Doctoral Degrees (Molecular Biology and Human Genetics)

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Browsing Doctoral Degrees (Molecular Biology and Human Genetics) by Subject "Antigens"

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    The evaluation of novel biomarkers and antigens for the diagnosis of Mycobacterium bovis infection in African buffaloes (Syncerus caffer).
    (Stellenbosch : Stellenbosch University, 2016-12) Goosen, Wynand Johan; Miller, Michele; Parsons, S. D. C.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics.
    ENGLISH SUMMARY: Mycobacterium bovis (M. bovis) forms part of the Mycobacterium tuberculosis complex (MTC), a group of genetically related bacteria that causes tuberculosis in humans and animals. African buffaloes (Syncerus caffer) are maintenance hosts of M. bovis, the causative organism of bovine tuberculosis (bTB). Since this species acts as a bTB reservoir for a wide range of domestic and wildlife species, the detection of M. bovis-infected animals is essential to control spread of the disease. However, diagnostic assays used for bTB management and control programmes, such as the single intradermal comparative tuberculin test (SICTT) and commercially available interferon-gamma release assays (IGRAs), are still believed to be sub-optimal for the diagnosis of bTB in bovids. A potential approach to improving detection of M. bovis infection could be the use of novel diagnostic antigens or the identification of alternative or ancillary biomarkers to interferon-gamma (IFN-γ). The studies presented in this dissertation aim to identify, develop and evaluate novel approaches for improving the detection of M. bovis infection in African buffaloes. The first objective was to evaluate the performance of two new commercially available IGRAs, the Bovigam® PC-EC assay and the Bovigam® PC-HP assay, for the first time in buffaloes and compare their performance to that of two versions of an adapted human IGRA, the modified QuantiFERON® TB-Gold (mQFT) assay. In addition, the effect of increased blood incubation time on sensitivity of the mQFT assay was assessed along with whether centrifugation was a necessary step prior to harvesting the plasma fraction. Furthermore, the relative sensitivities of Bovigam® assays, a modified Bovigam assay that contains an additional stimulation criteria with Mycobacterium fortuitum tuberculin and the SICTT were compared in identified M. bovis-infected buffaloes. Combinations of these assays were evaluated to identify the optimal test algorithm (i.e., highest sensitivity) for detection of bTB in African buffaloes. Commercially available bovine ELISAs as well as a human IP-10 ELISA were used to identify selected candidate biomarkers of M. bovis-specific immune activation and evaluate their diagnostic utility. Lastly, this study investigated what effect storing stimulated whole blood plasma under various conditions would have on the diagnostic performance of IP-10 assays in African buffaloes. Agreement between the Bovigam® PC-EC and the Bovigam® PC-HP assays was high (κ = 0.86, 95% CI 0.75–0.97) and these detected the greatest number of test-positive animals suggesting that they were the most sensitive assays. Agreement between two versions of the mQFT assay was also high (κ=0.88, 95% CI 0.77-0.98); however, all buffaloes with discordant mQFT results (n=6), including 3 confirmed M. bovis-infected animals, were positive at 30 hours incubation and negative at 20 hours. These results suggest that the mQFT assay is more sensitive using the longer incubation period (i.e., 30 hours). There were no significant differences in IFN-γ concentrations in plasma samples harvested from QFT tubes prior to and after centrifugation, a step which may facilitate plasma sampling over multiple incubation times to improve sensitivity. When the test performance of IFN-γ assays was compared in buffaloes, the Bovigam PPD assay had a relative sensitivity between 91-93% while the sensitivity of the modified PPD assay was between 90-91%. Diagnostic sensitivity was improved by