Masters Degrees (Molecular Biology and Human Genetics)

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Accessory gene components for an HIV-1 subtype C vaccine : functional analysis of mutated Tat, Rev and Nef antigens
(Stellenbosch : Stellenbosch University, 2002-12) Scriba, Thomas Jens; Van Rensburg, E. Janse; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Medicine.
ENGLISH ABSTRACT: HIV has attained a global distribution and the number of infected people reached an estimated 28.1 million in sub-Saharan Africa at the end of 2001. HIV-1 subtype C is overwhelmingly prevalent in Botswana and South Africa and to date no interventions have been successful enough to curb the rapid spread of the virus. A number of HIV-1 vaccine strategies are being developed, however the breadth and efficacy of such candidate vaccines, many of which are based on the HIV-1 structural genes pol, gag and env, have mostly been found to be inadequate. The HIV-1 accessory genes are attractive components of HIV vaccines due to their role in viral pathogenesis, early expression and the high ratio of conserved CTl epitopes. Yet, because of undesirable properties questions regarding their safety as vaccine components are raised. In this study candidate tat, rev and nefmutants were assessed for efficient expression and inactivation of undesirable functionality. / Plasmid constructs that encode the South African HIV-1 subtype C consensus Tat, Rev and Nef proteins were constructed. The coding sequences of the genes were codon-optimised for optimum protein expression and these synthetic genes were constructed using overlapping 50-mer oligonucleotides. Furthermore, the proteins were mutated at previously described sites by PCR-based site-directed mutagenesis to render them inactive for their respective functions. Corresponding wild-type Tat, Rev and Nef constructs were also made from viral isolates that were least dissimilar to the respective consensus amino acid sequences. tn vitro expression of the different constructs were assessed in 293 cells by Western blotting with polyclonal mouse sera, which were generated by DNA immunisation with one of the Tat, Rev and Nef constructs. The transactivation activity of Tat variants and Rev-mediated nuclear export activity of RRE-containing transcripts were studied in cotransfection experiments using reporter-gene-based assays while Nef functionality was assessed in a cotransfection assay with subsequent flow cytometric analysis of surface CD4 and MHC-I expression on 293 cells. Sequence analysis of the South African HIV-1 subtype C consensus sequences of Tat, Rev and Nef revealed a high degree of similarity with a consensus sequence that was drawn up from a large number of viruses from southern Africa. These consensus sequences were also closer to individual viral isolate sequences than any individual sequences were, indicating that the use of a consensus sequence may serve to reduce genetic diversity between a vaccine and circulating viruses. Expression levels of the sequence-modified tat and nef gene constructs were not significantly higher than the wild-type constructs, however, the codon-optimised rev mutant exhibited markedly higher expression than the wild-type rev construct. Immunoreactivity of the protein with the mouse sera demonstrates expression and immunogenicity of the Tat, Rev and Nef immunogens in mice. In the background of the subtype C Tat, a single C22 mutation was insufficient to inactivate l TRdependent CAT expression in 293T and Hela cells. Yet, this activity was significantly impaired using the single mutation, C3?, or the double mutation, C22C3? Compared to the wild-type Rev, the function of the Rev with a double mutation, M5M10, was completely abrogated. Similarly, while the wild-type Nef and native, codon-optimised consensus Nef proteins mediated CD4 and MHC-I downregulation, CD4 downregulation was completely abrogated in one of the mutants, while both Nef mutants were entirely deficient for MHC-I downregulation. These data demonstrate the high expression levels and impaired functionality of sequence-modified HIV-1 subtype C consensus Tat, Rev and Nef DNA immunogens that may be used as single-standing vaccine components or form part of a multicomponent HIV-1 vaccine.
Advancing the understanding of the PE/PPE protein families through PPE_MPTR protein expression and analysis of pe/ppe single nucleotide variants in differentially drug sensitive Mycobacterium tuberculosis.
(Stellenbosch : Stellenbosch University, 2022-11) Bartizal, Tom Jack; Sampson, Samantha; Kriel, Nastassja; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.
ENGLISH ABSTRACT: Mycobacterium tuberculosis is one of the leading causes of infection and death by a single microorganism. The M. tuberculosis genome contains two prominent gene families, encoding proteins known as the PE (proline-glutamate) and PPE (proline-proline-glutamate) families. Several of these are essential for M. tuberculosis pathogenesis, however, the majority of the PE/PPE proteins, especially those within the PPE_MPTR subfamily are understudied. This is apparent regarding the sub-cellular localisation of PPE_MPTR proteins which may interact at the host-pathogen interface. Some functional aspects have also been overlooked, such as the potential for pe/ppe variants to contribute to drug resistance in M. tuberculosis. These genes are often overlooked due to their polymorphic nature, as well as the challenges associated with short-read sequencing technologies to reliably assemble their highly repetitive and GC-rich sequences. For the first part of our study, we aimed to identify the sub-cellular localisation of select PPE_MPTR proteins (PPE_MPTR10, -24, -40, 42, -53 and -62) by expressing them as fluorescently tagged proteins in Mycobacterium smegmatis. We successfully amplified all six genes from M. tuberculosis H37Rv DNA, and successfully cloned ppe_mptr10 into the pCG expression vector. Unfortunately, no protein expression was detected and we were unable to determine the localisation of PPE_MPTR10 within M. smegmatis. For the second aim of our study, we made use of a newly developed analytical pipeline to screen whole-genome sequencing (WGS) data and identify high-confidence pe/ppe singlenucleotide variants (SNVs) associated with drug resistance profiles. Nine SNVs were predicted to be unique to either drug susceptible, multi- or extensively-drug resistant (DS, MDR or XDR) classes of M. tuberculosis. We aimed to verify these findings by PCR and Sanger sequencing of targeted SNVs in an independent set of clinical M. tuberculosis isolates. The SNVs were determined to be true variants identified by the pipeline present in clinical isolates but absent from the reference M. tuberculosis H37Rv. None of the target variants were found to be unique to the drug resistance class for which they were originally predicted. The successful identification of pe/ppe variants by genomic analysis demonstrates the potential to screen these repetitive GC-rich regions for genetic variants. Future studies will be required to establish the role of PPE_MPTR proteins in M. tuberculosis pathogenesis.
Alternative insulin mitogenic signaling pathways in immature osteoblast cell lines
(Stellenbosch : Stellenbosch University, 2002-03) Langeveldt, Carmen Ronel; Hulley, P. A.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Medicine.
