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The impact of nitrogen limitation on virulence and the quorum sensing response in Cryptococcus neoformans

dc.contributor.advisorVolschenk, Heinrichen_ZA
dc.contributor.advisorTrollope, Kimen_ZA
dc.contributor.authorSamie, Shakieren_ZA
dc.contributor.otherStellenbosch University. Faculty of Science. Dept. of Microbiology.en_ZA
dc.date.accessioned2020-02-26T08:00:12Z
dc.date.accessioned2020-04-28T15:15:51Z
dc.date.available2020-02-26T08:00:12Z
dc.date.available2020-04-28T15:15:51Z
dc.date.issued2020-04
dc.identifier.urihttp://hdl.handle.net/10019.1/108485
dc.descriptionThesis (MSc)--Stellenbosch University, 2020.en_ZA
dc.description.abstractENGLISH ABSTRACT: Cryptococcus neoformans may cause life-threatening meningitis in immune-compromised populations, with high mortality rates despite measures currently put in place to treat infections. It is, therefore, critical that new and effective anticryptococcal drugs and treatment strategies are developed. Using an antipathogenic approach, which does not directly kill the yeast but rather targets key virulence factors or quorum sensing (QS), is a promising, novel strategy. However, the regulation of cryptococcal QS, and how this process influences virulence factor expression, has not been characterised. In particular, the dynamics of these processes under nitrogen-limiting conditions, which the pathogen experiences in its natural environment and during host infection, has not been defined. This study aimed to characterise the expression of cryptococcal virulence factors during nitrogen limitation (NL) and whether this was being influenced by QS. Furthermore, this study aimed to engineer a C. neoformans QS biosensor to monitor QS responses during NL and to screen for potential compounds that could interfere with cryptococcal QS. After growth in nitrogen-limiting conditions relevant to the Cryptococcus natural niche, C. neoformans H99 displayed a thicker polysaccharide capsule, as well as higher urease and laccase activities. Melanisation and capsule thickness were specifically increased during both NL and temperature stress. Ergosterol biosynthesis also appeared to be induced during NL and temperature stress. A C. neoformans CK0289 QS mutant also expressed thicker capsules and increased urease activity during NL. However, capsule thickness, together with melanisation and ergosterol biosynthesis, were expressed notably weaker than C. neoformans H99. This suggests that NL does influence the virulence phenotype, but that QS may be required to signal for increased virulence factor and cell membrane ergosterol production during NL. Garlic-derived organosulfides and synthetic derivatives thereof were selected as compounds to screen for potential anti-QS activity against C. neoformans. The natural organosulfides showed potent antifungal activity and affected the expression of C. neoformans virulence factors. Specifically, capsule thickness was reduced and melanisation completely inhibited. Despite having no inhibitory effect on urease activity or ergosterol biosynthesis, these compounds still serve as possible candidates for the development of antipathogenic drugs against C. neoformans. To determine whether these compounds have quorum quenching activity, a biosensor responsive to cryptococcal QS and able to monitor changes in this process was designed. Although the biosensor was successfully constructed in Saccharomyces cerevisiae, no signal could be detected. This study has demonstrated that NL is an important signal that influences the virulence of C. neoformans, and that this relationship is potentially affected by QS. Further research is needed to comprehensively characterise this phenomenon as this would provide valuable information in understanding the physiology and pathogenicity of C. neoformans.en_ZA
dc.description.abstractAFRIKAANSE OPSOMMING: Ondanks maatreëls tans in plek om infeksies te behandel kan Cryptococcus neoformans lewensgevaarlike meningitis in immuun-gekompromitteerde populasies veroorsaak met hoë sterftesyfers. Daarom is dit krities om nuwe en effektiewe antikriptokokkale middels en behandelingstrategieë te ontwikkel. ‘n Belowende en ongewone strategie is die antipatogeniese benadering wat sleutel virulensie faktore of kworumwaarneming (KW) eerder as direkte gisdoding teiken. Die regulering van kriptokokkale KW en hoe die proses die uitdrukking van virulensie faktor affekteer is egter nog onbekend. Die dinamika tussen die prosesse onder stikstofbeperkende kondisies, soos wat die patogeen in sy natuurlike omgewing en tydens gasheerinfeksie ondervind, is veral nog nie omskryf nie. Hierdie studie stel ten doel om die uitdrukking van kriptokokkale virulensie faktore tydens stikstofbeperking (SB) te karakteriseer en die rol wat KW speel te ondersoek. Die studie beoog verder ook om ‘n C. neoformans KW biosensor te ontwikkel om KW reaksies tydens SB te kan meet en die indentifisering van potensiële kriptokokkale KW inmenging verbindings moontlik te maak. Tydens groei onder stikstofbeperkende kondisies, wat die natuurlike nis van Cryptococcus emuleer, vertoon C. neoformans H99 ‘n vergrote polisakkariedkapsule, asook hoër urease en lakkase ensiemaktiwiteit. Melanienvorming en kapsulegrootte word veral verhoog tydens beide SB en temperatuurstres. Ergosterolbiosintese word ook spesifiek geïnduseer tydens SB en temperatuurstres. Vergrote kapsule en verhoogte urease aktiwiteit word ook onder SB kondisies deur die C. neoformans CK0289 KW mutant vertoon, maar kapsulegrootte, melanienvorming en ergosterolbiosintese word beduidend swakker in vergelyking met C. neoformans H99 uitgedruk. Die resultate dui daarop dat SB die virulensie fenotiepe beïnvloed, en dat KW die nodige sein vir verhoogte virulensie faktore en selmembraan ergosterol produksie tydens SB verskaf. Knoffel-organosulfide en sintetiese derivate is geselekteer om vir potensiele anti-KW verbindings teen C. neoformans te sif. Die natuurlike organosulfide het kragtige swamdodende aktiwiteit vertoon en het die uitdrukking van C. neoformans virulensie fakore beïnvloed. Kapsulegrootte is spesifiek verlaag en melanienvorming volledig geïnhibeer. Hierdie verbindings dien steeds as moontlike antipatogeniese kandidaatmiddels teen C. neoformans, ten spyte van die afwesigheid van urease ensiemaktiwiteit en ergosterolbiosintese onderdrukking. ʼn Kriptokokkale KW-sensitiewe biosensor om die kworumsmorings aktiwiteit van die verbindings asook verandering in die KW proses te bepaal is ontwerp. Alhoewel die biosensor suksesvol in Saccharomyces cerevisiae gebou is, kon geen sein waargeneem word nie. Die studie demonstreer die belangrike rol van SB op C. neoformans virulensie faktore, en dat KW hierdie verhouding affekteer. Verdere navorsing word benodig om die fenomeen volledig te karakteriseer, om waardevolle inligting en ʼn dieper verstaan van die fisiologie en patogenisiteit van C. neoformans te bevorder.af_ZA
dc.format.extentxi,115 pages : illustrationsen_ZA
dc.language.isoen_ZAen_ZA
dc.publisherStellenbosch : Stellenbosch Universityen_ZA
dc.subjectCryptococcus neoformansen_ZA
dc.subjectNitrogen limitationen_ZA
dc.subjectVirulenceen_ZA
dc.subjectQuorum sensing (Microbiology)en_ZA
dc.subjectCryptococcus -- Effect of environment onen_ZA
dc.subjectUCTDen_ZA
dc.titleThe impact of nitrogen limitation on virulence and the quorum sensing response in Cryptococcus neoformansen_ZA
dc.typeThesisen_ZA
dc.description.versionMastersen_ZA
dc.rights.holderStellenbosch Universityen_ZA


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