The influence of small RNAs on the physiology of Mycobacterium tuberculosis

Zvinairo, Tawanda Kennedy (2015-12)

Thesis (MScMedSc)--Stellenbosch University, 2015.

Thesis

ENGLISH ABSTRACT: The role of bacterial small RNA (sRNA), i.e. RNA species between 50-500bp in size, in virulence, pathogenesis and drug resistance is gaining interest. In some bacterial species, it had been shown to play a crucial role in bacterial transcriptional and post-transcriptional regulation. sRNAs from various pathogenic bacteria were shown to modulate bacterial responses to the host and environment. In Mycobacterium tuberculosis, the causative agent of tuberculosis, more than 1000 sRNA species have been identified already; but the role of these sRNA in pathogenesis, virulence and stress responses is not well studied. Central dogma suggests that drug resistance in M. tuberculosis is associated with mutations in specific genes. However, a number of clinical drug resistant isolates do not harbour mutations in these genes, implicating other factors such as unknown mutations, as well as altered regulation of these resistance genes. Prediction of resistance, using molecular methods, can therefore be inaccurate in cases where known mutations are absent. In cases where known drug-resistance associated mutations are absent, mutations in other genes that regulate such resistance-associated genes might influence drug resistance. Growing evidence, in other bacteria and M. tuberculosis, hints at a role for mutations in intergenic regions and sRNAs species to play a role in bacterial growth and drug sensitivity. In light of this we hypothesised that mutations in sequences encoding sRNA or in sRNA target sequences influence the phenotype of M. tuberculosis clinical isolates. Using previously identified sRNA genes; we screened a genomic bank of clinical M. tuberculosis isolates for the presence of mutations in these sRNA encoding genes. A large number of isolates showed mutations in genes encoding for sRNAs. Furthermore, over-expression of sRNA using the plasmid pMV306 in Mycobacterium smegmatis showed differences in growth indicating that the presence of the extra copies of the three sRNA (mcr3, ASpks and mpr6) had a phenotypic effect on the bacterium. Overexpressed sRNAs did not affect the bacterial drug resistance phenotypes, although this requires further investigation before concluding the effect of sRNAs on drug resistance. We successfully modified a method to extract and purify sRNAs from Mycobacterium species, clean enough to perform Real Time Polymerase Chain Reaction even with small amounts. However challenges were faced in terms of quantification. Another challenge that still remains is obtaining reference genes specifically for sRNAs as we currently have none.

AFRIKAANSE OPSOMMING: Die rol van klein ribonukleïnsure ( m.a.w RNS spesies van ongeveer 50-250bp in grootte) in bakteriële virulensie, patogenese en antibiotika weerstandigheid word al hoe meer bevraagteken. 'n Rol vir hierdie nukleinsure in transkripsie en post-transkripsie regulering was voorheen gewys in verskeie bakteriële studies, waar dit gedemonstreer was dat hierdie RNA spesies n rol speel vir die bakterieë om aan te pas in die gasheer se omgewing 1–3. Meer as 1000 klein RNS spesies is voorheen in Mycobacterium tuberculosis (die bakterie wat tuberkulosis veroorsaak) geïdentifiseer, maar die rol van hierdie RNA in patogenese, virulensie en stress reaksies is nie bekend nie. Antibiotika weerstandigheid in M. tuberculosis word tans geassosieer met mutasies in spesifieke gene. Daar is wel n aantal weerstandige isolate waar hierdie bekende mutasies heeltemal afwesig is, wat suggereer dat ander rolspelers aanleiding kan gee to middelweerstandigheid. Byvoorbeeld, veranderde regulering van transkripsie patrone van gene (wat n bekende rol in weerstandigheid het) mag ook aanleiding gee tot weerstandigheid, maar sulke alternatiewe meganismes is nog nie goed ondersoek in die bakterium nie. Dis belangrik om al die rolspelers te identifiseer, want bestaande molekulere diagnostiese tegnieke fokus slegs op bekende gene; dus sal weerstandigheid gemis word in isolate waar bekende mutasies afwesig is en slegs molekulere tegnieke gebruik word. Die potensiële assosiasie van klein RNS in tuberkulose antibiotika weerstandigheid is voorheen in n paar studies gemaak. In lig van hierdie studies, is dit voorspel dat mutasies in klein RNA kan aanleiding gee tot verandering in die sensitiwiteit teenoor antibiotika in M. tuberculosis. Vir hierdie studie het ons n genoom bank, wat bestaan uit individuele genome van kliniese M.tuberculosis isolate, geanaliseer vir die teenwoordigheid van mutasies in klein RNS. Daar was spesifiek gefokus op die klein RNS spesies wat in vorige studies met antibiotika weerstandigheid geassosieer was. Hierdie bio-informatiese analise het mutasies in klein RNS spesies in n groot aantal weerstandige stamme geïdentifiseer. Hierdie mutasies was nie in sensitiewe isolate gevind nie, Om die rol van spesieke RNS spesies te ondersoek, was rekombinante plasmiede geskep wat bestaan het uit spesifieke klein RNS spesies van M. tuberculosis en die plasmied pMV306. Hierdie rekombinante was getransformeer in Mycobacterium smegmatis. Die teenwoordigheid van hierdie M. tuberculosis klein RNS kopieë in M.smegmatis het n negatiewe impak gehad op groei, en dui aan dat hierdie RNA spesies, naamlik mcr3, ASpks and mpr6, n potensiele belangrike rol het in die fenotipe van mikobakterieë het. Die ekstra kopieë het nie veranderinge veroorsaak in sensitiwiteit van M.smegmatis teenoor die antibiotika moksifloksasien en kanamisien nie, hoewel meer studies gedoen moet word voordat definitiewe konklusies gemaak kan word. In die finale deel van die studie, is n metode ontwerp om klein RNS op n makliker,vinner manier te isoleer van mikobakterieë. Hierdie metode was suksesvol aangewend om DNA-vry, hoë kwaliteit RNS, beide groter RNA en klein RNS spesies te isoleer. Die klein RNS was goeie kwaliteit, DNA-vry en kon omskep word in DNA met retrotranskripsie. Laasgenoemde DNA kon ook gebruik word in verder polymerase kettingreaksies. Dit het dus potential vir kwantitatiewe studies om die regulering van klein RNS te studeer.

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