Investigation of the expression and immunogenicity of Mycobacterium tuberculosis Rv1460

Thesis (PhD)--Stellenbosch University, 2021ENGLISH ABSTRACT: A challenge that has plagued humanity for centuries is the intracellular pathogen Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis. In 2018, over 9.6 million individuals fell ill with the disease and 1.5 million lost their lives. Not all infected individuals get sick, 95% remain healthy (asymptomatic) and are said to be latently infected. Only 3-10% of infected individuals develop tuberculosis (TB). The shortcomings of current immunodiagnostics include the failure to detect progression from latent infection to active tuberculosis disease, and the inability to monitor treatment efficacy. This highlights the need for new tuberculosis biomarkers. These biomarkers should be highly sensitive and specific diagnosing TB infection, specifically distinguishing between latent infection and active disease. One strategy to identify Mtb antigens with diagnostic potential is to identify genes that are specifically induced during infection or in specific disease stages. Despite the importance of iron homeostasis during TB infection, the utility of bacterial antigens expressed during the iron limitation experienced by Mtb within the host remains largely unexplored. The Rv1460 gene encodes a transcriptional regulator of the Rv1460-Rv1461-Rv1462-Rv1463-csd-Rv1465-Rv1466 mobilization of sulphur (SUF) operon, which encodes the primary iron-sulphur biogenesis system in Mtb. The SUF operon is induced during iron limitation and intracellular growth of Mtb, pointing to its importance during infection. This study therefore aimed to investigate the level of the Rv1460 protein during intracellular growth of Mtb, and to gain insight into the regulation involved in determining these levels. Furthermore, since Rv1460 levels are predicted to be elevated during intracellular growth and infection, we hypothesize that it is immunogenic and may induce an immune response in individuals infected with Mtb that has diagnostic potential. To facilitate rapid monitoring of Rv1460 expression at the single cell level during intracellular growth we sought to develop a fluorescent reporter for monitoring its expression. Two fluorescent reporter vectors were constructed by cloning two Rv1460 promoter regions (123 bp and 211 bp) upstream of a promoterless mCherry gene and transforming Mtb H37Rv with the resulting vector constructs. The expression of the mCherry protein had no effect on the growth of the fluorescent strains when compared to the wild type (wt) control. Florescence per cell was determined either by dividing fluorescence determined in a plate reader by colony forming units per well i.e., Relative fluorescent units (RFU)/Colony forming units (CFU) or by flow cytometry. The RFU/CFU measurements showed more than a 10x increase between day 4 and 10 for H37Rv attB::pMV306 (control strain), H37Rv attB::pMV306_123mCherry and H37Rv attB::pMV306_211mCherry (fluorescent reporter strains), while values remained constant between day 10 and 14. Flow cytometry showed no increase in fluorescence between day 4, 10 and 14 for the two reporter strains. The high background fluorescence observed in the control strain for the RFU/CFU measurements suggests that the increase observed between day 4 and 10 was due to autofluorescence of the cells or background fluorescence in the media. An increase in the Rv1460 transcript was observed between day 4 and 10, while between day 10 and 14 the transcript levels decreased. The mCherry transcript was significantly lower than the Rv1460 transcript and the ratio of the two transcripts did not remain constant in either strain over time. This suggests that the mCherry reporters did not accurately reflect changes in Rv1460 transcript levels over time. Monitoring fluorescence in the reporter strains during intracellular growth suggests that Rv1460 expression is induced in a subset of bacteria within macrophages, and this population increases over the course of a 72-hour infection. Rv1460 protein does not seem to be highly immunogenic under the conditions tested. Higher levels of Interleukin (IL)-8 (p = 0.08) and Matrix metallopeptidase (MMP)-9 (p = 0.00) was measured in the active TB group when compared to the Quantiferon (QFN) negative (neg) group for the short-term stimulation (12 hours) with Rv1460 suggestive of an effector or memory response. Short-term stimulation for all stimulation conditions showed significantly lower levels of Regulated and normal T cell expressed and secreted (RANTES) in the active TB group when compared to QFN positive (pos) and QFN neg groups. This cytokine selectively attracts monocyte/macrophages in the airways and strongly induces migration of T cells with the memory