ENGLISH ABSTRACT: Insulin is a mitogen for many cells and commonly signals through the classical, mitogenic Raf- MEK-ERK or metabolic PB-kinase pathways. Insulin deficiency or type I diabetes causes severe osteopenia. Obese patients with type II diabetes or insulin resistance, a disease associated with defective insulin signaling pathways and high levels of circulating insulin, have increased or normal bone mineral density. The question of whether hyperinsul inemia preserves bone mass is frequently raised. However, there is still a lot of controversy on the role of insulin as an osteoanabolic agent and this question still remains unanswered. A critical role for insulin signaling in bone building osteoblasts has recently been demonstrated with IRS-l knock-out mice. These mice developed low-turnover osteopenia due to impaired proliferation and differentiation, stressing the importance of osteoblastic IRS-l for maintaining normal bone formation. In the present study it was found that insulin does function in vitro as an osteoblast mitogen. This was illustrated in three relatively immature osteoblast (MBA-15.4, -15.6 mouse and MG- 63 human) cell lines, which responded to insulin with significant increases in proliferation. In the MBA -15.4 preosteoblasts insulin stimulation of proliferation was comparable to the welldescribed mitogen, TPA. The UMR-I06 cell line expresses markers of differentiated osteoblasts, and was much less responsive to insulin treatment. The difference in proliferative potential may be due to differences between spontaneously transformed cell lines, or the stage of cell differentiation. UOI26, a MEKI/2 inhibitor and wortmannin, a PB-kinase inhibitor, were used to investigate the pathway used by insulin to signal and activate ERK and osteoblast proliferation. In MBA-15.4 mouse preosteoblasts, GF-containing FCS was completely dependent on MEK for DNA synthesis. In contrast, in both MBA-15.4 and more mature MBA-15.6 osteoblasts, insulininduced proliferation was resistant to the inhibitors alone or in combination. Higher MEKinhibitor concentrations had no effect, and proliferation was also increased by the inhibitors in several experiments. This indicated that the classical, insulin mitogenic pathway was not involved in MBA-15.4 proliferation. Wortmannin had no effect on either insulin- or 20% FCSstimulated proliferation, but inhibited activation of Akt/PKB, the metabolic downstream target of PI3-kinase. Insul in signal ing to ERK was both MEK-and PI3-kinase- dependent, but this had no effect on proliferation. In contrast, FCS-stimulated ERK activation and proliferation was almost completely dependent on MEK-ERK activation. Proliferative signaling in the MG-63 human osteoblastic cell line in response to insulin was partially dependent on MEK and partially dependent on PB-kinase. In contrast, signaling in response to the phorbol ester, TPA, was partially dependent on PI3K but totally dependent on MEK-ERK. This indicates that the signal converges on ERK, suggesting the involvement of a PB-kinase upstream of a dominant MEK-ERK pathway. The differences found here between mouse and human insulin mitogenic signaling pathways indicate that there may be species differences between osteoblast signaling pathways, with mouse cells being independent and human cells being dependent on MEK for DNA synthesis in response to insulin. The effects of glucocorticoids on insulin mitogenic signaling in osteoblasts were also investigated, because chronic long-term steroid use results in excessive bone loss. The PTP inhibitor, sodium orthovanadate, reversed GC-impaired TPA- and FCS- induced proliferation in MBA-1SA and MG-63 preosteoblasts. PTPs, such as SHP-l and PTP-IB, dephosphorylate and inactivate phosphorylated kinases. Both SHP-l and PTPlB associated with kinases in the mitogenic signaling cascade of MBA-lS.4 preosteoblasts growing rapidly in 10% FCS. Further, SHP-I co-irnmunoprecipitated with active, tyrosine phosphorylated ERK, which may indicate that it can dephosphorylate and inactivate ERK. However, since the MEK-ERK or PB-kinase pathways are not important in insulin-induced proliferation in mouse osteoblasts, the PTPs are unlikely to be role players in the negative regulation of this signaling pathway. This was confirmed by the finding that vanadate was unable to reverse GC-induced decreases in insulinstimulated DNA synthesis. This suggests that vanadate-sensitive PTPs may not be important in the negative regulation of insulin-induced mouse osteoblast proliferation, and provides further evidence of a novel insulin mitogenic pathway in the MBA-lSA but not MG-63 osteoblastic cell line.
Analysis of a neurochip array dataset to study Parkinson’s disease in a South African study collection
(Stellenbosch : Stellenbosch University,, 2023-02) Step, Kathryn; Bardien, Soraya; Vorster, Alvera; Müller-Nedebock, Amica; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.
ENGLISH ABSTRACT: Parkinson’s disease (PD) is an incurable, and complex neurodegenerative disease. Both genetic and environmental factors likely contribute to disease onset. Notably, while several pathogenic variants and susceptibility factors have been described in populations of Asian and European ancestry, such variants have seldom been identified in individuals from sub-Saharan Africa (SSA). This could be due to the limited number of studies investigating the genetic etiology of PD in SSA. To address this knowledge gap, the present study undertook the largest, to date, PD-focused genomewide association study (GWAS), and pathogenic variant screening study in SSA to identify possible susceptibility variants and pathogenic variants in South African PD cases. For this, we used raw genotyping data generated from a large collaborative project known as COmprehensive Unbiased Risk Factor Assessment for Genetics and Environment in Parkinson’s Disease (Courage-PD), whose goal was to identify PD-associated variants. The NeuroChip array, used to genotype the study participants, contained a total of 306,670 tagging variants and 179,467 custom content variants, including 349 associated with PD. The South African dataset genotyped on the array comprised 452 cases and 280 controls. We hypothesised that these individuals would harbour susceptibility and pathogenic variants. To test this hypothesis, the NeuroChip genotyping data was analysed using various bioinformatic approaches. The quality control (QC) and association analysis were completed using PLINK, and the results were visualized using R software. After excluding 15 individuals during the QC stage, population stratification analysis identified two ‘broad’ ancestral groups, designated as ‘European’ (n=497) and ‘non-European’ (n=220). For the GWAS, no variants reached the genome-wide significance threshold of 5x10-8 , however, variants were found that met the ‘suggestive significance’ criteria (1x10-5 ). A total of 17 variants of interest were identified in the European ancestral group (in the KHDRBS2, FGF14, and PDXK genes) and 2 variants of interest were identified in the non-European ancestral group (in the SYNPR and PDE10A genes). These variants highlighted possible new PD genes that are plausible candidates, but that will need to be confirmed in future, much larger GWAS. Thereafter, a Polygenic Risk Score (PRS) analysis was performed, using PRSice software, on the European ancestral group where the most predictive PRS explained 4.5% of the phenotypic variation (the phenotype being PD). Furthermore, use of the NeuroChip data as a method of pathogenic variant screening, revealed that all 12 variants detected by our group previously were also detected by the array. Moreover, an additional 16 variants in 14 individuals were prioritized as being potentially pathogenic, and warrant further study. Finally, screening of p.G2019S in the LRRK2 gene, arguably the most prevalent PD pathogenic variant, using high-resolution melt analysis, revealed a relatively low frequency of 1.2% (n= 8/689) in our entire PD study collection. Notably, this variant has not been identified in any PD individuals of African ancestry, to date. Collectively, this study highlights the importance of screening and studying underrepresented populations to uncover additional genetic-related risks for PD development. However, future largescale whole-genome sequencing and association studies, including all South African ancestral groups, will likely be needed to identify the remaining, potentially novel genetic factors contributing to PD in our local populations.
Analysis of ex vivo host biomarkers in sputum samples for diagnosis of pulmonary tuberculosis
(Stellenbosch : Stellenbosch University, 2019-12) Mendy, Joseph F.; Chegou, Novel N.; Sutherland, Jayne; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics
Background Despite all the interventions deployed to control tuberculosis (TB), the disease still continues to be the principal cause of death from a single infectious agent in resource constrained settings. An estimated 60% of suspected TB patients do not have access to TB diagnostic tests. With the limitations of the current diagnostic tests and the importance of early diagnosis and initiation of treatment, biomarker diagnosis of TB would be an optimal option. For biomarkers are indicators of immune activity and state. Therefore, host or pathogen biomarker of TB disease would be ideal. Hence, the aim of this project was to profile a broad array of host markers for development of optimal signatures for detection of pulmonary tuberculosis from other respiratory disorders using ex vivo sputum samples Methods We recruited patients who were seeking medical attention at the MRCG at LSHTM outpatients department and Tuberculosis clinic with symptoms suggestive of TB, prior to clinical or microbiological diagnosis. All age groups were recruited. Sputa were collected at baseline from all participants and at 1 and 2 months from the confirmed TB cases. The sputa were digested with Sputolysin and the supernatant analysed using Luminex arrays while RNA extracted from the pellet were analysed with RT-qPCR. Statistical analyses and graphs were generated using R programming Language and GraphPad Prism, with a q value ≤ 0.05 considered significant. A receiver operating curve (ROC) was used to assess the diagnostic performance of individual and combination markers. Results Confirmed TB (428) and ORD (313) patients were analysed, 70 markers were assessed for diagnostic potential and treatment response. Of these, 37 were significantly different between TB and ORD. The best single marker was MMP-2 with an AUC of 0.73. An eight-marker signature (IFNү, IL-1β, IL-8, IL-10, IL-12p70, MIP-1β, RANTES and VEGF) was able to diagnose smear and culture positive TB from ORD with an AUC of 0.77, sensitivity of 78% and specificity of 70%, while a threemarker signature (IL-1β, IL-7 and VEGF) classified smear negative but culture positive TB from ORD with an AUC of 0.74, sensitivity of 86% and specificity of 60%. Among children who had TB, a fourmarker signature (FGF, IL-4, MIP-1a and RANTES) differentiated those with TB from ORD, with an AUC of 0.87, sensitivity of 82% and specificity of 87% and a five-marker signature consisting of BAFF, C3L1, IL-22, MMP-3 and sTNFR1 was able to discriminate TB and HIV co-infected from ORD with an AUC of 0.90, sensitivity of 88% and specificity of 85%. We also found a four-marker signature consisting of EGF, IL-15, MIP-1β and TNF-β that could predict slow versus fast treatment responders at baseline with an AUC of 0.74, sensitivity of 75% and specificity of 80%. Conclusion We have discovered novel sputum host biomarkers and biosignatures for screening of tuberculosis and treatment response. The data is promising for potential translation into a user friendly device as a rapid screening test for pulmonary TB. However, this markers and signatures require further investigations to authenticate their usefulness.
Analyzing genetic factors contributing to dysmotility in Hirschsprung’s disease and African Degenerative Leiomyopathy in a South African population
(Stellenbosch : Stellenbosch University, 2019-03) Maluleke, Twananani; Moore, Samuel; Kinnear, Craig; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics.
Introduction Hirschsprung’s disease (HSCR) and African degenerative leiomyopathy (ADL) are rare gastrointestinal disorders affecting neonates and young children. HSCR is characterised by the absence of intrinsic ganglion cells in the distal segment of the intestine; its aetiology has been linked to cellular and molecular mechanisms associated with the enteric nervous system (ENS) development, ADL on the other hand is a distinctive form of visceral myopathy (VSCM) of uncertain aetiology affecting enteric smooth muscles (ESM) of the distal intestine. Gut motility is a result of highly coordinated contractions by muscle layers, neural network and pacemaker intestinal cells of Cajal whose development are controlled by genetic factors. The aetiology of HSCR has been associated with 15 genes linked to ENS development meanwhile ADL has been linked to environmental factors. Actin gamma 2 (ACTG2) is a gene that encodes the ACTG2 protein which is involved in ESM development. Studying the ACTG2 in HSCR patients may ascertain whether individuals affected by HSCR also display muscular dysfunction thereby providing a possible factor in the recurrence of dysmotility post-surgical resection. Additionally, ACTG2 has been identified as the genetic factor in VSCM pathology; therefore, the study may provide novel information regarding the genetic factors of ADL. Aim This project aims to study the genes associated with the development of enteric nervous system (RET, NRG1, SOX10, EDNRB) and smooth muscle cells (ACTG2) that contribute to HSCR and ADL in the South African neonate population. Methods Seventeen whole blood samples were collected from HSCR participants after informed consent; of which only 14 samples were included for genotyping and 9 samples were selected for RNA analysis based on the quality of extracted DNA and RNA respectively. Five whole blood samples were also collected from ADL patients after informed consent. RNA samples from the HSCR cohort were reverse transcribed and quantitative polymerase chain reaction was performed. DNA samples from HSCR and ADL samples were screened for variants in the ACTG2 exons through bidirectional Sanger sequencing. Novel variants were analysed in silico to ascertain their pathogenicity. Results and Discussion In both HSCR and ADL cohorts the variant K119E/R was observed in 64% (9/14) and 60% (3/5) of the study population respectively; K119E/R is likely to function as a disease modifier as it was also observed in the control samples six out nine individuals. Variants S345L and W357G in exon 10 with probable significant effect in the pathogenesis of ESM were identified in the HSCR cohort only. The ADL cohort had polymorphic intronic variants predicted to shift the exonic splice sites namely g>c -IVS12 exon 3 and c>t -IVS3 exon 5. Differential expression of ENS genes EDNRB, RET, SOX10 and NRG1 associated with ENS development in the HSCR cohort was not achieved due to experimental factors. Conclusion ACTG2 encodes an enteric smooth muscle γ-2 actin which plays a pivotal role in the contractile proteins of ESM, thus the data suggests that a muscular component may exist in HSCR aetiology that should be investigated further in vitro and provides further insights into genetic factors that may contribute to ADL pathogenesis.
The antimycobacterial activity of phytocannabinoids
(Stellenbosch : Stellenbosch University, 2023-02) Williams, Ricquelle Daphne; Mavumengwana, Vuyo; Loxton, Andre; Smith, Liezel; Allie, Nasiema; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.
ENGLISH ABSTRACT: Tuberculosis (TB) is a deadly and communicable disease that is caused by the bacterium, Mycobacterium tuberculosis (M.tb). M.tb is skilled at manipulating and evading hostdefense mechanisms by alveolar macrophages, allowing its survival and replication intracellularly. Since the current treatment regimen makes use of antibiotics, the everincreasing number of multi-drug resistant (MDR) M.tb strains has resulted in a need for research into alternative options. Although most anti-TB drug discovery strategies target the actual pathogen, slow pace in discovering new antimycobacterials is testament to the need to change strategies. Host directed therapy (HDT) is an intervention where the eradication of the intracellular pathogen is mediated by the host immune response modulated by small molecules. Cannabis sativa L. (C. sativa) is valued for its psychoactive potentials and varied ethnobotanical medicinal properties due to a plethora of bioactive constituents. In addition to the plant’s utility as a treasure trove for medicinal applications, this research study aims to present a case for the use of small molecules derived from C. sativa as an alternative HDT against TB. We aimed to evaluate the antimycobacterial effect of crude extracts of two C. sativa plants, one grown outdoor (C. sativa plant 1 or P1) and one grown under controlled indoor conditions (C. sativa plant 2 or P2) and evaluated their bioactive organic extracts activity in THP-1 macrophages infected with mycobacteria. In addition, we isolated endophytic fungi from C. sativa and evaluated their antimycobacterial activity and whether they produce similar compounds to the host plant. Herein, it was demonstrated that the dichloromethane (DCM) extract of C. sativa plant 1 (DP1) and the methanol and ethyl acetate extracts of C. sativa plant 2 (MP2 and EP2) stimulated THP-1 macrophages in the killing of Mycobacterium smegmatis (M. smegmatis) mc2155 compared to untreated macrophages. In addition, the methanol extract of C. sativa plant 2 (MP2) displayed the best activity with a percentage survival of 14.31% (p = 0.000009) at 6-hours post treatment; 2.63% at 12-hours post treatment (p = 0.0001) and 0% survival at 24-hours post treatment (p = 0.0005). The metabolite profile showed that these three extracts (DP1, MP2 and EP2) share three compounds, cannabinol (CBN), cannabigerol (CBG), and cannabielsoin (CBE), which could be the cause of macrophage stimulation. However, MP2, which showed the best activity, contains cannabidiol (CBD), which could be the cannabinoid causing the increased activity. Although the actual bioactivities of CBN, CBG, CBE and CBD remain speculative until further purification, characterization and reevaluations are carried out, a cautious judgement can be made that these compounds likely play a beneficial role. Fungal endophytes Alternaria alternata (A. alternata), Alternaria infectoria (A. infectoria), Fusarium incarnatum (F. incarnatum), and Fusarium chlamydosporum (F. chlamydosporum) were isolated from surface sterilized buds of C. sativa and identified using molecular and phylogenetic methods. A. alternata showed some compounds (via LC-QTOF-MS) which were previously isolated in C. sativa, with its extracts exhibiting immunomodulatory activity against THP-1 macrophages infected with M. smegmatis mc2155. The percentage survival for treated THP-1 cells was found to be 51.81% at 6 hours (p = 0.0003) compared to 81.91% untreated. Overall, these results establish that cannabinoids are able to influence THP-1 infected cells’ ability to clear mycobacterial infection albeit a low percentage cell survival. To develop specialized HDT strategies targeting macrophages, a deeper comprehension of the mutual interaction between cannabis and immunity is required. Future studies to isolate and characterize compound(s) from the endophytic fungi and plant extracts could result in lead development of naturally sourced drugs for host-directed TB treatment.
Application of Becton Dickinson FACSTM Combinatorial Antibody Profile (FACSTM CAP) technology to the identification of efficiency of tuberculosis therapy
(Stellenbosch : Stellenbosch University, 2015-03) Smith, Bronwyn Kerry; Walzl, Gerhard; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics
ENGLISH ABSTRACT : Currently the treatment of individuals with active Mycobacterium tuberculosis (Mtb) infection involves a standard six-month multi-drug regimen, impacting negatively on treatment adherence, which in turn fuels multi- and extensive drug resistant TB. However, some patients may not require the full six-month regimen due to less extensive disease or rapid early treatment response. The identification of these patients has been problematic but would allow significant cost savings and may impact positively on treatment adherence if treatment duration could be shortened and if this subgroup constituted a significant portion of patients. The aim of this project was to identify peripheral blood lymphocyte surface markers through a proprietary technology, FACSTM CAP by Becton Dickinson Technologies, to investigate the change in expression during the course of treatment with potential treatment monitoring utility. Peripheral blood mononuclear cells (PBMCs) were isolated from TB patients (n=33), healthy community controls (n=11) and other lung disease controls (OLD, n=9) at diagnosis of disease, week 4 (after commencement of treatment) and week 24 (end of treatment, EOT). Antibodies to 252 surface markers were used to stain PBMCs, the cells were fixed in 2% paraformaldehyde and data acquired on a FACS Calibur flow cytometer. Post-acquisition compensation and analysis was performed using FlowJo software. The analysis was performed by gating on the lymphocytes and overlaying sample plots on isotype controls. Statistics analysis included repeated measures ANOVA, paired t-test and independent t-test. Comparisons were made between the expression levels of patient time points (diagnosis, week 4 and week 24) and participant groups (TB, healthy community controls and OLD controls). Sample wells that provided an uncertain demarcation of the positive and negative expression population were flagged and excluded from analysis. After the application of the Bonferroni correction, results revealed five overall treatment response markers (CD120b, CD126, CD62L, CD48 and CD29) that were significantly different (p-value <0.0002) when comparing expression levels at TB diagnosis and EOT (week 24) samples. A comparison of expression between TB at diagnosis and healthy community controls showed a significant difference for four markers (CD48, CD18, CD126 and fMLPr). Due to the application of the stringent Bonferroni correction, only these few markers were found to be statistically significant therefore all markers with a p-value <0.01 prior to Bonferroni correction, were included for analysis with Ingenuity Pathway Analysis (IPA) and Qlucore Omics Explorer software. IPA identified 23 biological pathways that were associated with two or more markers with significant changes during treatment. The top nine pathways are discussed and included the inflammatory response, cell migration, differentiation and maturation and crosstalk between cells of the innate and adaptive immune responses. In conclusion, this project resulted in the identification of three promising biologically significant surface markers that require further validation as candidates for biomarkers of TB treatment response. Future studies will investigate the most promising markers, including those that showed a trend for differences after the Bonferroni correction, in a candidate biomarker project with a new cohort of TB patients undergoing treatment.
Bio-profiling of TB patients with and without Type 2 Diabetes before and during anti-tuberculosis drug (ATD) therapy
(Stellenbosch : Stellenbosch University, 2017-03) Selamolela, Mosa; Ronacher, Katharina; Kleynhans, Leanie; Stellenbosch University. Faculty of Medicine and Health Sciences. Biomedical Sciences: Molecular Biology and Human Genetics.
Background Patients with type 2 diabetes (DM2) are three times more likely to acquire active tuberculosis (TB) compared to otherwise healthy people. TB-DM2 comorbidity is characterized by poor TB treatment outcomes, increased risk of failure and relapse. The exact mechanisms of increased susceptibility of diabetics to TB are not well understood, but it is thought that hyperglycaemia is associated with an impairment of the innate and adaptive immune response. Aim To assess soluble (cytokines) and cellular (T-cell subsets) immunological markers of treatment response in TB patients with and without DM2 and their association with glycaemic control. Materials and Methods Serum cytokine concentrations were measured by means of the Luminex assay in 14 TB and 11 TB-DM2 patients at Baseline, Month 2 and Month 6. The HO-1 levels were measured from serum samples by means of ELISA in 40 TB and 20 TB-DM2 patients at Baseline, Week 2, Month 2 and Month 6. The frequency of different T-cell subsets (central memory, naïve, effector memory and terminally differentiated effector memory T cells) was established in 8 healthy controls, 13 DM2, 18 TB and 23 TB-DM2 patients at Baseline, Month 2 and Month 6 using flow cytometry. Results Throughout treatment pro- and anti-inflammatory cytokine concentrations were increased in serum of TB-DM2 patients when compared to TB patients. Fibrinogen and Procalcitonin were higher in TB patients compared to the TB-DM2 patients at the end of treatment. Various cytokines (IL-1β, IL-4, IL-6, IL-7, IL-8, IL-9, G-CSF, GM-CSF, MCP-1(MCAF) and IFN-γ) and growth factors (VEGF and PDGF) exhibited positive correlation at baseline with HbA1c and random blood glucose, respectively. The frequencies of CD4+ naïve T cells increased from Baseline and Month 2 in TB patients. In contrast CD4+ and CD8+ naïve T cells decreased over time in the TB-DM2 patients. CD4+ T CM cells increased over time in TB-DM2 patients. Activated CD8+ naïve T cells increased over time in both TB and TB-DM2 patients while the terminally differentiated effector memory T cells decreased in both groups of study patients over time. No significant changes were observed in any CD8+ T cell subset in both groups of study patients. Conclusion The present study reveals that TB-DM2 patients have altered cytokine production at baseline and throughout treatment which may be linked to chronic inflammation associated with obesity and diabetes. Glycaemic control displays an influence in cytokine production shown in TB-DM2 patients. The frequencies of T cell subsets is altered in TB-DM2 patients and changes throughout treatment. These results show that TB-DM2 patients are characterised by changes in the adaptive immune system, which may contribute to poor treatment outcomes.
Bioinformatics-based strategies to identify PFHBII-causing and HCM main locus and/or HCM modifying mutations
(Stellenbosch : University of Stellenbosch, 2004-12) Yako, Yandiswa; Corfield, Valerie A.; Moolman-Smook, Johanna C.; University of Stellenbosch. Faculty of Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.
ENGLISH ABSTRACT: Progressive familial heart block type II (PFHBII) is an inherited cardiac conduction disorder of unknown aetiology, which has been described in a South African family. The disorder was mapped to a 2.9 centimorgan (cM) locus on chromosome 1q32.2-32.3. Clinically, PFHBII manifests cardiac conduction aberrations, that progress to a disease of the heart muscle, dilated cardiomyopathy (DCM). DCM is also reported as an end phase in hypertrophic cardiomyopathy (HCM), another heart muscle disorder. These cardiomyopathies are genetically heterogeneous with some of the genes reported as causes of both disorders. Therefore, genes identified as causes of HCM and DCM were considered plausible candidates for PFHBII mutation analysis. Additionally, this study provided an opportunity to assess potential modifiers of HCM. HCM exhibits marked phenotypic variability, observed within and between families harbouring the same causative mutation. Genes within the PFHBII locus were selected for PCR-SSCP analysis based on homology to genes previously reported as causing conduction system disorders associated with arrhythmias, DCM and/or HCM. Results were confirmed by direct sequencing and association between the detected variants and HCM parameters was assessed using a quantitative transmission disequilibrium test (QTDT). Eleven plausible candidate genes were selected within the PFHBII locus and two of the genes, PFKFB2 and ATF3, that encode for 6-phosphofructo-2,6-bisphosphatase (PFK-2/FBPase-2) and activating transcription factor 3 (ATF3), respectively, were analysed for PFHBII-causing and HCM main locus and/or HCM modifying mutations. Mutation analysis of PFKFB2 and ATF3 in the PFHBII family revealed no PFHBII causal mutation. PFKFB2 and ATF3 were later localised outside the PFHBII locus, and, therefore, were excluded as PFHBII plausible candidates. Further analysis of the two genes for HCM main locus and/or HCM modifying mutations in the HCM panel identified several sequence variants. QTDT analysis of these variants showed no significant association. Completion of the Human Genome Project (HGP) and annotation of new genes within the PFHBII locus allowed the identification of more PFHBII plausible candidate genes. Identification of causal mutations in plausible PFHBII candidate genes will allow molecular diagnosis of PFHBII pathophysiology. Furthermore, identification of both HCM-modifying and HCM-causing genes will give insight into the phenotypic variability noted among South African HCM-affected individuals and into the molecular cause of the disease among individuals with HCM-like clinical features.
