The identification of novel proteins involved in iron-sulphur cluster biogenesis in mycobacteria

Thesis (MSc)--Stellenbosch University, 2016ENGLISH ABSTRACT : Mycobacterium tuberculosis remains one of the world’s deadliest pathogens, and although decades of research have been dedicated to the discovery and development of anti-tuberculosis agents and vaccines, tuberculosis (TB) remains an ongoing global public health threat. Given the emergence of drug resistance and the poor treatment outcomes associated with this disease, it is imperative that we improve our understanding of the pathogenesis of M. tuberculosis in order to develop new anti-TB drugs and strategies for shortening drug treatment. Iron-sulphur (Fe-S) clusters are ubiquitous cofactors that are required for the maturation of various proteins, many of which are involved in essential biological processes. Multiprotein complexes are required for the in vivo assembly of Fe-S clusters, and the SUF system, encoded by the Rv1460-Rv1461-Rv1462-Rv1463-csd-Rv1465- Rv1466 operon in M. tuberculosis, is thought to be the major Fe-S cluster biogenesis machinery in this organism. This process is poorly understood in mycobacteria, and it is currently unclear if proteins outside of this operon are involved in Fe-S cluster biogenesis. In this study, we sought to identify novel proteins involved in Fe-S cluster assembly in mycobacteria using various approaches. Initially we optimised blue-native PAGE for the analysis of mycobacterial cytoplasmic protein complexes, and demonstrated that protein complexes could be identified by immunodetection of BN-PAGE and subsequent gel fraction analysis by mass spectrometry. However, due to the complexity of the cytoplasmic fraction and poor resolution of this technique, we were unable to use it to identify complexes containing the Fe-S cluster biogenesis machinery. In a second approach, we set out to generate an antibody against Csd, an Fe-S cluster assembly protein, to facilitate immunodectection of the Fe-S cluster biogenesis machinery. However, heterologous expression of the M. tuberculosis Csd in Escherichia coli proved unsuccessful due to insolubility of the recombinant protein. In an attempt to achieve soluble expression of Csd, a 6xHis-tag fusion was expressed in the closely related organism Mycobacterium smegmatis. Soluble expression of the fusion protein was optimised and confirmed by immunodetection. We then employed affinity enrichment using nickel-charged magnetic beads and mass spectrometry to identify potential interacting partners for Csd. Strains expressing a 6xHis- tag SufC fusion and a 6xHis-tag mCherry fusion were also included for comparison. Utilising this method, we were able to demonstrate affinity enrichment for Fe-S cluster assembly proteins, and Fe-S cluster-containing enzymes, as well as identify potential novel interacting partners. This work has established the methodology for identifying novel protein-protein interactions in mycobacteria, and lays the foundation for elucidating the process of Fe-S cluster assembly in mycobacteria.AFRIKAANSE OPSOMMING : Mycobacterium tuberculosis is nog steeds een van die mensdom se dodelikste patogene ten spyte van intensiewe navorsing wat fokus op die ontdekking en ontwikkeling van antimikrobiese middels en vaksiene om tuberkulose (TB) te bekamp. Die toename in antimikrobiese weerstandbiedendheid in hierdie patogeen sowel as die onbereidwilligheid van pasiënte om die streng medisynekursus te volg, beklemtoon die noodsaaklikheid om die organisme en sy oorlewingsmeganismes beter te verstaan. Dit kan lei tot die ontwikkeling van nuwe teen-TB middels en die verkorting van middelbehandeling. Yster-swawel (Fe-S) komplekse dien as kofaktore vir verskeie proteïene, waarvan baie noodsaaklike rolle verig in sekere biologiese prosesse. Multiproteïen komplekse is nodig sodat Fe-S komplekse in vivo gesintetiseer kan word – ‘n proses wat nog nie goed beskryf is vir mikobakterieë nie. Die SUF sisteem, wat in M. tuberculosis deur die geenoperon Rv1460-Rv1461-Rv1462-Rv1463- csd-Rv1465-Rv1466 geënkodeer word, word beskou as die hoofkomponent in die biogenese van Fe-S komplekse. Onsekerheid heers of ander proteïene buite dié van die operon ook ‘n rol speel in biogenese. In hierdie studie het ons gepoog om nuwe proteïene te identifiseer wat betrokke kan wees in die biogenese van Fe-S komplekse deur verskeie benaderings te volg. Aanvanklik het ons die tegniek van blou-natuurlike poliakrielamied jelelektroforese (blue-native PAGE) geöptimiseer om mikobakteriële sitoplamiese proteïenkomplekse te analiseer. Ons het daarmee geslaag om proteïenkomplekse te identifiseer met behulp van immuno-opsporing gevolg deur jelfraksie-analise met massa-spektrometrie. Vanweë die kompleksiteit van die sitoplasmiese fraksie en die swak resolusie van die tegniek is geen komplekse wat Fe-S komplekse bevat, geïdentifiseer nie. In die tweede benadering is daar gepoog om ‘n teenliggaam te genereer vir Csd, ‘n proteïen betrokke in die biogenese van Fe- S komplekse. Dit is ten doel om die immuno-opsporing van die Fe-S komplekse te vergemaklik. Heteroloë uitdrukking van Csd in E. coli het gelei tot die produksie van ‘n onoplosbare proteïen. Deur die proteïen uit te druk in die nabyverwante M. smegmatis, tesame met ‘n 6xHis fusie, het gelei tot ‘n oplosbare produk wat later bevestig is deur immuno-opsporing. Ons het daarna affiniteit-verryking gebruik met behulp van nikkel- gelaaide magnetiese krale en massa-spektrometrie om proteïene wat moontlike interaksies met Csd het, te identifiseer. Die nodige kontroles (rasse wat ‘n 6xHis fusie het met onderskeidelik ‘n sufc en ‘n mcherry geen) is ingesluit ter vergelyking. Met die gebruik van hierdie metode het ons affiniteit-verryking vir proteïene betrokke in die biogenese van Fe-S komplekse gewys asook Fe-S kompleksbevattende ensieme. Ons het terselfdetyd nuwe potensiële proteïene geïdentifiseer wat interaksie toon met Fe-S komplekse. Hierdie studie het ‘n metode daargestel vir die identifikasie van nuwe proteïen-proteïen interaksies in mikobakterieë en lê ‘n fondasie vir die toeligting van die proses van die biogenese van Fe-S komplekse in mikobakterieë