Browsing by Department "Division of Chemical and Systems Biology"

Now showing 1 - 7 of 7
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    Open Access
    Comprehensive definition of Ser/Thr/Tyr phosphorylation in mycobacteria: towards understanding reprogramming of normal macrophage function by pathogenic mycobacteria
    (2018) Nakedi, Kehilwe Confidence; Blackburn, Jonathan M; Da Cruz Soares, Nelson
    Mycobacterium tuberculosis, the causative agent for the disease Tuberculosis, is a serious public health problem that is responsible for 1.6 million deaths each year. The WHO’s recent report on Tuberculosis estimates that a third of the world’s population is latently infected with the bacteria, and, of those, 10% will progress to active disease. M. tuberculosis is a successful pathogen mainly due to its ability to adapt and survive in changing environments. It can survive a dormant state with limited metabolic activity during latent infection, while also being able to escape the macrophage and disseminate into active disease. Efforts to eradicate the disease must be based on understanding the biology of this organism, and the mechanisms it uses to infect, colonize, and evade the immune system. Understanding the behaviour of pathogenic mycobacteria in the macrophage is also important to the discovery of new drug targets. In this thesis, we employed state of the art mass spectrometry techniques, which allowed us to unpack the biology of this bacterium in different growth environments and expand our understanding of the mechanisms it employs to adapt and survive. We investigated protein regulation by the process of phosphorylation, through sensory kinases, which add a phosphate group to a protein of interest, thereby regulating its function. First, we interrogated the phosphoproteomic landscape between M. bovis BCG and M. smegmatis to explain how differential protein regulation results in the differences between slow and fast growth of mycobacteria. Second, we focused on Protein Kinase G (PknG), which plays an important role in bacterial survival by blocking phagosome/lysosome fusion. We identified the in vivo physiological substrates of this kinase in actively growing M.bovis BCG culture. Our results revealed that this kinase is a regulator of protein synthesis. We then examined the mechanisms of survival in murine RAW 246.7 macrophages mediated by PknG, using M. bovis BCG reference strain and PknG knock-out mutant. Our results indicated strong evidence that pathogenic mycobacteria disrupt the macrophagic cytoskeleton, through phosphorylation of proteins that are involved in cytoskeleton rearrangement. These results explain the strategies that pathogenic mycobacteria employ mediated by PknG to block phagosome-lysosome fusion and evade the host immune system and survive for prolonged periods in the macrophages. The findings of this thesis contribute to our understanding of the physiology of pathogenic mycobacteria and their interaction with the host.
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    Development of SNAP-tag-based fusion proteins targeting HIV-1 viral reservoirs
    (2020) Cingo, Siphelele Sanele; Barth, Stefan
    Background Globally, the HIV/AIDS epidemic has cost over 35 million lives and approximately a further 37 million people are currently infected with HIV. In South Africa alone, more than 7 million people are HIV positive. Since the initiation of combination antiretroviral therapy (cART), viral replication can be supressed below the limit of detection by conventional testing. There is, however, no approved therapy for the cure of HIV. This is because HIV establishes viral reservoirs in memory CD4+ T-cells, where replication is low or arrested, allowing prolonged survival. Since there is little or no replication, a therapeutic strategy which targets the viral production and replication becomes ineffective and upon cessation of antiretroviral therapy a dramatic viral relapse occurs. The eradication of