Characterisation of a high copy number mutant pAL5000 origin of replication
(Stellenbosch : Stellenbosch University, 2001-12) Jansen, Yvette; Bourn, W. R.; Van Helden, P. D.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.
ENGLISH ABSTRACT: The plasmid pAL5000 is a mycobacterial plasmid isolated from Mycobacterium fortuitum. It is a low copy number plasmid, which replicates in both fast growing (e.g. M. smegmatis) and slow growing (e.g. M. bovis BCG) mycobacteria. Most mycobacterial-E. coli shuttle vectors utilise the pAL5000 origin of replication. The minimum replicon consists of ORF1 (RepA), ORF2 (RepB) and the origin of replication. Dr W.R. Bourn created an E. coli-mycobacterial vector based on the pAL5000 origin of replication (pORI) and then subjected it to semi-random mutagenesis. A high copy number mutant was identified (pHIGH) and the causative mutation was tentatively identified as a 3bp deletion situated just upstream of repB. This work describes the further characterisation of the mutant plasmid. Firstly, it was shown by retransforming M. smegmatis with both the original and mutant plasmids (pORI and pHIGH), that the mutation causing the increased copy number was plasmid-encoded and not on the chromosome. Following this, it was demonstrated by simple subcloning of the region that carries the 3 bp deletion, that other pAL5000-based vectors could be converted to high copy number. In addition to this, the subcloned region was sequenced and the nature of the mutations was confirmed. The subcloning experiment confirmed that the 3bp deletion caused the high copy number phenotype. Following this, the exact copy number of pHIGH and the relative increase in copy number was determined. From this, the copy number of pORI could also be determined. The plasmid pHIGH has a copy number of approximately 54, compared to the 8 of pORI (a relative increase by a factor of 7). Because it is important for researchers to know the characteristics of the vectors that they use, especially the influence it will have on its host, stability tests and growth curves were also performed. It was seen that the higher copy number did not markedly increase the stability, however, this is because pORI is already extremely, and unexpectedly, stable in the host M. smegmatis. According to the growth curves, the increased copy number has little effect on the growth of the host M. smegmatis. Possible mechanisms for the increased copy number were then investigated. By using a promoter probe vector, the possible existence of a promoter situated between the two open reading frames of pAL5000 (repA and repB) was investigated. It was thought that the mutation might have created, or changed an existing promoter, situated between repA and repB. The results showed, however, that in both pORI and pHIGH there might be a very weak promoter upstream of repB, but the mutation did not cause any change that was measurable by the method that was used. A further possibility was that the mutation caused a change in the RNA secondary structure, which might then have an effect on the translational efficiency of RepB. It was found that the 3bp deletion in pHIGH causes a change in the local RNA secondary structure around the ribosomal binding site and the start codon, when compared to pORI (wild type). This change may cause the translation initiation rate of RepB to be different between pHIGH and pORI. Ultimately it would lead to a different ratio of RepA and RepB in the cell.
Characterisation of lesions and associated immune cell populations in the lung of African buffalo (Syncerus cafe) infected with Mycobacterium Boris
(Stellenbosch : Stellenbosch University, 2021-03) Goldswain, Samantha; Miller, Michele; Kleynhans, Leanie; Jennifer, Landolfi; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics.
ENGLISH ABSTRACT: A vast array of species is susceptible toMycobacterium bovis (M. bovis) infection, with varying pathogenesis and disease outcomes. Understanding the pathogenesis of disease is important for informing epidemiologic concerns and disease management strategies, especially in reservoir hosts, such as African buffaloes (Syncerus caffer). Therefore, this project is focused on characterisation of histologic morphology and associated immune cell populations in lung granulomas from M. bovis infected African buffalo to advance knowledge of disease development in buffaloes. A scoring system was developed to compare numbers and distributions of different immune cells, as well as other pathologic changes in bovine tuberculosis (bTB) pulmonary granulomas. In addition, an immunohistochemistry (IHC) staining technique was optimised for immune cell surface marker detection in buffalo lung tissues. Formalin-fixed and frozen tissues were available from M. bovis naturally infected buffaloes from Hluhluwe-iMfolozi Park, South Africa. Formalin-fixed lung tissues were selected based on gross lesion scores that represented a range of severity. Lung sections from 14 buffaloes were stained with haematoxylin and eosin (H&E) to assess the histologic morphology of granulomas. Microscopic characteristics were then scored based on six categories. In addition, immunohistochemical techniques were optimised using antibodies that detected immune cell surface antigens (CD3, CD4, CD21, CD163, NCR1). This study documented characteristics of a low histologic stage granuloma to include macrophages/multinucleated giant cells (MNGCs) at the core, lymphocytic infiltration surrounding the macrophages, and minimal to no necrosis and fibrosis. Granulomas compatible with more advanced disease were necrotic, with macrophages surrounding the necrotic core, lymphocytes located peripherally, and were encapsulated by a fibrous capsule. More developed granulomas were not always mineralised. Based on IHC, CD3+ T lymphocytes and CD163+ macrophages/MNGCs were present in all granulomas examined; B cells (CD21+) were only present in higher stage granulomas; and natural killer (NCR1+) cells were not abundant in any of granulomas. The CD4 antibody did not stain buffalo tissues and therefore, distribution of this subset of T lymphocytes could not be evaluated. Ziehl-Neelsen staining was performed to detect the presence of acid-fast bacilli, however, no bacilli were visible in the slides analysed. The appearance of the buffalo pulmonary granulomas did not completely fit with the stages described in cattle (Wangoo et al., 2005). Therefore, a scoring system for categorising granulomas, adapted for buffaloes, should be investigated to provide a species-specific description that would be beneficial for understanding bTB pathogenesis. In summary, findings showed that lymphocytes and macrophages/MNGCs appear to be the predominant immune cell types present and their distribution and relative numbers appear to change as pulmonary granulomas develop. Characteristics such as increased fibrous encapsulation and development of a necrotic core appear to be similar to granulomas in cattle. However, mineralisation may not be a consistent feature, suggesting some species-specific differences that should be further investigated. This study also demonstrated that immunohistochemistry is a practical method for further characterisation of local immune responses to M. bovisinfection in buffalo. Further research with a larger sample set will be informative for understanding local and associating systemic immune responses to bTB in buffaloes.
Characterising persister growth in response to environmental conditions using a biofilm model
(Stellenbosch : Stellenbosch University, 2023-03) Mathee, Raadhiyah; Sampson, Samantha Leigh; Mouton, Jomien; Bagheri, Bahareh; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.
