Investigating the induction of autophagy by different Mycobacterium tuberculosis strains: Do strain-specific differences exist?

Polson, Alma (2017-03)

Thesis (MSc)--Stellenbosch University, 2017.

Thesis

ENGLISH ABSTRACT: The pathogenicity of Mycobacterium tuberculosis (M.tb) is determined by its ability to survive within host macrophages. The mammalian autophagy pathway is now recognised as a major contributor to disease pathogenesis. Autophagy, a destructive catabolic process, plays a significant role in the destruction of intracellular pathogens. Clearer understanding of the natural range of autophagic responses elicited by different mycobacteria is required. Autophagy induction has been shown to differ in magnitude depending on the mycobacterial species. However, no study has investigated the specific autophagic capacities of different M.tb strains. We aim to investigate the host autophagic response to different M.tb strains (and clades within strains) responsible for the tuberculosis epidemic in South Africa. THP-1 cells were infected with seven different M.tb clinical isolates, representing six different lineages and the lab strain H37Rv. After RNA extraction, gene expression analysis of 84 autophagy-related genes was performed using the RT2 Profiler™ autophagy array. Our results revealed that all seven strains influenced the autophagy pathway in various ways and different magnitudes. Infection with the LAM 1 and CAS/Kili strains resulted in significant down-regulation of interferon gamma (IFNG) gene expression compared to the other stains. Since IFNG is a potent inducer of autophagy, we conclude that these two strains are weak inducers of autophagy. The autophagosome formation is regulated through the ATG1-10, ATG12-14, ATG16-18, ATG29 and ATG31 genes. The LAM 1, Atypical Beijing, H37Rv, CAS/Kili and LCC strains have the ability to inhibit autophagosome formation, whereas Typical Beijing and Haarlem 3 induces the formation of autophagosomes. Differential expression of genes involved with fusion of autophagosomes to lysosomes, LAMP1, DRAM, GABARAP and NPC1, showed that all the investigated strains impaired autophagolysosomal fusion. This result is not unexpected, since it is known that M.tb is able to block autophagolysomal fusion. Furthermore, the LCC and LAM 1 impede the formation of the autophagic vacuole, while LAM 1 also influences protein transport, protein targeting to membrane/vacuole and protease activity. The top 30 differentially expressed genes were subsequently investigated as potential TB susceptibility genes by analysing single nucleotide polymorphism(s) (SNPs) data generated using the Illumina Multi-ethnic Genotyping Array (MEGA) in a cohort of South African Coloured TB patients and control individuals. After conducting a case-control association study, none of the variants in the top 30 differentially expressed autophagy associated genes were associated with TB susceptibility following Bonferonni correction for multiple testing. This study improves our understanding of how M.tb manages to overcome the host immune system and points to genes exploited by specific strains during this process.

AFRIKAANSE OPSOMMING: Die patogenesiteit van Mycobacterium tuberculosis (M.tb) word bepaal deur die vermoë om te oorleef binne die gasheer-makrofage. Die soogdier-gasheer se outofagie-meganisme word nou erken as 'n belangrike bydraende faktor wat die siekte se patologiese uitkoms bepaal. Outofagie, 'n vernietigende kataboliese proses, speel 'n belangrike rol in die vernietiging van intrasellulêre patogene. ‘n Beter begrip van die natuurlike verskeidenheid van outofagie-reaksies, wat deur verskillende mikobakterieë ontlok word, is nodig. Dit is reeds bewys dat die induksie van outofagie verskil na gelang van die mikobakteriële spesies. Daar is egter geen vorige studies wat die spesifieke outofagie-vermoëns van verskillende M.tb-stamme ondersoek het nie. In die huidige studie was ons doel om die gasheer se outofagie-reaksie op verskillende M.tb-stamme (en families binne stamme), wat verantwoordelik is vir die tuberkulose-epidemie in Suid-Afrika, te ondersoek. THP-1-selle is met sewe verskillende M.tb-kliniese isolate geïnfekteer, wat ses verskillende families en die laboratorium-stam H37Rv insluit. Na RNA-isolasie, is die uitdrukking van 84 outofagie-verwante gene met behulp van die “RT2 Profiler™ autophagy array” ontleed. Ons resultate het aangetoon dat al sewe stamme die outofagie-pad op verskeie wyses en met verskillende grade beïnvloed. Dit het getoon dat infeksie met die stamme LAM 1 en CAS/Kili die vermoë het om betekenisvolle afregulering van die interferon gamma (IFNG)-geen in vergelyking met die ander stamme teweeg te bring. Aangesien IFNG 'n kragtige induseerder van outofagie is, kan ons aflei dat hierdie twee stamme swak induseerders van outofagie is. Die vorming van outofagosome word deur die ATG1-10-, ATG12-14-, ATG16-18-, ATG29- en ATG31-gene gereguleer. Die LAM 1-, Atipiese Beijing-, H37Rv-, CAS/Kili- en LCC-stamme het die vermoë om outofagosome se vorming te inhibeer, terwyl Tipiese Beijing- en Haarlem 3-stamme die vorming van outofagosome induseer. Die differensiёle uitdrukking van gene betrokke by die samesmelting van outofagosome en lisosome, naamlik LAMP1, DRAM, GABARAP en NPC1, het aangedui dat al die stamme, wat ondersoek is, outofagolisosomale fusie kan ontwrig. Hierdie resultaat was te wagte, aangesien dit bekend is dat M.tb in staat is om outofagolisosomale vorming te blokkeer. In aansluiting hierby het LCC en LAM 1 verder die vermoë om die vorming van die outofagie-vakuool te blokkeer, terwyl LAM 1 ook proteïen-transport, proteïen-teikening van die membraan/vakuool en protease-aktiwiteit beïnvloed. Die eerste 30 gene, wat differensieel uitgedruk word, is daarna ondersoek as moontlike gene, wat TB-vatbaarheid verhoog deur die ontleding van data aangaande enkel-nukleotied-polimorfisme(s) (SNPs), wat gegenereer is deur gebruik van die “Illumina Multi-ethnic Genotyping Array (MEGA)” op die DNS van 'n studie-groep Suid-Afrikaanse Kleurling TB-pasiënte en gesonde individue. Na die uitvoering van ‘n gevalle-kontrole – assosiasie-studie is geeneen van die eerste 30 diferensieёl-uitgedrukte gene geïdentifiseer wat kandidaat-gene vir TB-vatbaarheid, ná Bonferroni-korreksie vir meervoudige toetse, was nie. Hierdie studie sal bydrae om ons begrip van hoe M.tb daarin slaag om die gasheer se immuunstelsel te oorkom, te verbreed. Dit dui ook aan watter gene deur spesifieke stamme gedurende hierdie proses uitgebuit word.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/101367
This item appears in the following collections: