Investigating the Human-M. tuberculosis interactome to identify the host targets of ESAT-6 and other mycobacterial antigens

Thesis (PhD)--Stellenbosch University, 2012.ENGLISH ABSTRACT: The causative agent of human tuberculosis, Mycobacterium tuberculosis, is an intracellular pathogen that secretes virulence factors, namely ESAT-6 and CFP-10, as substrates of the ESX-1 secretion system. It is hypothesised that these substrates interact with host proteins in a targeted manner in order to elicit a required immune response, and they have been shown to be involved in processes related to pro-inflammatory responses, necrosis, apoptosis, membrane lysis and cytolysis. However, the biological function of ESX-1 substrates during host-pathogen interactions remains poorly and incompletely understood. Therefore, the present study was designed to gain insight into the role of the ESX-1 secretion system substrates in host-pathogen interactions and to identify how M. tuberculosis mediates the response of the human host. In this study, a cDNA yeast two-hybrid library was constructed from human lung mRNA, to identify mycobacterial-host protein-protein interactions that occur within the lung alveoli. The ESX-1 secretion system substrates, ESAT-6 and CFP-10, were cloned in-frame into the pGBKT7 vector, which was used in the yeast two-hybrid system to screen the lung cDNA library in Saccharomyces cerevisiae. The ESAT-6 and CFP-10 screens identified 79 and 19 positive colonies, respectively. Of the total number of clones characterised, only two in-frame inserts were identified with the ESAT-6 screen, corresponding to the human proteins filamin A and complement component 1, q subcomponent, A chain (C1QA). In addition, the screen with CFP-10 also identified C1QA as binding partner. Subsequent in vitro and in vivo experiments were unable to confirm the putative interactions of C1QA with ESAT-6 and CFP-10. However, the interaction between filamin A and ESAT-6 was demonstrated and confirmed by both in vivo co-localisation and co-immunoprecipitation. Furthermore, the degradation of filamin A in the presence of ESAT-6 was shown to be reflective of cytoskeleton remodelling and the induction of cell death. The work presented here suggests that as ESAT-6 gains access to the cytosol, it initiates cell death by inducing destabilisation of the cytoskeleton cell structure. This may possibly be driven by the interaction of ESAT-6 and filamin A. Finally, we also initiated an investigation of the identified putative binding partners (filamin A and C1QA) as possible genetic markers for genetic susceptibility studies to tuberculosis. A case-control analysis was performed involving 604 cases, of which 109 were Tuberculous Meningitis (TBM), and 486 were controls from the South African Coloured (SAC) population within the Ravensmead-Uitsig catchment area. The results of this analysis demonstrated a novel association of a regulatory variant (rs587585) located upstream of the C1QA gene and demonstrated an increasing trend towards increased values in tuberculosis patients with the associated genotype. This study has contributed significantly to our understanding of human-mycobacterial hostpathogen protein-protein interactions and has opened the way for future studies further exploring the consequences and function of the identified ESAT-6-filamin A interaction. It has also led to the identification of a novel genetic association with tuberculosis. Finally, it demonstrates the usefulness of the yeast two-hybrid system to identify potential proteinprotein (host-pathogen) interactions that can lead to additional important and exciting research.AFRIKAANSE OPSOMMING: Die organisme