HIV, therefore, requires the targeted killing of the reservoir cells, or latency reversal followed by the prevention of further infection using cART. Targeting of cell-surface antigens for therapeutic purposes is the basis of immunotherapy. FDA-approved monoclonal antibodies such as Trastuzumab have been used to treat breast cancer via the human epidermal growth factor 2 (HER2) receptor. Immunotoxins (ITs) composed of an antibody fragment fused to apoptosis-inducing protein toxins targeting cellsurface antigens have been used for therapy of refractory leukaemia. The anti-CD22 recombinant IT Moxetumomab pasudotox based on Pseudomonas aeruginosa exotoxin A (ETA) has been FDA approved to treat hairy cell leukaemia. Moxetumomab pasudotox targets the antigen CD22 found on the surface of tumour cells. The HIV neutralizing VHH-nanobody J3, isolated from an immunised Llama has demonstrated anti-HIV properties against more than 95 % of HIV strains in vitro. As part of an ongoing project to develop a J3-ETA IT, this work sought to produce a J3-SNAP fusion protein by osmotic stress expression in the presence of compatible solutes in the periplasmic space of E. coli. SNAP-tag is a self-labelling protein that covalently binds benzylguanine (BG)-modified substrates in a 1:1 stoichiometric ratio. When recombinantly fused to any protein of interest, SNAP-tag allows the stable labelling of the protein of interest of in vitro and in vivo imaging. The periplasmic space of bacteria has been reported as a dedicated compartment to express functional proteins of interest. Furthermore, osmotic stress expression in the presence of compatible solutes has been reported to result in up to a thousand-fold increase in protein yield for difficult to express proteins. This study ultimately aimed to understand whether a functional J3-SNAP or J3-ETA can be expressed under osmotic stress in the presence of compatible solutes, in the periplasmic space of E. coli. 11 Experimental work In this study, a SNAP-tag-based fusion protein and an ETA-based IT were designed using J3, an anti-HIV-1 Env VHH-nanobody isolated from an immunised llama. Using the SnapGene® software (v.5.0.8, GSL Biotech LLC, USA), in silico design and cloning of an ETA-based IT J3-ETA and SNAP-tag-based fusion protein J3-SNAP was performed. Molecular cloning of designed open reading frames (ORFs) was performed into appropriate bacterial expression plasmid vectors. Plasmid vectors confirmed to contain the required ORFs by Sanger sequencing were transformed into E. coli BL21-DE3. Histidine-tagged J3-SNAP was expressed by osmotic stress in the presence of compatible solutes. J3-SNAP was purified by IMAC and assessed by SDS-PAGE and Western blot analysis. To ascertain the binding of J3- SNAP to cells expressing HIV-1 Env in vitro, recombinant Env protein was transiently transfected into HEK293T-cells to generate an Env expressing cell line. Cell-surface binding of SNAP-Surface® Alexa Fluor® 488 -conjugated J3-SNAP on Env expressing HEK293Tcells was assessed by confocal microscopy analysis. Results Successful expression of J3-SNAP in E. coli BL21-DE3 was confirmed by SDS-PAGE and Western blot analysis. The J3-SNAP fusion protein was subsequently purified by IMAC. Purified J3-SNAP was conjugated to the benzyl guanine-modified fluorophore SNAPSurface® Alexa Fluor® 488 and full-length conjugated protein was confirmed by combinations of SDS-PAGE and Western blot analysis. Cell-surface binding of J3-SNAP to HIV-1 Env-expressing HEK293T-cells was demonstrated in vitro by confocal microscopy analysis. These results prompted the generation of the IT, J3-ETA, by replacing SNAP-tag with ETA. Conclusion Successful binding studies suggest using J3 to target HIV-1 Env. Accessing patient probes would allow for the confirmation of these results for future human applications. Future in vitro studies would need to confirm the selective elimination of Env expressing T-cells by J3-ETA and thereafter confirmed on Env-positive patient probes.