combining one or more IGRA together with the SICTT (95-100%). Investigation of alternative biomarkers to IFN-γ found that IP-10 levels were significantly increased in antigen-stimulated blood samples from M. bovis-infected buffaloes (p < 0.0001). In addition, IP-10 was produced in far greater abundance than IFN-γ, demonstrating its potential as a novel biomarker of bTB in buffaloes. Moreover, using IP-10, agreement between the mQFT assay and the Bovigam assays was increased while the excellent agreement between the Bovigam assays was retained. Since transport and storage of buffalo blood samples are important considerations for development of diagnostic tests for bTB, the effects of heat-inactivation and storage on protein saver cards (PSCs) on IP-10 performance were assessed. Incubation of plasma at 65 °C for 20 min caused no statistically significant loss of IP-10 and this protein could be quantified in plasma stored on PSCs for 2 and 8 weeks. Moreover, for all storage conditions, IP-10 retained its excellent diagnostic characteristics. Diagnostic tests for bTB in wildlife are limited by the lack of species-specific immunological reagents. Logistical constraints make validation of tests, using novel biomarkers such as IP-10, more difficult. These limitations may preclude definitive conclusions about the diagnostic utility of IP-10 in buffaloes. Some of the factors that may have influenced our conclusions include: 1) limited sample size which could affect calculation of appropriate IP-10 test cut-off values; 2) use of antigen-specific whole-blood incubation assay protocols which had been optimized for measurement of IFN-γ rather than IP-10, and 3) lack of post-mortem samples to confirm M. bovis infection status in Bovigam-negative IP-10-positive buffaloes. In conclusion, both the Bovigam® PC-EC assay and the Bovigam® PC-HP assay were shown to be more sensitive than either the SICTT or mQFT assay in its current format. Plasma collected from the QFT tubes prior to centrifugation could be reliably utilized in this assay. Moreover, increasing the blood incubation time from 20h to 30h increased the mQFT assay’s sensitivity. In an additional study, both the Bovigam PPD assay and modified PPD assay displayed greatest sensitivity for the detection of M. bovis-infected buffaloes. The SICTT detected additional IGRA-negative animals and maximum sensitivity was attained when these assays were used in combination. In addition to IFN-γ, IP-10 appears to be a useful marker of immune activation in buffaloes when using a commercially available IP-10 bovine ELISA. IP-10 shows promise as a diagnostic biomarker in M. bovis-infected buffaloes and measurement of IP-10 increased the sensitivity over conventional IGRAs. IP-10 could be measured in plasma stored on PSCs; however, the sensitivity of tests utilizing such samples was reduced with increased storage time. Plasma samples could be heated to 65 ºC for 20 min with no degradation of IP-10, demonstrating the thermal stability of this cytokine. These findings are supported by previous cattle studies that advocate the parallel use of the SICTT and the Bovigam PPD assay. Moreover, these findings also highlight the potential application of IP-10 for the diagnosis of bTB in African buffaloes. Improved sensitivity of M. bovis-specific IGRAs is a significant advantage of using IP-10 as a preferred biomarker in this species. Other advantages of IP-10 are its thermal tolerance and stability on PSCs. These characteristics facilitate movement of diagnostic samples by permitting heat-inactivation of potential pathogens in plasma, and transport of samples by conventional delivery methods, respectively.
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    The mycosins, a family of secreted subtilisin-like serine proteases associated with the immunologically-important ESAT-6 gene clusters of Mycobacterium tuberculosis
    (Stellenbosch : Stellenbosch University, 2002-12) Gey van Pittius, Nicolaas Claudius; Beyers, A. D.; Warren, R. M.; Van Helden, P. D.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.