ENGLISH ABSTRACT: The rise in antibiotic therapy failure worldwide poses a serious threat to successful tuberculosis (TB) treatment. This is partly due to a subpopulation of cells known as persisters. Persisters possess the ability to switch to a temporary drug tolerant state, avoiding the action of antimicrobials thereby contributing to the lengthy TB treatment regimen and high treatment failure rates. Persister formation is thought to be influenced by various environmental conditions. Biofilms offer a model system to study persister formation and physiology since cells harboured within biofilms are exposed to heterogeneous environments with varying levels of oxygen and nutrients. Moreover, biofilms are enriched for persister cells and can withstand antibiotic treatment and can thus serve as an appropriate model to study the nature of persisters. Previously the isolation and characterisation of these persisters proved to be challenging using standard culture techniques, slowing the progress in research. However, with the development and validation of techniques using fluorescence dilution and flow cytometry, we are now offered insight into the microbial lifestyle at a single cell level. The fluorescence dilution reporter system carries a constitutive green fluorescent protein used as a marker of viability and an inducible red fluorescent protein used to monitor bacterial replication. We attempted to utilise a hydrodynamic flow cell system as an in vitro biofilm model to investigate bacterial growth and replication dynamics. However, further use of this system was abandoned as bacteria failed to adhere to the substratum, preventing further applications. The widely used microtiter plate assay was then employed to carry out all objectives of this study. Protocol modification and optimisation were performed to incorporate the use of the reporter strain, and to remove and disperse the cells. Results showed changes in cell morphology after exposure to biofilm microenvironments, particularly the presence of shorter rods and more circular shapes. The bacterial replication dynamics of biofilm cell populations revealed a subpopulation of cells that retained high levels of red fluorescent intensity, potentially indicative of persisters/persister like cells. We then explored spatial growth patterns in biofilms to determine cell localisation in response to induction using Confocal Laser Scanning Microscopy. This provided overall mapping of the pellicle structure and showed evidence of individual cells expressing red fluorescent intensity. The findings of this study provide insight into the use of a biofilm model to study persisters by Imaging Flow Cytometry and a starting point for further study incorporating quantitative assessment of intact biofilm structures.
Characterization of Biomarkers of Immunological Activation in African Elephants (Loxodonta africana)
(Stellenbosch : Stellenbosch University, 2021-03) De Waal, Candice Raquel; Miller, Michele Ann; Kerr, Tanya Jane; Kleynhans, Leanie; Landolfi, Jennifer; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics.
ENGLISH ABSTRACT: African elephants (Loxodonta africana) are considered priority species within conservation areas because of their aesthetic value, ecological importance, and economic contribution to the ecotourism industry. Conservation efforts have focused on protecting habitat, but there are fewstudiesinvestigating the role of disease. The recent discovery of tuberculosis (TB) in a free-ranging African elephant in Kruger National Park (KNP), South Africa, has resulted in movement restrictions, preventing the translocation of elephants from this population. Since diagnostic tests for TB in wildlife are limited, the development of blood-based teststo detect Mycobacteriumtuberculosiscomplex (MTBC) infection in African elephants isneeded. These antigen-specific immune assays would have a significant beneficial impact on current practices in wildlife and zoological medicine. Therefore, the aim of this project was to identify blood-based host biomarkers that can be used to detect immune responses of African elephants. Cytokine gene expression assays (GEAs) have been employed to measure cell-mediated immune responses in a variety of species. These GEAs use real-time, reverse-transcription quantitative PCR (RT-qPCR) to measure changes in gene expression of immune cells,following stimulation ofwhole blood. In this study, wholeblood from African elephants from KNP, a Mycobacteriumbovis-endemic area,wasstimulated using pokeweed mitogen and mycobacterial antigens. Newlydesigned primers, as well as modified primers originally developed for useinother species, were used to amplifyand sequence African elephant mRNA transcripts of selected target(CXCL9, CXCL10, IFNγ, IL4, IL10, IL12, TGFβ,andTNF)and reference genes(ACTB, B2M, GAPDH, YWHAZ). These mRNA transcriptswere used to design sequencespecific primers and develop a RT-qPCR to determine changes in cytokine expression as a measure of general immune activation and antigen-specific responses. Confirmed mRNA transcriptsfor African elephants were used to develop real-time RT-qPCRs forIL10, TNF,andTGFβ,relative to GAPDHas the optimal reference gene.Thesecytokine GEAsdemonstrated the use of identified biomarkers to measure immune responses in this species. To our knowledge, this was the first study that has investigatedcytokine biomarkers in African elephants using real-time RT-qPCR. Results of the cytokine GEAs showed up-regulation of IL10and TNF, as well as down-regulation of TGFβ,in response to mitogen stimulation. When expression of these cytokines wasevaluated in response to mycobacterial antigen stimulation, asignificantup-regulation of IL10was observed following PPDa and PPDb stimulation. However, following stimulation with ESAT6/CFP10, as well as calculated differential PPD response, a very slight down-regulation of IL10was observed, as expected in TB uninfected elephants. Similarly, a slight down-regulation of TNFand TGFβwas observedfollowing all mycobacterial antigen stimulations. Findingsin this study provide novel insights into the African elephant immune system. The generated mRNA transcripts provide a basis for development of immunological assays for TB, as well asother diseases. Finally, evaluation ofgene expression following antigen stimulation provided insight into the use of PPDa, PPDb and ESAT6/CFP10 as stimulantsof antigen-specific TB responses. This will aid in the development of tools to improve disease detection and diagnosisin African elephants.
Characterization of mycobacterium bovis persisters in South African wildlife
(Stellenbosch : Stellenbosch University, 2020-12) Ncube, Pamela; Sampson, Samantha Leigh; Miller, Michele Ann; Bagheri, Bahar; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences.
ENGLISH ABSTRACT: Mycobacterium tuberculosis infection in humans can either progress to disease, or in many patients, be contained and lead to a latent infection without evidence of active disease. However, it is unknown whether a similar scenario exists in animals infected with Mycobacterium bovis. Knowledge gaps in the pathogenesis and progression of disease in the wide spectrum of livestock and wildlife species that can be infected by M. bovis also complicate our understanding of this disease. Since management strategies may differ for latently infected animals, it is important to evaluate whether M. bovis has the potential to form persisters, which are believed to underlie latent infection. In this study, we aimed to characterize M. bovis persister formation upon in vitro acid stress which mimics the macrophage phagolysosome microenvironment. A total of 23 samples from naturally infected M. bovis wildlife species were successfully decontaminated, purified, and genotyped to the strain level. Spoligotyping identified a total of 5 different M. bovis spoligotypes, namely SB0121 (16 isolates), SB0130 (4 isolates), SB0140 (1 isolate), SB1474 (1 isolate), and SB1275 (1 isolate). In preparation for persister assay experiments, 22 of the 23 isolates were successfully transformed with the Fluorescent Dilution (FD) reporter plasmid pTiGc. Thereafter, growth curves confirmed that there were no growth defects of the transformants due to the carriage of the reporter plasmid. Five M. bovis strains were selected for acid sensitivity and persister assay experiments, using the acid stress in vitro model. This model enriches for persisters, as reflected by a sub-population of viable but non- or slowly replicating (VBNR) bacteria. Laboratory strains, SAMMtb::pTiGc and BCG::pTiGc, had the highest average percentage of VBNR cells at 12.2 ± 1.5% (± SD) and 7.2 ± 0.6%, respectively on day 4. In contrast, the clinical strain with the highest VBNR average percentage was PN18067_1::pTiGc at 1.3 ± 0.1%, while PNMP20_1::pTiGc and PN18062_1::pTiGc had a similar average percentage of 0.2 ± 0.0%. These data suggest that upon acid stress: (i) laboratory strains seem to have a higher propensity to form VBNR populations than three clinical isolates examined, (ii) M. bovis may demonstrate VBNR populations following acid stress, although these are very small, (iii) VBNR formation may vary depending on strain genotype. However, it is important to acknowledge that the VBNR populations detected under the conditions employed in this study were very small (0.5 ± 0.6 %), and may not be indicative of significant persister formation. We have, therefore, not definitively demonstrated persister formation, but note that we only investigated a single stressor, a single time point, and only 3 clinical isolates, leaving this an open question to be further investigated in the future. This is the first study to use clinical strains coupled with fluorescent reporters to investigate the ability of M. bovis to persist and provides some insights into a long-standing question on latent M. bovis in animals and describes proof of concept data for future investigation. Future work will need to validate these findings using more complex in vitro and in vivo models and additional clinical isolates.
Characterization of tuberculous lesions in naturally infected African buffalo (Syncerus caffer)
(Stellenbosch : University of Stellenbosch, 2010-12) Laisse, Claudio Joao Mourao; Van Helden, David Paul; Gavier-Widen, Dolores; Agren, Erik O.; University of Stellenbosch. Faculty of Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.