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    Differential Lipidomic and Proteomic Responses Induced by Sub-lethal Drug Challenge in Susceptible and Drug Resistant Mycobacterium smegmatis
    (2021) Giddey, Alexander D; Blackburn, Jonathan
    Tuberculosis remains a major global health challenge and the increasing strength and prevalence of drug resistance threaten to undo much of the good progress made. As one of the primary, frontline anti-tuberculous drugs, growing resistance to rifampicin in particular is concerning. Sub-lethal drug exposure and the development of adaptive phenotypic drug resistance, represent an important avenue by which genetic resistance and treatment failure or relapse may occur. Proteins and general metabolites are molecular classes that are highly dynamic, responsive and essential to understanding the state of an organism, while mass spectrometry-based proteomics and metabolomics are powerful tools by which these can be examined. For mycobacteria in particular, the lipidome and cell wall are compartments of major importance with respect to virulence, adaptation, host-pathogen interactions and persistence. As such, we sought to determine the effect of sub-lethal rifampicin exposure upon the model organism Mycobacterium smegmatis over time and determine what phenotypic adaptations might be observed and explained by alterations in the proteome and lipidome, with special focus on the cell wall sub-proteome. From these data we formed several new hypotheses with respect to virulence and mechanisms of both drug resistance and sensing, which were investigated further. Finally, we examined the effect of sub-lethal rifampicin exposure, and consequent proteomic alterations, upon the M. smegmatis lipidome and propose a model by which mycobacteria respond to sub-lethal challenge with rifampicin: Upon initial insult, drug-susceptible mycobacterial growth slows and stress response networks, including the SOS response, are temporarily activated. For both susceptible and resistant bacteria, cell wall remodelling begins early through dysregulation of cell wall and lipid synthesis enzymes — such as MtrAB, Mur proteins and PimB — resulting in ultimate accumulation of lipids with composition such as to impede passive diffusion of rifampicin into the cell. Some of this lipid accumulation, namely with PIMs, may take place rapidly and so ultimately reveal extremely large increases in abundance, which possibly necessitates downregulation of enzymes such as PimB by ~4 hours post treatment. In concert with ongoing lipid dysregulation, the cell wall proteome is altered as ABC transporter proteins are generally downregulated as an additional mechanism by which to control cell wall permeability through altered cell wall composition — through removal of cell wall penetrating transport proteins — and by limiting controlled influx of exogenous compounds. Bacterial efforts to resume normal growth and adapt to rifamipicin stress involves the dysregulation of numerous virulence factors, such as PknG, which results in impaired virulence. Transcriptional and translational machinery are also gradually upregulated so as to compensate for intracellular rifampicin's inhibition of RpoB, with transcriptional activity regulated separately to that of translational machinery. Ultimately, the combination of increased transcription, translation, and cell wall impermeability allows mycobacteria to overcome rifampicin challenge and resume normal growth. In M. smegmatis specifically, all this is accompanied by the gradual upregulation of the chromosomal resistance factor Arr which, at a later timepoint, modifies extracellular rifampicin to alleviate drug pressure.
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    Isolation and characterisation of novel DNA aptamers against Mycobacterium tuberculosis biomarkers: new tools for tuberculosis diagnostics
    (2018) Amos-Brown, Bianca; Blackburn, Jonathan
    Tuberculosis is a curable disease with an average treatment success rate of 86 %. Despite this, there were an estimated 1.5 million deaths due to tuberculosis in 2013, most of which occurred in low and middle income countries. In order to overcome tuberculosis in developing countries innovation in diagnostics is key to administering treatment. While detection of whole mycobacteria has been favoured in the past to diagnose tuberculosis, culturing mycobacteria is costly and microscopy is often not sensitive enough due to low bacterial loads. Detection of Mycobacterium tuberculosis biomarkers in urine, a safe and easy specimen to test, could offer a cost effective and simple solution to identify patients with tuberculosis. Enzyme linked immunosorbent assays (ELISAs) were performed on concentrated tuberculosis patient urine to detect two M. tuberculosis biomarkers: lipoarabinomannan (LAM) and early secreted antigen-6 kDa (ESAT-6). Concentrating urine improved the detection of LAM in human immunodeficiency virus (HIV) negative patients and patients with a CD4 count > 200 cells/µl. ESAT-6 was not detected by ELISA due to a high background signal caused by the available antibodies cross reacting with a human protein present in urine which was identified by western blot and mass spectrometry. Targeted mass spectrometry did not detect ESAT-6 or its dimer partner, culture filtrate protein-10 kDa (CFP-10) in tuberculosis positive patient urine. Since concentrating urine samples is impractical in a clinical setting a more sensitive diagnostic is needed to detect LAM in urine and ESAT-6 or CFP-10 in other samples. Aptamers can be packed more densely on biosensor surfaces increasing the dynamic range of detection while matching the affinity that an antibody has for a biomarker. Chemically modified DNA aptamers were isolated for LAM and the ESAT-6.CFP-10 dimer. The aptamers were characterised by enzyme linked oligonucleotide assays (ELONAs) and biolayer interferometry. One aptamer bound with high affinity to ESAT-6 while one aptamer bound with low affinity to LAM. The use of aptamers as capture agents for detecting biomarkers in biological specimens thus appears to be a viable option for diagnosing tuberculosis, although availability and concentration of individual biomarkers seems likely to remain key to the choice of specimen in which to make diagnostic measurements.