    ENGLISH ABSTRACT: Pathogenic organisms frequently utilize proteases to perform specific functions related to virulence. There is little information regarding the role of proteolysis in Mycobacterium tuberculosis and no studies on the potential involvement of these enzymes in the pathogenesis of tuberculosis. The present study initially focused on the characterization of a family of membrane anchored, cell wall associated, subtilisin-like serine proteases (mycosins-1 to 5) of Mycobacterium tuberculosis. These proteases were shown to be constitutively expressed in M. tuberculosis, to be located in the cell wall of the organism and to be potentially shed (either actively or passively) from the wall. Relatively high levels of gamma interferon secretion by T-cells in response to these proteases were observed in Mantoux positive individuals. The absence of any detectable protease activity lead to a protein sequence analysis which indicated that the mycosins are probable mycobacterial-specific proprotein processing proteases. To identify possible substrates for these proteases, the genome sequence regions surrounding the mycosin genes were analyzed. This revealed that the mycosin genes are in fact part of a cluster of 6 to 12 genes which have been duplicated multiple times in the genome of M. tuberculosis. Due to the presence of members of the previously described ESAT-6 T-cell antigen family within this duplicated region, the five gene cluster regions were named the ESAT-6 loci. In silico analysis of finished and unfinished genome sequencing data revealed the presence of orthologues of the Mycobacterium tuberculosis H37Rv ESAT-6 loci in the genomes of other mycobacteria, e.g. M. tuberculosis CDC1551, M. tuberculosis 210, M. bovis, M. leprae, M. avium, and the avirulent strain M. smegmatis. Phylogenetic analyses done on the resulting sequences have established the duplication order of the gene clusters and demonstrated that gene cluster region 4 (Rv3444c-3450c) is ancestral. Region 4 is also the only region for which an orthologue could be found in the genomes of Corynebacterium diptheriae and Streptomyces coelicoior. Thus, the comparative genomic analyses revealed that the presence of the ESAT-6 gene cluster seems to be a unique characteristic shared by members of the high G+C gram-positive bacteria and that multiple duplications of this cluster have occurred and have been maintained only within the genomes of members of the genus Mycobacterium. The ESAT-6 gene cluster regions were shown to consist of the members of the ESAT-6 gene family (encoding secreted T-cell antigens that lack detectable secretion signals), the mycosins (secreted, cell wall-associated subtilisin-like serine proteases) as well as genes encoding putative ABC transporters, ATP-binding proteins, and other membrane-associated proteins. Thus, from the observation that members of the ESAT-6 family are secreted without the normal sec-dependent secretion signals, it was hypothesized that the membrane-associated and energy-providing proteins function together to form a transport system for the secretion of the members of the ESAT-6 protein family. Supporting this hypothesis, one of the ESAT-6 gene clusters was shown to be expressed as a single polycistronic RNA, forming an operon structure. The promoter for this operon, P e s r e g 3. was also identified and its activity characterized. Subsequent secretion analyses results have shown that secretion of members of the ESAT-6 protein family is dependent on the presence of the proteins encoded by the ESAT-6 gene cluster regions, confirming the putative transport-associated functions of the ESAT-6 gene cluster-encoded proteins. The mycobacterial ESAT-6 gene clusters contain a number of features of quorum sensing and lantibiotic operons, and an extensive review of the literature have led to the hypothesis that the members of the ESAT-6 family may be secreted as signaling molecules and may be involved in the regulation of expression of genes during intracellular residence of the bacterium. In the final part of this study, the evolutionary history of the PE and PPE gene families (members of which is found situated in the ESAT-6 gene clusters) were investigated. This investigation revealed that the expansion of these families are linked to the duplications of the ESAT-6 gene clusters, which is supported by the absence of the multiple copies of the PE and PPE families in the genome of the fast-growing mycobacterium M. smegmatis. Furthermore, dot blot analyses showed that the PPE gene present in ESAT-6 gene cluster region 5 is able to distinguish between mycobacteria belonging to the slow-growing or fast-growing species, indicating a function for the genes of these two families and/or the ESAT-6 gene clusters in the phenotypical differences distinguishing these two groups of mycobacteria. In conclusion, this study has highlighted numerous important aspects of mycobacterial genomics and has greatly contributed to the current body of knowledge concerning the role of proteases, gene duplication and mechanisms of antigen expression and secretion in M. tuberculosis.