ENGLISH ABSTRACT: Mycobacterium bovis has a wide host range and infects many wild and domestic animal species as well as humans. African buffalo (Syncerus caffer) is considered to be a wildlife reservoir of M. bovis in certain environments in South Africa, such as in the Kruger National Park (KNP) and Hluhluwe-iMfolozi Park (HiP). A detailed pathological study was conducted on 19 African buffalos (Syncerus caffer) from a herd in the HiP in South Africa. The animals tested positive to the intradermal bovine tuberculin test and were euthanazed during a test-and-cull operation to decrease the prevalence of bovine tuberculosis (bTB) in the park. The superficial, head, thoraxic and abdominal lymph nodes and the lungs were examined grossly for presence of tuberculous lesions and were scored on a 1-5 scale for macroscopic changes. The gross lesions were examined histologically and scored I-IV according to a grading system used for bTB lesions in domestic cattle. Macroscopical lesions were limited to the retropharyngeal, bronchial, and mediastinal lymph nodes and the lungs. The most frequently affected lymph nodes were the bronchial (16/19) and mediastinal (11/19). All four grades of microscopic lesions were observed, although grade II lesions were the most frequent. Acid-fast bacilli were observed only rarely. Bovine tuberculosis was confirmed by PCR analyses. All animals were in good body condition and most of the lesions were in an early stage of development, indicating an early stage of the disease. The absence of lesions in the mesenteric lymph nodes and the high frequency of lesions in respiratory tract associated lymph nodes suggest that the main route of M. bovis infection in African buffalo is inhalatory rather than alimentary. This study presents a systematic evaluation and semiquantification of the severity and stages of development of tuberculous lesions in buffalo. The results may contribute to i) the understanding of the pathogenesis of the disease, ii) the evaluation of experimental models of M. bovis infection in Syncerus caffer, and iii) the interpretation of pathological data from vaccination trials.
Characterizing the proteomes of selected members of the Mycobacterium tuberculosis complex
(Stellenbosch : Stellenbosch University, 2014-04) Botha, Louise; Warren, R. M.; Gey van Pittius, N. C.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences, Molecular Biology and Human Genetics.
ENGLISH ABSTRACT: Mycobacterium tuberculosis is a pathogenic organism that infects a third of the world’s population and causes approximately 2 million deaths per year. This pathogen is a member of the Mycobacterium tuberculosis complex (MTBC) which constitutes eleven members that share 99.9% similarity at nucleotide level and have near identical 16S rRNA. MTBC members cause Tuberculosis in a variety of host species. M. bovis and M. caprae form part of the animal-adapted MTBC members that cause disease in a variety of animal hosts (primarily bovidae) and goats, respectively. Extensive genetic analyses have been done to try and explain virulence, phenotype and host-preferences of these members with no success. Recent advances in mass spectrometry techniques enable us to analyse thousands of proteins simultaneously and explore the possible proteomic variation between these members that could contribute to the phenotypic, virulence and host-specificity characteristics of the MTBC members. In this study, we aimed to characterize the proteomes of M. bovis and M. caprae by analysing the high and or low abundance proteins, relative to M. tuberculosis H37Rv, which could possibly explain virulence mechanisms and host-specificity of these MTBC members. Whole cell lysate protein extracts were extracted from mid-log phase cultures of M. tuberculosis H37Rv (A600 = 0.7), M. bovis (A600 = 0.65) and M. caprae (A600 = 0.7). Proteins were fractionated by SDS-PAGE and in gel reduction/alkylation and trypsin digests were done. Peptides were identified using LC-MS/MS on the Orbitrap Velos mass spectrometer and their corresponding proteins were identified by searching peptide databases. Protein functional groups were assigned according to TubercuList. To provide an integrated overview of the overall network of protein expression (rather than just limit analysis to individual proteins), pathway analysis was done on the differentially expressed proteins of M. bovis and M. caprae using PATRIC (Pathosystems Resource Integration Center) and pathways were visualized using iTUBY (Interactive Pathway Explorer database). We detected 2199, 2367 and 2350 proteins for M. tuberculosis H37Rv, M. bovis and M. caprae which correlate to 60% of the proposed M. tuberculosis proteins being expressed during log-phase. Considering similarities between genomes, it was no surprise that the functional distribution of the detected proteins extracted was similar. Metabolic pathways affected by the proteins which were in higher abundance in M. bovis and M. caprae included amino acid and lipid metabolism, oxidative phosphorylation and xenobiotic degradation. The over-abundant proteins in M. bovis and M. caprae were also involved in ribosomal proteins and carbohydrate metabolism, respectively. Lower abundance proteins in these species were found in lipid and pyrimidine metabolism. These affected pathways can be associated with the ability of M. bovis and M. caprae to adapt to their environment more readily which helps them to survive inside the hosts and cause severe pathogenesis. In this study the proteomes of M. tuberculosis H37Rv, M. bovis and M. caprae were characterized and the variation between detected proteins and protein abundances explored in order to describe differences between these closely related strains. Future research on animaladapted Mycobacterial species will address knowledge gaps that are needed to prevent transmission and spread of the disease. Understanding the mechanisms of virulence and pathogenicity could lead to development of efficient vaccines and diagnostic tests for a variety of animal hosts.
Chemokine and cytokine measurements using Luminex and Selected Reaction Monitoring (SRM): comparing technologies
(Stellenbosch : Stellenbosch University, 2020-12) Nkonyane, Tshepiso Ruth; Chegou, Novel N.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences.
ENGLISH ABSTRACT: Background: There is an urgent need for tools for the rapid and accurate diagnosis of tuberculosis (TB) disease in resource-constrained settings. Tests based on host immunological biomarkers may be useful, especially if based on easily available samples. Objectives: To evaluate the usefulness of plasma proteins as biomarkers for diagnosing TB, to validate previously identified biosignatures [4-marker biosignature (CCL1+CRP+IP10+NCAM), 2-marker biosignature (CCL1+CRP) and 3-marker biosignatures; [(CCL1+NCAM+SAA), (CRP+IP-10+SAA), (CCL1+CRP+NCAM), (CCL1+CRP+SAP), (CRP+IP-10+MIG) and (CRP+SAP+MIG)] in a new cohort of adults suspected of having TB, and to develop a Selected Reaction Monitoring (SRM) for detecting TB protein biomarkers. Methods and Materials: We collected plasma samples from 151 individuals that presented with symptoms requiring investigation for TB disease at a primary health care clinic in the outskirts of Cape Town, South Africa, prior to assessment for TB disease. We evaluated the concentrations of 18 host markers in stored plasma samples using a multiplex platform. Using a combination of clinical, radiological and laboratory diagnosis, patients were later classified as having TB disease or other respiratory diseases (ORD). The diagnostic potential of individual analytes was analyzed using the receiver operating characteristic curve while the predictive abilities of a combination of analytes for TB disease were analyzed using the general discriminant analysis. Selected Reaction Monitoring (SRM) was used to develop an assay for the diagnosis of TB. Skyline software was used for the development of the SRM assay and analysis of the generated data. Results: Out of the 151 individuals that enrolled, 56 were pulmonary TB cases. Out of the 18 host markers that were evaluated in Chapter 3, there were significant differences in the levels of 9 host markers between patients with TB disease and those with ORDs. The concentrations of VEGF-A, IP-10, MMP-3, ferritin, SAA, CFH and CCL 1 were significantly higher in the TB cases than individuals with ORD, whereas NCAM and Apo A1 levels were significantly higher in the ORD group. When the diagnostic accuracies of individual host markers were investigated by ROC curve analysis, two markers (IP-10 and CCL 1) showed strong potential with AUC ≥0.85. When the data obtained from all study participants were fitted into GDA models regardless of HIV status, combinations between up to seven different host markers showed potential in the diagnosis of TB disease. However, an optimal diagnostic biosignature comprising of five proteins (transthyretin+MMP-3+IP-10+CFH+CCL1) diagnosed TB with an AUC of 0.90 (0.83- 0.97). Eight (8) previously identified biosignatures were assessed in study participants regardless of HIV status. A 4-marker biosignature (CCL1+CRP+IP-10+NCAM), 2-marker biosignature (CCL1+CRP) and 3-marker biosignatures [(CCL1+NCAM+SAA), (CRP+IP10+SAA), (CCL1+CRP+NCAM), (CCL1+CRP+SAP) and (CRP+IP-10+MIG)] all diagnosed TB with AUC ≥0.85, while one 3-marker signature (CRP+SAP+MIG) only diagnosed TB with AUC of 0.62. The most optimal diagnostic biosignature in all participants regardless of HIV status was the 4-marker (CCL1+CRP+IP-10+NCAM) signature which diagnosed TB with a sensitivity of 0.76(0.50-0.92) and specificity of 0.85(0.65-0.95). We further evaluated these biosignatures in HIV negative participants only and these biosignatures showed promise with areas under the ROC curves (AUC) ≥0.85 for all, except one 3-marker (CRP+SAP+MIG) biosignature. The most optimal diagnostic biosignature in HIV negative participants only was the 3-marker biosignature (CCL1+NCAM+SAA) that diagnosed TB with a sensitivity of 0.79(0.49-0.94) and specificity of 0.84(0.63-0.95). An SRM assay consisting of 83 proteins was developed, based on cytokines and chemokines that were previously investigated in diagnosing TB and reported in chapter 4. Conclusion We have shown that different candidate plasma biosignatures have potential in the diagnosis of TB disease. The observed results require further validation in a larger study. The previously identified biosignatures showed promise when validated in the current study. The biosignatures both identified and validated in this thesis hold promise as good candidate biomarkers/ biosignatures to be considered for the future development of point of-care TB tests. The developed SRM- based assay may be a useful alternative tool to diagnose TB. The assay requires further validation and optimization.