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    The chromatin landscape of colorectal cancer cells
    (2020) Magagula, Loretta Qinisile; Mhlanga, Musa; Skok, Jane
    Chromatin organization is at the heart of deciphering gene regulation as it is instructive to transcription. Current technological advances in next-generation sequencing approaches have offered unprecedented opportunities to interrogate the genomic landscape in multiple pathological and clinical presentations. Historically, mutations and alterations at the genomic loci of protein-coding genes were thought to be exclusively causal to many human diseases. However, the non-coding genome has emerged as the master regulator of chromatin dynamics and transcriptional activity. With cancer increasingly becoming the greatest health epidemic of our time, the comprehensive genomic characterization of tumor genotypes has become central to current therapeutic approaches. Functioning as the basic unit of chromatin organisation, chromatin loops and topologically associating domains (TADs) compartmentalize genomic loci and their corresponding molecular transcriptional elements in three-dimensional space. Transcription of the human genome is proximity-dependent requiring the cooperative engagement of non-coding elements and epigenetic modifiers to create permissive topological chromatin contacts and structures. The repertoire of chromatin contacts at any given time is regulated by the threedimensional structure and organization of the chromatin. TAD structures are formed and maintained by chromatin insulating proteins such as CTCF (CCCTC-binding factor) and multiprotein complex, cohesin. The dysfunction of which, through mutational and epigenetic aberrations, directly impacts a plethora of chromatin contacts and the resultant transcriptional profiles within each cell. Loops and TADs are formed by the binding of CTCF on the conserved 19 bp CTCF binding motif as the chromatin is protruded through the "ring-like" multi-protein complex, cohesin. When two convergently oriented and CTCF enriched CTCF-binding sites (CBSs) come into contact within the ring, cohesin is thought to "hand-cuff" the chromatin resulting in the formation a chromatin loop. These loop structures then serve to compartmentalize and restrict the chromatin contacts and their frequency within each loop. Promoter-resident CBSs can also function as "docking sites” for tissue- and context-specific enhancers. The dysregulation of CTCF binding has been repeatedly demonstrated to directly alter chromatin contacts in a vast array of cellular contexts including cancer. Fundamentally, CTCF functions as a potent regulator of chromatin contacts, which directly instruct transcriptional status. Thus, CTCF binding has become an attractive regulatory target for manipulating the topological and transcriptional activity of chromatin. In this study, we sought to identify CBS swith differential, specifically abrogated CTCF enrichment that may be hijacked by oncogenes in an attempt to modify transcriptional programmes to favour cancer progression. To this end, we developed an integrated bioinformatic pipeline to identify promoter-associated lower-CTCF enrichment sites (PA-LCes) in colorectal cancer (CRC) cell lines as compared to primary colonic tissue from CTCF ChIP-Seq data. With ever-growing catalogues of nextgeneration sequencing datasets, including ChIP-Seq, in the public domain, the use of ENCODE datasets proved to be an economical option and added layer of standardization in our analysis. Briefly the pipeline developed in this study takes ENCODE ChIP-Seq FASTQ files from the NCBI SRA using fastqdump as input files. The FASTQ files undergo a quality control and dataset filtration with FASTQC. The filtered datasets are then aligned to the hg38 human genome and fed back into FASTQC to ensure aligned reads pass quality control metrics. The mapped reads are then processed using samtools and duplicate reads are marked with the picard markduplicates argument. Narrow peaks are then called from processed reads using MACS2 and processed using bedtools. Called peaks then undergo a final quality control step using ChIPQCr and are visualized using IGV before undergoing differential enrichment analysis. Differential CTCF enrichment analysis between the peaks in primary sigmoidal colon cells and CRC cell lines is then conducted using DeSeq2 within DiffBind. Lower CTCF enrichment peaks are then used for the discovery of the canonical CTCF MA00139.1 motif using homer and compared to similar annotations in the primary consensus peakset. The resultant lower CTCF enrichment peaks are then annotated using homer and ChiPpeakAnno to determine their genomic locations and extract