest in cells of the urease- deficient mutant. The latter finding was a suggested explanation for the elevated urea levels that were observed for this strain. Additional aspects linked to arginase metabolism were also explored and it was found that compared to the wild-type, urease-deficient cells differed with respect to agmatinase activity, polyamine biosynthesis and intracellular levels of proline and reactive oxygen species. Very importantly, the urease-negative strain also showed differences in melanin production, which provided the first evidence that urease may affect the functioning of another cryptococcal virulence mechanism. Overall, the results provided strong evidence that the urease enzyme of C. neoformans is linked to vital metabolic pathways within the yeast cell, of which many are known to be involved in polyamine metabolism. Thus, using a polyamine-free growth medium, cryptococcal polyamine biosynthesis was subsequently explored further. Mass spectrometry analysis showed that agmatine and spermidine are the major extra- and intracellular polyamines of C. neoformans at both 26 and 37 °C. Cadaverine and putrescine were identified as minor polyamines and spermine was undetected. Temperature was found to influence the results obtained for all three strains as significant differences were detected in the relative percentages of extra- and intracellular polyamines between 26 and 37 °C. Strain differences in polyamine metabolism were observed at both tested temperatures where, compared to the wild- type, relative percentages of extracellular putrescine and spermidine were found to be significantly lower and agmatine significantly higher in cultures of the urease-deficient mutant. The inverse was true for intracellular spermidine and agmatine. All three strains were more susceptible to treatment with the polyamine inhibitor cyclohexylamine compared to DL-α-difluoromethylornithine and all inhibitory effects were more pronounced at 37 °C than at 26 °C. At both tested temperatures, the urease-deficient mutant was less susceptible to cyclohexylamine compared to the wild-type. Growth inhibition by cyclohexylamine and DL-α-difluoromethylornithine was reduced with spermidine and putrescine supplementation, respectively. Taken together, the results provided novel insight into polyamine metabolism by C. neoformans, highlighting for the first time the involvement of urease in biogenic amine production. Based on our findings, cryptococcal urease plays a complex cellular role that extends far beyond the use of exogenous urea as a nitrogen source. AFRIKAANSE OPSOMMING: Urease ensiemproduksie is ’n goed-bestudeerde virulensie meganisme van die opportunistiese patogeen Cryptococcus neoformans. Daar word geglo dat dit die hooffunksie van urease is om hierdie giste van 'n maklik-assimileerbare stikstofbron in die vorm van ammoniak te voorsien, deur die hidrolise van eksogene ureum. Cryptococci vind laasgenoemde binne die menslike gasheer asook in die natuurlike omgewing. Hoewel dit nog selde in C. neoformans bestudeer is, kan urease ook ureum hidroliseer wat oorspronklik ontstaan het as gevolg van metaboliese reaksies binne die gissel, soos dié wat betrokke is by poliamien biosintese. Die doel van hierdie studie was daarom om te bepaal of kriptokokkale urease gekoppel is aan metaboliese weë wat verband hou met die endogene ureumproduksie van die gis. Biochemiese ontledings is derhalwe gedurende die hele studie uitgevoer op wilde-tipe, urease- gebrekkige en urease-gerekonstitueerde stamme van C. neoformans H99. Om kriptokokkale fisiologie by die liggaamstemperatuur van soogdiere te ondersoek, is alle eksperimente nie net by 26 °C uitgevoer nie, maar ook by 37 °C. Die resultate het getoon dat urease 'n rol speel by kriptokokkale groei en dat in vergelyking met nutriënt-ryke toestande en 'n temperatuur van 26 °C, urease ensiem- aktiwiteit die hoogste is onder nutriënt-beperkte toestande by 37 °C. Laasgenoemde resultate is verkry in die afwesigheid van eksogene ureum, wat saam met die waarneming van verhoogde ureumvlakke in ru-ekstrakte van urease-gebrekkige selle daarop dui dat ureum endogeen deur C. neoformans geproduseer word. Daar is verder gevind dat arginase-ensiemaktiwiteit, 'n moontlike bron van intrasellulêre ureum, die hoogste was in selle van die urease-gebrekkige mutant. Laasgenoemde