wat tuberkulose veroorsaak, Mycobacterium tuberculosis, is `n intrasellulȇre patogeen wat virulensie faktore afskei, naamlik ESAT-6 en CFP-10, as substrate van die ESX-1 sekresiesisteem. Daar word vermoed dat hierdie substrate met gasheerproteïene in „n teiken wyse interaksie het om `n vereiste immuunreaksie voort te bring. Hierdie substrate is betrokke by prosesse soos pro-inflammatoriese reaksies, nekrose, apoptose, membraanlise en sitolise. Die biologiese funksie van die ESX-1 substrate tydens gasheer-patogeen interaksies word egter tans swak en onvolledig verstaan. Daarom was die huidige studie ontwerp om insig te bekom oor die rol hiervan in gasheer-patogeen interaksies en om te identifiseer hoe M. tuberculosis die reaksie teenoor die gasheer bemiddel. In hierdie studie was `n komplementȇre deoksiribonukleïensuur (kDNS) gis twee-hibried biblioteek gemaak vanaf long boodskapper ribonukleïensuur (bRNS) om proteïen-proteïen interaksies wat in die long plaasvind, te identifiseer. Die substrate van die ESX-1 sekresiesisteem, ESAT-6 en CFP-10, is in volgorde gekloneer in die pGBKT7 vektor en is gebruik om die long kDNS biblioteek in Saccharomyces cerevisiae te ondersoek. In die soeke na interaksies met ESAT-6 and CFP-10, was 79 en 19 positiewe kolonies onderskeidelik geïdentifiseer. Van die aantal klone, was slegs twee volgordes in-leesraam geïdentifiseer met ESAT-6. Hierdie proteïene het ooreengestem met filamin A en “complement component 1, q subcomponent, A chain” (C1QA). Bykomend hiertoe, is C1QA ook geïdentifiseer as „n bindende vennoot met CFP-10. Daaropvolgende in vitro and in vivo eksperimente kon nie die vermeende interaksie van C1QA met ESAT-6 en CFP-10 bevestig nie. Maar die interaksie tussen filamin A en ESAT-6 kon wel gedemonstreer word deur die gebruik van mede-lokalisering en medeimunopresipitasie. Die afbreek van filamin A in die teenwoordigheid van ESAT-6 is ook aangetoon en blyk „n weerspieëling te wees van sitoskelet hermodellering en die induksie van seldood. Die werk wat hier aangebied word, dui daarop dat soos ESAT-6 toegang kry tot die sitosol, inisieër dit seldood deur die destabilisaisie van die sitoskelet selstruktuur. Dit word moontlik aangedryf deur die interaksie van ESAT-6 met filamin A. Laastens het ons `n ondersoek van die geïdentifiseerde bindingsvennote (filamin A and C1QA) as moontlike genetiese merkers vir genetiese vatbaarheidsstudies vir tuberkulose uitgevoer. `n Pasiënt-kontrole studie is gedoen waarby 604 individue ingesluit is, waarvan 109 gediagnoseer is met Tuberculosis Meningitis (TBM), en die ander 486 kontrole individue was van die Suid Afrikaanse Kleurling (SAC) bevolking binne die Ravenmead-Uitsig opvanggebied. Die resultate het „n nuwe assosiasie van „n regulerende variant (rs587585) wat stroomop van die C1QA geen gelokaliseer is, getoon. Hierdie variant het `n verhoogde neiging in tuberkulose pasiënte met die geassosieërde genotipe getoon. Hierdie studie het `n beduidende bydrae gemaak tot ons begrip van menslike-mikobakteriese gasheer-patogeen proteïen-proteïen interaksies. Hierdie resultate het die weg oopgemaak om die gevolge en funksie van die geïdentifiseerde ESAT-6-filamin A interaksie verder te ondersoek. Dit het ook aanleiding gegee tot die identifikasie van `n genetiese assosiasie met tuberkulose. Om saam te vat, hierdie werk bewys die bruikbaarheid van die gis twee-hibriede sisteem, om potensiële proteïen-proteïen interaksies te ontdek wat die moontlikheid het om aanleiding te gee tot addisionele navorsingsvrae.The National Research Foundation,Harry Crossley FoundationMedical Research Council of South AfricaStellenbosch University Postgraduate bursaryProf. Paul van Helde