phenotype.AFRIKAANS OPSOMMING: ‘n Uitdaging wat die mensdom al vir eeue teister is die intrasellulêre patogeen Mycobacterium tuberculosis, die bakterieë wat tuberkulose (TB) veroorsaak. In 2018 het meer as 9.6 miljoen individue tuberkulose opgedoen en 1.5 miljoen het hulle lewens as gevolg daarvan verloor. Nie alle individue wat met met die bakterium besmet is raak siek nie, 95% van besmette individue bly gesond (asimptomaties) en word beskryf as latente infeksie. Slegs 3-10% van die wat met die bakterium besmet is ontwikkel TB. Die tekortkominge van huidige immunodiagnostieke tegnieke sluit in mislukte pogings om vordering van latente infeksie na aktiewe TB siekte te voorspel en die onvermoeë om die doeltreffenheid van behandeling vastestel. Hierdie gebreke beklemtoon die behoefte aan nuwe TB biomerkers. Hierdie biomerkers moet hoog sensitief en baie spesifiek vir die diagnosering van TB infeksie wees, en moet spesifiek tussen latente infeksie en aktiewe siekte kan onderskei. Een strategie wat gebruik kan word is om M. tuberculosis antigene met diagnostiese potensiaal te identifiseer. Die wat tydens die uitdrukking van spefieke gene gedurende infeksie en spesifieke siekte fases uitgedruk word. Ondanks die belangrikheid van yster homeostase tydens TB-infeksie, is die gebruik van bakteriële antigene wat die bakterium tydens yster beperkings in die gasheer ervaar grootliks onder bestudeer. Die Rv1460 geen kodeer ‘n transkripsionele reguleerder van die Rv1460-Rv1461-Rv1462-Rv1463-csd-Rv1465-Rv1466 (SUF) operon, wat die primêre yster-swawel (Fe-S) biogenese stelsel in M. tuberculosis is. Die SUF-operon word geïnduseer tydens yster beperkings en intrasellulêre groei van M. tuberculosis, wat daarop dui dat dit belangrik is tydens infeksie. Met hierdie studie het ons be-oog om die vlakke van Rv1460 proteïen tydens intrasellulêre groei van M. tuberculosis te ondersoek, en om insig te verkry van die regulering van die proteïen en bepaling van proteïen vlakke. Aangesien daar voorpel word day die Rv1460-vlakke verhoog sal word tydens intrasellulêre groeie en infeksie, reken ons dat dit immunogenies is en ‘n immuunrespons kan veroorsaak by individue wat met M. tuberculosis besmet is, end dat dit diagnostiese potensiaal het. In ‘n poging om die Rv1460-uitdrukking op enkel sel vlak tydens intrasellulêre groei vastestel, het ons ‘n fluoressensie verslaggewende proteïen ontwikkel om uitdrukking vas te stel. Twee fluoressensie vektore is gekonstrueer deur twee Rv1460-promotor gebiede (123 en 211 bp) stroomop van ‘n promotorlose mCherry geen te kloon en M. tuberculosis H37Rv met die resulterende vektore konstrukte te transformeer. Die uitdrukking van die mCherry proteïen in die fluoresserende stamme het geen effek op die groei van die fluoresserende bakteriële stamme gehad nie in vergelyking met die wt-kontrole stamme Fluoressensie per sel is bepaal deur die fluoressensie wat deur ‘n plaatleser of vloei-sitometrie bepaal was deur die kolonie vormerende eenhede per putjie (RFU/CFU) te deel. Die RFU/CFU lesings het meer as ‘n 10x toename getoon tussen dag 4 en 10 vir for H37Rv attB::pMV306 (kontrole stam), H37Rv attB::pMV306_123mCherry and H37Rv attB::pMV306_211mCherry (fluoresserende stamme), terwyl die waardes konstant gebly het tuseen dag 10 en 14. Vloei-sitometrie het geen toename in fluoressensie getoon tussen dag 4, 10 en 14 vir die twee fluoresserende stamme nie. Die hoë agterground fluoressensie (RFU/CFU lesings) wat waargeneem is tussen dag 4 en 10 in die kontrole stamme kan toegeskryf word aan die outofluoresensie van die sel of agterground fluoressensie van die media. ‘n Toename in die Rv1460-transkripsie vlakke was waargeneem tussen dag 4 en 10, terwyl ‘n afname tussen dag 10 en 14 waargeneem was. Die mCherry transkripsie vlakke was aansienlik laer as die Rv1460 transkripsie vlakke en die verhouding van die twee transkripsie vlakke het gewissel met tyd. Dit dui daarop dat die mCherry verslaggewende proteïen nie die Rv1460 veranderinge oortyd akkuraat weerspieël nie. Fluoressensie bepalings tydens intrasellulêre groeie dui daarop dat ‘n seker populasie bakterieë in die makrofage Rv1460 uitdruk en dat hierdie populasie toeneem oor die 72-uur infeksie periode. Rv1460 proteiën blyk nie om hoogs immunogenies te wees onder die toestande waarin dit getoets was nie. Hoër vlakke van IL-8 (p = 0.08) and MMP-9 (p = 0.00) was gemeet in die aktiewe TB group in vergelyking met die QFN negatiewe groep vir die kortermyn Rv1460 stimulasie (12 ure). Kortemyn stimulaie, vir al die stimulaie kondisies gee aansienlike laer vlakke van RANTES in die aktiewe TB groep in vergelyking met die QFN positiewe en QFN negatiewe groepe. RANTES het ‘n selektiese aksie as monosiet/makrofaag lokmiddel in die lugweë wat die migrasie van T selle met ‘n geheue fenotiepe induseer.Doctorat