Advancing the understanding of the PE/PPE protein families through PPE_MPTR protein expression and analysis of pe/ppe single nucleotide variants in differentially drug sensitive Mycobacterium tuberculosis.

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dc.contributor.advisorSampson, Samanthaen_ZA
dc.contributor.advisorKriel, Nastassjaen_ZA
dc.contributor.authorBartizal, Tom Jacken_ZA
dc.contributor.otherStellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.en_ZA
dc.date.accessioned2022-11-14T10:34:59Zen_ZA
dc.date.accessioned2023-01-23T06:52:18Zen_ZA
dc.date.available2022-11-14T10:34:59Zen_ZA
dc.date.issued2022-11en_ZA
dc.descriptionThesis (MSc)--Stellenbosch University, 2022.en_ZA
dc.description.abstractENGLISH ABSTRACT: Mycobacterium tuberculosis is one of the leading causes of infection and death by a single microorganism. The M. tuberculosis genome contains two prominent gene families, encoding proteins known as the PE (proline-glutamate) and PPE (proline-proline-glutamate) families. Several of these are essential for M. tuberculosis pathogenesis, however, the majority of the PE/PPE proteins, especially those within the PPE_MPTR subfamily are understudied. This is apparent regarding the sub-cellular localisation of PPE_MPTR proteins which may interact at the host-pathogen interface. Some functional aspects have also been overlooked, such as the potential for pe/ppe variants to contribute to drug resistance in M. tuberculosis. These genes are often overlooked due to their polymorphic nature, as well as the challenges associated with short-read sequencing technologies to reliably assemble their highly repetitive and GC-rich sequences. For the first part of our study, we aimed to identify the sub-cellular localisation of select PPE_MPTR proteins (PPE_MPTR10, -24, -40, 42, -53 and -62) by expressing them as fluorescently tagged proteins in Mycobacterium smegmatis. We successfully amplified all six genes from M. tuberculosis H37Rv DNA, and successfully cloned ppe_mptr10 into the pCG expression vector. Unfortunately, no protein expression was detected and we were unable to determine the localisation of PPE_MPTR10 within M. smegmatis. For the second aim of our study, we made use of a newly developed analytical pipeline to screen whole-genome sequencing (WGS) data and identify high-confidence pe/ppe singlenucleotide variants (SNVs) associated with drug resistance profiles. Nine SNVs were predicted to be unique to either drug susceptible, multi- or extensively-drug resistant (DS, MDR or XDR) classes of M. tuberculosis. We aimed to verify these findings by PCR and Sanger sequencing of targeted SNVs in an independent set of clinical M. tuberculosis isolates. The SNVs were determined to be true variants identified by the pipeline present in clinical isolates but absent from the reference M. tuberculosis H37Rv. None of the target variants were found to be unique to the drug resistance class for which they were originally predicted. The successful identification of pe/ppe variants by genomic analysis demonstrates the potential to screen these repetitive GC-rich regions for genetic variants. Future studies will be required to establish the role of PPE_MPTR proteins in M. tuberculosis pathogenesis.en_ZA
dc.description.abstractAFRIKAANSE OPSOMMING: Mycobacterium tuberculosis word beskou as een van die hoofoorsake van infeksie en dood deur 'n enkele mikro-organisme. Die M. tuberculosis-genoom bevat koderende streke vir twee prominente geenfamilies, wat kodeer vir proteïene bekend as die PE (prolien-glutamaat) en PPE (prolien-prolien-glutamaat) families. Daar is voorgestel dat verskeie van hierdie proteïene noodsaaklik is vir M. tuberculosis patogenese, maar die meerderheid van die PE/PPE proteïene, veral dié binne die PPE_MPTR subfamilie, word onderbestudeer. Dit is duidelik met betrekking tot die sub-sellulêre lokalisering van PPE_MPTR proteïene wat moontlik tot interaksies by die gasheer-patogeen koppelvlak betrokke mag wees. Sommige funksionele aspekte is ook oor die hoof gesien, soos die potensiaal vir pe/ppe-variante om by te dra tot ontwikkeling van middelweerstand in M. tuberculosis. Hierdie gene word dikwels geïgnoreer as gevolg van hul polimorfiese aard, sowel as die uitdagings met kortleesvolgordebepalingstegnologieë om hul hoogs herhalende en GC-ryke streke te onthul. Vir die eerste doel van ons studie het ons daarop gemik om die sub-sellulêre lokalisering van PPE_MPTR proteïene (PPE_MPTR10, -24, -40, 42, -53 en -62) te identifiseer deur hulle uit te druk as fluoresserende proteïene in Mycobacterium smegmatis. Al ses gene van M. tuberculosis H37Rv DNA is geamplifiseer en ppe_mptr10 in die pCG uitdrukkingsvektor gekloneer. Ongelukkig kon geen proteïenuitdrukking bevestig word nie en kon ons nie die lokalisering van PPE_MPTR10 binne M. smegmatis bepaal nie. Vir die tweede doel van ons studie, het ons gebruik gemaak van 'n nuwe analitiese pyplyn om heelgenoom data te gbruik om hoë-vertroue pe/ppe enkelnukleotied variante (SNVs) wat met geneesmiddelweerstandsprofiele geassosieer word, te identifiseer. Ons het nege SNVs geïdentifiseer wat uniek was aan óf dwelm vatbare, multi- of ekstensief-middel weerstandbiedende (DS, MDR of XDR) klasse van M. tuberculosis. Ons het ten doel gehad om hierdie bevindinge te verifieer deur PCR en Sanger-volgordebepaling van geteikende SNVs in 'n onafhanklike stel kliniese M. tuberculosis-isolate. Die SNVs is vasgestel as ware variante wat geïdentifiseer is deur die pyplyn teenwoordig in kliniese isolate maar afwesig van die M. tuberculosis H37Rv verwysing. Daar is gevind dat geen van die teikenvariante uniek is aan die geneesmiddelweerstandsklas waarvoor dit oorspronklik voorspel is nie. Die suksesvolle identifikasie van pe/ppe-variante deur genomiese analise demonstreer die potensiaal om hierdie herhalende GC-ryke streke vir genetiese variante te keur. Toekomstige studies sal vereis word om die rol van PPE_MPTR proteïene in M. tuberculosis patogenese vas te stel.af_ZA
dc.description.versionMastersen_ZA
dc.embargo.lift2023-12-31en_ZA
dc.embargo.terms2023-12-31en_ZA
dc.format.extentxi, 96 pages : illustrationsen_ZA
dc.identifier.urihttp://hdl.handle.net/10019.1/126325en_ZA
dc.language.isoen_ZAen_ZA
dc.publisherStellenbosch : Stellenbosch Universityen_ZA
dc.rights.holderStellenbosch Universityen_ZA
dc.subject.lcshMycobacterium tuberculosis -- Pathogenesisen_ZA
dc.subject.lcshProline-glutamateen_ZA
dc.subject.lcshProline-proline-glutamate)en_ZA
dc.subject.lcshDrug resistanceen_ZA
dc.subject.lcshUCTDen_ZA
dc.titleAdvancing the understanding of the PE/PPE protein families through PPE_MPTR protein expression and analysis of pe/ppe single nucleotide variants in differentially drug sensitive Mycobacterium tuberculosis.en_ZA
dc.typeThesisen_ZA
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Masters Degrees (Molecular Biology and Human Genetics)