Molecular genetic analysis of preterm labour

Thesis (MSc)--University of Stellenbosch, 2007.ENGLISH ABSTRACT: The World Health Organisation (WHO) has defined preterm labour as the onset of labour before 37 completed weeks of gestation with an incidence ranging between 5-10%. Although patient care has improved, the rate of preterm birth has slowly been increasing and currently impacts significantly on maternal and fetal mortality and morbidity. The complex condition of preterm labour involves multiple etiologies and risk factors, which complicates the search for candidate markers and / or biomarkers. The aim of this prospective study was to investigate potential genetic associations with preterm labour. The study cohort consisted of consecutive first-time booking, low-risk primigravid pregnant women from a restricted geographical region. The study cohort comprised 421 [306 Coloured and 115 Black] pregnant women presenting at the Paarl Hospital Obstetric clinic. Subsequently, DNA was extracted from whole blood and investigated for a range of known polymorphisms in pro-inflammatory and anti-inflammatory cytokines, as well as the novel LGALS13 gene, for potential variants that may impact on pregnancy outcome. Screening techniques involve combinations of allele-specific PCR amplification, Multiphor SSCP/HD analysis, restriction enzyme analyses and DNA sequencing. A significant association was demonstrated between the IL-1RN*2-allele and adverse pregnancy outcome, mainly in the preterm labour and hypertension group. The presence TNFα-308 A-allele was associated with overall adverse pregnancy outcome and preterm labour. In addition to this, a novel IL-1RN allele was identified in the control group. Mutation screening and subsequent statistical methods revealed an association between a novel LGALS13 exonic variant, 221delT, and preterm labour in Coloured women. Two previouslydocumented intronic variants (IVS2-22A/G and IVS3+72T/A) demonstrated linkage disequilibrium, signifying evolutionary conservation of exon three. Additionally, two novel intronic variants, IVS2-36 G/A and IVS2-15 G/A, demonstrated no association with adverse pregnancy outcome. In this study we identified rare novel exonic variants; two non-synonymous variants in exon three (M44V, [N=2] and K87R, [N=1]) and a silent variant in exon four (P117P, [N=1]) - all identified in individuals from the control cohort. Within coding exon three, an interesting variant [“hotspot”] was identified, which represents six polymorphic bases within an 11bp stretch. No associations were demonstrated with these variants and pregnancy outcome. Furthermore, a previously documented 5' “‘promoter” variant, -98 A/C, was identified and demonstrated no association with adverse pregnancy outcome. However, subdivision of lateonset pre-eclamptic cases revealed a significant association with the A-allele and late-onset preeclampsia. Genotype-phenotype investigation demonstrated association between the IL-10 -1082 A/G, IL-4 C/T and 221delT loci and poor pregnancy progress which manifested as (i) delivery of infants weighing <2000g, (ii) before 37 weeks of gestation. The findings of this study will strengthen our understanding of the pathophysiology underlying pregnancy complications and facilitate the further development of effective treatment strategies to reduce maternal and fetal morbidity and mortality.AFRIKAANSE OPSOMMING: Die Wêreld Gesondheid Organisasie (WHO) klassifiseer voortydse kraam as kontraksie voor 37 volledige weke, met ‘n insidensie tussen 5-10%. Alhoewel pasiënte-sorg verbeter het, neem die tempo van voortydse geboorte steeds toe, wat ‘n groot impak het op moederstrefte en fetale mortaliteit en morbiditeit. Die komplekse kondisie van voortydse kraam sluit veelvoudige oorsake en risiko faktore in, wat die navorsing van kandidaat en / of biologiese merkers kompliseer. Die doel van hierdie prospektiewe studie, was die potensiële navorsing van genetiese assosiasies met voortydse kraam. Die studie kohort bevat opeenvolgende eerste bespreking van lae risiko primigravida swanger vrouens vanaf ‘n beperkte geografiese omgewing. Die studie kohort beslaan 421 [306 Kleurling en 115 Swart] swanger vrouens teenwoordig by die Paarl Hospitaal Verloskunde kliniek. Vervolgens was DNS geëkstraeer van bloedmonsters en geondersoek vir ‘n verskeidenheid van bekende polimorfismes in pro-inflammatoriese en antiinflammatoriese sitokiene, insluitend die nuwe sifting van die LGALS13 geen potensiaal vir variante wat ‘n impak op swangerskap uitkomste sal hê. Die siftings tegnieke toegepas, sluit in ‘n kombinasie van alleel-spesifieke amplifikasie, Multiphor enkelstring konformasie polimorfisme / heterodupleks analise, restriksie ensiem verterings en volgorde bepalings tegnieke. ‘n Betekenisvolle assosiasie was gedemonstreer tussen die IL-1RN*2-alleel en nadelige swangerskap, beperk tot voortydse kraam en die hipertensie groep. Die teenwoordigheid van die TNFα-308 A-alleel was geassosieer met algehele nadelige uitkomste en voortydse kraam. Daarby, was ‘n nuwe IL-1RN alleel geïdentifiseer in die kontrole groep. Mutasie sifting en opeenvolgende statistiese metodes, het ‘n assosiasie getoon tussen ‘n nuwe LGALS13 koderende variant, 221delT, en voortydse kraam in Kleurling vrouens. Twee voorafbeskryfde introniese variante (IVS2-22 A/G en IVS3+72 T/A), het ‘n betekenisvolle bewys opgelewer dat daar koppelings-onewewig bestaan tussen hierdie variante, en toon evolusionêre konservasie van ekson drie. Addisioneel was twee nuwe introniese variante ontdek, IVS2-36 G/A en IVS2-15 G/A, wat geen assosiasie getoon nie. In hierdie studie het ons ‘n nuwe seldsame koderende variante geïdentifiseer in die kontrole groep, waarvan twee nie-sinonieme variante was in ekson drie (M44V, N=2 en K87R, N=1) en ‘n stil variasie in ekson vier (P117P, N=1). Geleë in die koderende area van ekson drie, was ’n interessante variant [“hotspot’] ontdek, waarvan ses basisse in ‘n 11 basis paar area polimorfies is. Geen assosiasie was getoon met hierdie variante en swangerskap uitkomste nie. Verder was ‘n voorafbeskryfde 5' ‘promotor’ variant, -98 A/C, geïdentifiseer wat geen assosiasie getoon met nadelige swangerskap uitkomste nie. Onderverdeling van laat-aanvangs preeklampsie, het getoon dat die A-alleel ‘n betekenisvolle assosiasie getoon het met die ontwikkeling van laat pre-eklampsie. Genotipe-fenotipe interaksies het ’n assosiasie getoon tussen die IL-10 -1082 A/G, IL-4 C/T en 221delT lokusse en nadelige swangerskap uitkomste, wat manifesteer as (i) kraam van suigelinge wat <2000g weeg, (ii) geboorte voor 37 weke. Die bevindings van hierdie studie sal ons basiese kennis verbeter oor die patologie beskrywend aan swangerskap komplikasies, asook die fasilitering en ontwikkeling van effektiewe behandelings strategieë, om moederstrefte en fetale mortaliteit en morbiditeit te verminder