bevinding kon potensieel die verhoogde ureumvlakke, wat in hierdie stam waargeneem is, verklaar. Ander aspekte wat ook aan arginase metabolisme gekoppel word, is ook ondersoek en daar is gevind dat in vergelyking met die wilde-tipe, urease- gebrekkige selle verskil ten opsigte van agmatinase aktiwiteit, poliamien biosintese en intrasellulêre vlakke van prolien en reaktiewe suurstofspesies. Dit is belangrik om daarop te let dat die urease-negatiewe stam ook verskille in melanien-produksie getoon het, wat die eerste bewys gelewer het dat urease die funksionering van 'n ander kriptokokkale virulensiemeganisme mag beïnvloed. Die resultate lewer origens sterk bewys dat die urease ensiem van C. neoformans gekoppel is aan lewensbelangrike metaboliese weë in die gissel, waarvan baie daarvoor bekend is om betrokke te wees by poliamien-metabolisme. Deur dus van 'n poliamienvrye medium gebruik te maak kon kriptokokkale poliamien-biosintese gevolglik verder bestudeer word. Massaspektrometrie-analise het getoon dat agmatien en spermidien die belangrikste ekstra- en intrasellulêre poliamiene van C. neoformans by beide 26 en 37 °C is. Kadawerien en putresien is geïdentifiseer as geringe polyamiene en geen spermien is opgemerk nie. Daar is gevind dat die temperatuur die resultate vir al drie stamme beïnvloed, aangesien beduidende verskille in die relatiewe persentasies ekstra- en intrasellulêre poliamiene tussen 26 en 37 °C waargeneem is. Stamverskille in poliamien-metabolisme is by albei temperature waargeneem, waar in vergelyking met die wilde tipe, daar gevind is dat die relatiewe persentasies van ekstrasellulêre putresien en spermidien beduidend laer is, en agmatien beduidend hoër is in kulture van die urease-gebrekkige mutant. Die teenoorgestelde was waar vir intrasellulêre spermidien en agmatien. Al drie stamme was meer vatbaar vir behandeling met die poliamien inhibeerder sikloheksilamien in vergelyking met DL-α-difluorometielornitien, en alle inhiberende effekte was duideliker by 37 °C as by 26 °C. Die urease-gebrekkige mutant was by beide toetstemperature minder vatbaar vir sikloheksilamien as die wilde tipe. Groei-inhibisie deur sikloheksilamien en DL-α-difluorometielornitien was verminder deur die onderskeidelike byvoeging van spermidien and putresien. Gesamentlik lewer die resultate nuwe insig in die poliamien metabolisme van C. neoformans, en lig dit vir die eerste keer die betrokkenheid van urease in biogeniese amienproduksie toe. Gegrond op ons bevindings speel kriptokokkale urease 'n komplekse sellulêre rol, wat ver verby die gebruik van eksogene ureum as stikstofbron strek. Doctorate 2020-11-13T12:58:00Z 2021-02-01T07:55:41Z 2020-11-13T12:58:00Z 2021-02-01T07:55:41Z 2020-12 Thesis http://hdl.handle.net/10019.1/109450 en_ZA Stellenbosch University xxiv, 130 pages : illustrations (some color) application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Cryptococcus neoformans Urease -- Inhibitors Enzymes -- Regulation Cryptococcus -- Effect of environment on Cryptococcus -- Physiology Urease enzyme -- Effect of temperature on UCTD Toplis, Barbra-Ann A deeper understanding of the urease enzyme in the opportunistic yeast Cryptococcus neoformans |
| title | A deeper understanding of the urease enzyme in the opportunistic yeast Cryptococcus neoformans |
| title_full | A deeper understanding of the urease enzyme in the opportunistic yeast Cryptococcus neoformans |
| title_fullStr | A deeper understanding of the urease enzyme in the opportunistic yeast Cryptococcus neoformans |
| title_full_unstemmed | A deeper understanding of the urease enzyme in the opportunistic yeast Cryptococcus neoformans |
| title_short | A deeper understanding of the urease enzyme in the opportunistic yeast Cryptococcus neoformans |
| title_sort | deeper understanding of the urease enzyme in the opportunistic yeast cryptococcus neoformans |
| topic | Cryptococcus neoformans Urease -- Inhibitors Enzymes -- Regulation Cryptococcus -- Effect of environment on Cryptococcus -- Physiology Urease enzyme -- Effect of temperature on UCTD |
| url | http://hdl.handle.net/10019.1/109450 |
| work_keys_str_mv | AT toplisbarbraann adeeperunderstandingoftheureaseenzymeintheopportunisticyeastcryptococcusneoformans AT toplisbarbraann deeperunderstandingoftheureaseenzymeintheopportunisticyeastcryptococcusneoformans |