A comparative analysis of fungal agents with anti-glioblastoma properties
(Stellenbosch : Stellenbosch University, 2023-12) Lai, Daanyaal; Baatjies, Lucinda; Mavumengwana, Vuyo; Mabasa, Lawrence; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.
ENGLISH ABSTRACT: Cancer can be defined as a disease where cells within the body grow uncontrollably and may spread throughout the body. Globally cancers have been found to amass a high mortality rate, with central nervous system (CNS) tumors increasing substantially over the past 30 years more specifically within the elderly. Primary treatment regimens for CNS tumors include but are not limited to chemotherapy, radiation therapy and surgical resection. However, these treatments carry a lot of toxic side effects, thus highlighting the need to develop alternative treatment strategies. In the current study, fungal crude extracts were evaluated for their anti-glioblastoma (GBM) potential. The fungal crude extracts were derived from different fungal species which were obtained from crustose lichens, plants, and marine vertebrates such as ascidians. A total of 45 different fungi were grown with 15 fungi from each source undergoing sequencing to identify their genus and species. The fungi which were successfully identified via ITS and cultured for metabolite extraction. The metabolites were extracted by soaking the fungal mat in methanol and consequently concentrating using a rotary evaporator. Fungal endophytes Mucor janssenii (P13) and Diaporthe rudis (R3.10) which came from a lichen and a plant respectfully, showed the greatest cell growth inhibitory activity when screened against the GBM cancer cell line. Fungal crude extracts of Mucor janssenii (P13) showed inhibition of 55% at 250 µg/mL and Diaporthe rudis (R3.10) showed a 50% inhibition at 83 µg/mL against the U251 GBM cell line. These extracts showed no cytotoxicity against the Vero cell line, which was used as the normal control cell line. Nanoparticles (NPs) were synthesized, functionalized with crude extracts Mucor janssenii (P13) and Diaporthe rudis (R3.10) and their inhibitory effects against the GBM cell line assessed. U251 GBM cell line was treated with the functionalized NPs at concentration range of 100 µg/mL to 12.5 µg/mL. The functionalized NPs displayed inhibition only at 100 µg/mL with both Mucor janssenii (P13) and Diaporthe rudis (R3.10) functionalized NPs displaying inhibition of 60% and 65% respectfully. Untargeted metabolite profiling was conducted with both crude extracts P13 and R3.10 along with their “spent” fractions from NP functionalization. The present study has concluded and highlighted the importance of fungi as a reservoir of natural products.
Computational analysis of the immunogenicity and sequence diversity of Mycobacterium tuberculosis PPE_MPTR proteins
(Stellenbosch : Stellenbosch University, 2017-03) Colic, Antoinette; Sampson, Samantha; Christoffels, Alan; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics.
ENGLISH ABSTRACT: Mycobacterium tuberculosis presents a substantial health risk to humans, particularly in Africa. Prevention of infectious diseases via vaccination is the most effective strategy in decreasing prevalence; however the current BCG vaccine against tuberculosis has shown varying levels of efficacy. M. tuberculosis infection represents an on-going interaction between the host and the bacteria, of which we do not yet fully understand all the mechanisms contributing to the pathogenesis at a molecular level. A deeper understanding of host-pathogen interactions is an important step towards developing new and more effective vaccines and therefore combating the disease. Protective immunity against M. tuberculosis is induced by stimulating antigen specific T-cells, which recognise peptide antigens presented by HLA molecules on infected cells. Identifying epitopes that are capable of binding to HLA molecules and eliciting T-cell responses form part of the development of subunit vaccines. An area of mycobacterial biology that is poorly understood is the function of the PE/PPE proteins. These proteins are a large, genetically diverse family of immunogenic proteins that are predicted to play a role in modulating host immune responses. In particular, the PPE major polymorphic tandem repeat (PPE_MPTR) proteins are a subgroup of the PE/PPE proteins which are restricted to pathogenic mycobacterial species and represent one of the most genetically diverse set of proteins within the M. tuberculosis proteome. While many studies have investigated the presence of T-cell epitopes within the PE/PPE family of proteins, no studies have focused specifically on the PPE_MPTR subfamily. Based on the extreme variation in both the length and genetic diversity of the PPE_MPTR proteins, it has been speculated that they may represent a source of antigenic variation which allows the organism to escape antigen-specific host responses. Given the hyper-variable nature of the PPE_MPTR proteins and their possible role in host-pathogen interactions, genetic diversity within the PPE_MPTR proteins may differentially modulate human immune response. Furthermore, epitopes within the PPE_MPTR proteins may be possible subunit vaccine candidates for M. tuberculosis. Conventional experimental techniques used to identify potential epitopes can often be time consuming and expensive. Various computational tools exist to predict binding of peptide sequences to various HLA alleles. Using a collection of known M. tuberculosis epitopes from the Immune Epitope Database (IEDB), an evaluation of the current open source HLA class II prediction tools has been performed, with the results used to inform an in silico identification of human CD4+ T-cell epitopes within the PPE_MPTR proteins. Characterisation of the genetic diversity of these proteins is also an essential step in improving our understanding of this protein family. Publically available whole genome sequence data from strains belonging to various lineages has been used to investigate the level of sequence diversity within these ppe_mptr genes, and the impact of genetic variants on epitope density has been investigated. To date, this study is the most comprehensive analysis of the genetic variation of the ppe_mptr genes. Predicted epitopes have been filtered using a reverse vaccinology approach in order to identify possible subunit vaccine candidates for M. tuberculosis. Findings from epitope prediction analysis support the hypothesis of host-pathogen interactions for the PPE_MPTR proteins. Genetic variation results indicate that certain PPE_MPTR proteins are highly variable while others are relatively conserved across strains, and that genetically diverse regions are less likely to contain epitopes. Therefore no evidence to support antigenic variation was found. Areas of high and low epitope density are correlated to areas of non-repeat and repeat regions within the genome respectively, and therefore epitopes within the PPE_MPTR proteins are conserved non-repeating peptides. This is consistent with previous literature on the conservation of reported M. tuberculosis epitopes within clinical strains. Further studies are therefore needed to determine the role of the variable copy number of repeats found within the PPE_MPTR proteins. Possible vaccine candidates with high predicted population coverage in African countries within the PPE_MPTR proteins have been identified.

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