LCes located proximal (<1kb) to annotated TSS or promoter regions i.e. PA-LCes. The PA-LCe discovery pipeline developed in this study is highly robust, resulting in some previously validated CBSs implicated in oncogenesis. Intriguingly, the PA-LCe sites identified in this study emanate from bidirectional promoters at oncogenes with differential methylation and transcriptional patterns in cancer. Additionally these PA-LCes transcribe antisense lncRNAs such as the tumor-suppressive aslncRNA ZNF582-AS1. This data adds to the recent body of evidence that suggests that disruption of promoter-associated CBSs leads to fluctuations in promoter activity. Recent studies have implicated the requirement of CTCFlncRNA complexes at promoter regions in facilitating and regulating CTCF docking on chromatin which subsequently influences transcriptional activity. In accordance with this, our data suggests that the lncRNAs at PA-LCe loci may be molecular targets for the regulation ofCTCF binding and transcriptional activity in CRC. Perturbation of CTCF enrichment at PALCes in CRC result in differential chromatin contacts, epigenetic context and, the transcriptional activity of the promoters in which they reside. As CTCF binding at CBSs sites is highly modular, the use of targeted CRISPR-mediated gene-editing and DNA methylation at PA-LCe CBSs may represent viable and druggable oncogenic targets.
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    The Gut Mucosal Microbiome of HIV- exposed Uninfected Infants in Africa
    (2020) Khomunala, Phumudzo; Blackburn, Jonathan; Fortuin, S
    Background: South Africa has a large HIV disease burden, with the highest rate of infection occurring in young women of the childbearing age. This gave impetus to the Prevention-of-Mother-To-Child-Transmission (PMTCT) program, that has been successfully implemented. Due to the success of the PMTCT program, HIVexposed-uninfected (HEU) infants represent a growing population in South Africa. However, these infants have been found to have increased morbidity and mortality rates compared to their HIV Unexposed Uninfected (HUU) peers, as well as altered immune and vaccine responses. The reasons for this remain unclear, but one hypothesis is that altered gut microbiomes in HEU adversely affect the developing infant immune system. The microbiome (a collection of an array of microorganisms, their genes, genomes, proteomes, and metabolites) is an area of emerging research interest; dysbiosis of the gut microbiome has recently been associated with disease outcomes and progression in several disease areas. The microbial colonisation of the infant begins in utero and continues after birth. It is affected by several factors: birth mode, age of gestation, feeding mode, maternal health status as well as environmental factors. Aim: To elucidate the microbiomes of HEU infants in Africa, compared to HUU controls Design: Ultra-high-performance liquid chromatography mass spectrometry was used to analyse and characterize a subset of existing stool samples stored from the InFANT cohort study. The infant gut metaproteome of 34 HEU versus 29 HUU infants, from the South African arm of the study was analysed. Cross-sectional samples were collected and analysed at two-time points, namely at birth and within the first week of life (between 4 to 7 days after birth). Results: Comparative analysis of the HEU and HUU reveal differences in the microbial composition between the two groups at birth and day 4-7, with the most apparent difference occurring at birth. In our comparison we found that the relative abundances of Bacteroidetes and Firmicutes were different between the HEU and HUU at both birth and day 4-7. There was a dramatic shift in the microbial composition within the first week of life. Conclusion: It is evident from our analysis that the HEU infant has a different gut microbiome to that of the HUU infant at birth. The HEU microbiome is characterised by a high microbial diversity at birth. This could be associated with more severe outcomes from childhood ailments. The human breast milk (HBM) microbiome greatly influences and mitigates the differences upon subsequent breastfeeding, but differences in the measured microbiomes of HEU and HUU nonetheless remain. Recommendations: A longitudinal study should be carried out to better monitor the long-term effects of the microbiome on infant immune priming. A study of the HBM microbiome should also be investigated to better understand the role of HBM in mediating and priming the infant's immune system. Further, a study of the metabolome of the infant gut and the matching HBM of the mother may identify potential metabolites that could be used as biomarkers for vaccine responses.