Tuberculosis State Is Associated with Expression of Toll-Like Receptor 2 in Sputum Macrophages

ABSTRACT During tuberculosis, macrophages are critical for both pathogen survival and host immune activation. Since expression of particular cell surface markers reflects cell function, we used flow cytometry to measure the abundance of surface markers associated with polarity, lipid uptake, or pattern recognition on macrophages found in induced sputum. Nine macrophage surface markers were examined from three groups of donors: infection-free, latent tuberculosis infection, and active pulmonary tuberculosis. Using a trend test, we found that expression of Toll-like receptor 2 was greater from absence of infection to latent infection and from latent infection to active tuberculosis. The results point to the possibility that innate immune cell phenotypes be used to distinguish among tuberculosis infection stages. Moreover, this study shows that readily accessible sputum macrophages have potential for tuberculosis diagnosis and prognosis. IMPORTANCE Mycobacterium tuberculosis is an intracellular pathogen that parasitizes the host macrophage. While approximately two billion people are infected worldwide, only 5 to 10% become diseased with pulmonary tuberculosis, at least in the absence of comorbidities. Tuberculosis control requires development of noninvasive methods probing the host immune status to help distinguish latent infection from active tuberculosis. With such methods, high-risk individuals could be targeted for treatment before disease manifestation. Previous investigations have been based on examination of peripheral blood cells or, more rarely, lung macrophages obtained with invasive procedures, such as bronchoalveolar lavages. Here we show that differences exist in the expression of a surface protein (Toll-like receptor 2) between macrophages recovered from the sputum of individuals in different diagnostic groups: i.e., infection free, latent tuberculosis infection, and active pulmonary tuberculosis. Thus, phenotypic analysis of local macrophages obtained with noninvasive procedures can help distinguish among tuberculosis infection stages

Human Immunodeficiency Viruses Pseudotyped with SARS-CoV-2 Spike Proteins Infect a Broad Spectrum of Human Cell Lines through Multiple Entry Mechanisms

Severe acute respiratory syndrome-related coronavirus (SARS-CoV-2), the causative agent of coronavirus disease 19 (COVID-19), enters cells through attachment to the human angiotensin converting enzyme 2 (hACE2) via the receptor-binding domain (RBD) in the surface/spike (S) protein. Several pseudotyped viruses expressing SARS-CoV-2 S proteins are available, but many of these can only infect hACE2-overexpressing cell lines. Here, we report the use of a simple, two-plasmid, pseudotyped virus system comprising a SARS-CoV-2 spike-expressing plasmid and an HIV vector with or without vpr to investigate the SARS-CoV-2 entry event in various cell lines. When an HIV vector without vpr was used, pseudotyped SARS-CoV-2 viruses produced in the presence of fetal bovine serum (FBS) were able to infect only engineered hACE2-overexpressing cell lines, whereas viruses produced under serum-free conditions were able to infect a broader range of cells, including cells without hACE2 overexpression. When an HIV vector containing vpr was used, pseudotyped viruses were able to infect a broad spectrum of cell types regardless of whether viruses were produced in the presence or absence of FBS. Infection sensitivities of various cell types did not correlate with mRNA abundance of hACE2, TMPRSS2, or TMPRSS4. Pseudotyped SARS-CoV-2 viruses and replication-competent SARS-CoV-2 virus were equally sensitive to neutralization by an anti-spike RBD antibody in cells with high abundance of hACE2. However, the anti-spike RBD antibody did not block pseudotyped viral entry into cell lines with low abundance of hACE2. We further found that CD147 was involved in viral entry in A549 cells with low abundance of hACE2. Thus, our assay is useful for drug and antibody screening as well as for investigating cellular receptors, including hACE2, CD147, and tyrosine-protein kinase receptor UFO (AXL), for the SARS-CoV-2 entry event in various cell lines

Storage lipid studies in tuberculosis reveal that foam cell biogenesis is disease-specific.

Foam cells are lipid-laden macrophages that contribute to the inflammation and tissue damage associated with many chronic inflammatory disorders. Although foam cell biogenesis has been extensively studied in atherosclerosis, how these cells form during a chronic infectious disease such as tuberculosis is unknown. Here we report that, unlike the cholesterol-laden cells of atherosclerosis, foam cells in tuberculous lung lesions accumulate triglycerides. Consequently, the biogenesis of foam cells varies with the underlying disease. In vitro mechanistic studies showed that triglyceride accumulation in human macrophages infected with Mycobacterium tuberculosis is mediated by TNF receptor signaling through downstream activation of the caspase cascade and the mammalian target of rapamycin complex 1 (mTORC1). These features are distinct from the known biogenesis of atherogenic foam cells and establish a new paradigm for non-atherogenic foam cell formation. Moreover, they reveal novel targets for disease-specific pharmacological interventions against maladaptive macrophage responses