Urease activity of the systemic fungal pathogen Emergomyces africanus

Lerm, Barbra-Ann (2017-03)

Thesis (MSc)--Stellenbosch University, 2017.

Thesis

ENGLISH ABSTRACT: A novel species of an Emmonsia-like fungus, recently named Emergomyces africanus Dukik, Kenyon, Govender et de Hoog, was identified as a cause of disseminated mycosis in HIV-infected persons in South Africa. During the disease process, the yeast-like cells of E. africanus are known to disseminate and colonize various organs of the human body. The ability to cause disseminated AIDS-related mycosis is also characteristic of another unrelated fungus, Cryptococcus neoformans, an opportunistic pathogen responsible for the AIDS-defining illness, cryptococcal meningitis. During the latter disease progression, entry of cryptococcal cells into the brain is facilitated by virulence factors that include urease enzyme activity. However, in contrast to C. neoformans, the enzymes produced by E. africanus, some of which may be involved in pathogenesis, have not been described. Using a clinical isolate of C. neoformans as a reference, the aim of this study was to confirm, characterize and quantify the urease activity of E. africanus clinical isolates. Urease activity was tested using Christensen's urea agar, after which the presence of a urease gene in the genome of E. africanus was confirmed by gene sequence analysis. Subsequent evaluation of colorimetric enzyme assay data, using Michaelis-Menten enzyme kinetics, revealed similarities between the substrate affinity of the urease enzyme produced by E. africanus (Km ca. 26.0 mM) and that of C. neoformans (Km ca. 20.6 mM). However, nutrient conditions were found to affect the urease activity of these two fungi differently. Colorimetric enzyme assays revealed that the addition of 2.5 g/l urea to the culture medium stimulated the urease activity of E. africanus, whereas nutrient limitation notably increased cryptococcal urease activity. The significant enhancement of urease activity in C. neoformans CAB 1055 under conditions of nutrient limitation was also confirmed by relative real-time quantitative PCR (RT-qPCR) analyses. Unlike that observed for E. africanus JX398293, the urease gene expression of C. neoformans CAB 1055 was significantly up-regulated by a mean fold change of 2 ± 0.15 in response to nutrient-limited conditions. In addition to the work on the effect of different nutrient conditions, a preliminary study was conducted to investigate the effect of pH on the urease activity of E. africanus JX398293 and C. neoformans CAB 1055. For both fungi, analysis of colorimetric enzyme assay data revealed that there was no significant difference in the urease activity of crude protein extracts originating from yeast cells exposed to an environment with a pH of 5 and 8, respectively. Thus, indications are that environmental pH does not play a role in the regulation of urease activity in E. africanus JX398293 and C. neoformans CAB 1055. Overall, this study has confirmed the presence of an active urease enzyme in the novel E. africanus species. Furthermore, we demonstrated novel mechanisms of urease regulation in both E. africanus JX398293 and C. neoformans CAB 1055. Future studies should aim at the development of a urease knock-out mutant strain of E. africanus, which can be used to investigate the potential role of urease in the pathogenesis of E. africanus and its survival in the natural environment.

AFRIKAANSE OPSOMMING: ‘n Nuut-ontdekte spesie van ‘n Emmonsia-agtige fungus, wat onlangs die naam Emergomyces africanus Dukik, Kenyon, Govender et de Hoog ontvang het, is as ‘n oorsaak van verspreide mikose in HIV-geïnfekteerde persone in Suid-Afrika geïdentifiseer. Dit is bekend dat die gis-agtige selle van E. africanus gedurende die verloop van die siekte versprei en verskillende organe van die menslike liggaam koloniseer. Die vermoë om verspreide VIGS-verwante mikose te veroorsaak is ook kenmerkend van ‘n ander onverwante fungus, Cryptococcus neoformans, ‘n opportunistiese patogeen verantwoordelik vir die VIGS-definiërende siekte, cryptokokale meningitis. Gedurende die verloop van dié siekte word die binnedringing van cryptokokkale selle in die brein gefasiliteer deur virulensiefaktore wat urease ensiemaktiwiteit insluit. In teenstelling met C. neoformans, is die ensieme wat deur E. africanus geproduseer word - sommige wat by patogenese betrokke mag wees - egter nog nie beskryf nie. Deur ‘n kliniese isolaat van C. neoformans as verwysing te gebruik, was die doel van dié studie om urease-aktiwiteit in kliniese isolate van E. africanus te bevestig, te karakteriseer en te kwantifiseer. Ureaseaktiwiteit is met behulp van Christensen se ureum-agar getoets, waarna die teenwoordigheid van 'n ureasegeen in die genoom van E. africanus deur geenvolgordeanalise bevestig is. ‘n Daaropvolgende evaluasie van kolorimetriese ensiemtoetsdata, wat deur Michaelis-Menten ensiemkinetika verkry is, toon ooreenkomste tussen die substraataffiniteit van die urease-ensiem wat deur E. africanus geproduseer word (Km ca. 26.0 mM), en dié wat deur C. neoformans geproduseer word (Km ca. 20.6 mM). Daar is egter vasgestel dat nutriëntkondisies die urease-aktiwiteit van hierdie twee fungusse verskillend affekteer. Kolorimetriese ensiemtoetse het getoon dat die byvoeging van 2.5 g/l ureum by die kultuurmedium die urease-aktiwiteit van E. africanus stimuleer, terwyl nutriëntbeperking die cryptokokkale urease-aktiwiteit merkbaar verhoog. Die beduidende bevordering van urease-aktiwiteit in C. neoformans CAB 1055 onder nutriëntbeperkende omstandighede is deur relatief-intydse-kwantitatiewe- PKR (RTkPKR) analise bevestig. In teenstelling met E. africanus JX398293, is die ureasegeenuitdrukking van C. neoformans CAB 1055 beduidend in reaksie op nutriëntbeperkte toestande verhoog deur 'n gemiddelde verandering van 2 ± 0.15. Benewens die navorsing op die uitwerking van verskillende nutriëntkondisies, is ‘n voorlopige studie gedoen om die uitwerking van pH op die urease-aktiwiteit van E. africanus JX398293 en C. neoformans CAB 1055 te bepaal. Analises van kolometriese ensiemtoetsdata van beide fungusse het gewys dat daar geen beduidende verskil in die urease-aktiwiteit van ru-proteïenekstrakte van gisselle wat aan omgewings met ‘n pH 5 en 8 blootgestel is nie. Daar is dus aanduidings dat omgewings-pH nie ‘n rol in die regulering van urease-aktiwiteit in E. africanus JX398293 en C. neoformans CAB 1055 speel nie. In geheel bevestig hierdie studie die teenwoordigheid van ‘n aktiewe urease-ensiem in die nuwe E. africanus spesie. Ons het verder nuwe meganismes vir die regulering van urease-aktiwiteit in beide E. africanus JX398293 en C. neoformans CAB 1055 gedemonstreer. Toekomstige studies moet fokus op die ontwikkeling van ‘n mutante stam van E. africanus waarvan die ureasegeen uitgeslaan is en wat gebruik kan word om die moontlike rol van urease, in die patogenese en oorlewing van E. africanus in die natuurlike omgewing, te ondersoek.

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