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    Uncovering the hidden mechanisms governing the transcriptional regulation of inflammation
    (2020) Fok, Ezio T; Mhlanga, Musa; Fanucchi, Stephanie
    Inflammation provides broad immunological protection that is essential for our survival. This cellular response is characterised by a biphasic cycle consisting of an initial acute pro-inflammatory phase and a subsequent resolving anti-inflammatory phase. Underlying each of these phases are changes in the expression of hundreds of immune genes, which encode for inflammatory mediators called cytokines. Importantly, the biphasic nature of inflammation requires cytokine expression to be highly regulated and coordinated to different timescales during each phase of inflammation. For the initial proinflammatory response, cytokine expression needs to be rapid and robust to efficiently initiate host defence mechanisms and provide effective immunological protection. In contrast, the expression of anti-inflammatory cytokines is temporally delayed to ensure that anti-inflammation always follows pro-inflammation. In order to choreograph the expression of these cytokines during inflammation, numerous mechanisms within the cell serve to regulate and coordinate cytokine transcription. Within the eukaryotic nucleus, multiple modes of transcriptional regulation function cooperatively to provide the regulatory capacity that is required for complex transcription patterns to emerge. These include the organisation of the genome, which confine cognate chromosomal contacts that are causal to transcription, and long-non coding RNAs (lncRNAs) that function to discretely fine tune transcriptional activity. Although many of the mechanisms that regulate transcription have been well described, their role in cytokine expression during inflammation remains largely unknown. In particular, the mechanisms that facilitate rapid and robust cytokine expression during proinflammation and the regulatory networks that coordinate the biphasic regulation of inflammation are unresolved. In this work, two novel lncRNAs were discovered to transcriptionally regulate these key features of cytokine expression during inflammation. The first, UMLILO (Upstream Master LncRNA of the Inflammatory chemokine LOcus), was found to emanate from the ELR+ CXCL chemokine TAD and regulate the transcriptional activation of the pro-inflammatory ELR+ CXCL chemokines (IL-8, CXCL1, CXCL2 and CXCL3). By exploiting the pre-formed local 3D topology, UMLILO is able to epigenetically prime the chemokines for transcriptional activation. This involves the discrete deposition of H3K4me3 onto the promoters of the chemokines, which allows for the pre-loading of transcriptional machinery prior to their signal-dependent activation. This reveals a fundamental mechanism for the epigenetic priming and rapid activation of pro-inflammatory cytokine genes. The second lncRNA, called AMANZI (A MAster Non-coding RNA antagoniZing Inflammation), was found to coordinate the transcription of two functionally opposed cytokines: the master pro-inflammatory IL-1β and the broad antiinflammatory IL-37. AMANZI is encoded in the promoter of IL-1β, which results in its concomitant expression when IL-1β is transcriptionally active. Functionally, AMANZI mediates the formation of a dynamic chromosomal contact between IL-1β and IL-37. This leads to the delayed transcriptional activation of IL-37 ensuring that the pro-inflammatory function of IL-1β precedes IL-37 mediated anti-inflammation. This revealed a novel biphasic circuit that coordinated the expression of IL-1β and IL-37, through the activity of AMANZI, to regulate the two functionally opposed states of inflammation. Clinical observations in healthy individuals revealed that a polymorphism occurring in AMANZI (rs16944) was able to augment the state of this genetic circuit and shift the relative levels of IL-1β and IL-37 to influence an individual's inflammatory capacity. This affected the establishment of innate immunological memory, which is involved in the progression of many inflammatory conditions and the efficacy of certain vaccines. The work described here uncovers novel mechanisms that transcriptionally regulate key features of the inflammatory response. Importantly, this work implicates the role of two novel lncRNAs in inflammation, essentially contributing to the functional annotation to the genome and providing novel targets for the modulation of pathogenic inflammation.