Browsing by Author "Sampson, Samantha Leigh"

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    Characterization of mechanisms leading to Mycobacterium tuberculosis strain diversity
    (Stellenbosch : Stellenbosch University, 1997) Sampson, Samantha Leigh; Stellenbosch University. Faculty of . Dept. of .
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    Molecular epidemiology of drug resistant Mycobacterium tuberculosis in Africa : a systematic review
    (BMC (part of Springer Nature), 2020) Chisompola, Namaunga Kasumu; Streicher, Elizabeth Maria; Muchemwa, Chishala Miriam Kapambwe; Warren, Robin Mark; Sampson, Samantha Leigh
    Background: The burden of drug resistant tuberculosis in Africa is largely driven by the emergence and spread of multidrug resistant (MDR) and extensively drug resistant (XDR) Mycobacterium tuberculosis strains. MDR-TB is defined as resistance to isoniazid and rifampicin, while XDR-TB is defined as MDR-TB with added resistance to any of the second line injectable drugs and any fluoroquinolone. The highest burden of drug resistant TB is seen in countries further experiencing an HIV epidemic. The molecular mechanisms of drug resistance as well as the evolution of drug resistant TB strains have been widely studied using various genotyping tools. The study aimed to analyse the drug resistant lineages in circulation and transmission dynamics of these lineages in Africa by describing outbreaks, nosocomial transmission and migration. Viewed as a whole, this can give a better insight into the transmission dynamics of drug resistant TB in Africa. Methods: A systematic review was performed on peer reviewed original research extracted from PubMed reporting on the lineages associated with drug resistant TB from African countries, and their association with outbreaks, nosocomial transmission and migration. The search terms “Tuberculosis AND drug resistance AND Africa AND (spoligotyping OR molecular epidemiology OR IS6110 OR MIRU OR DNA fingerprinting OR RFLP OR VNTR OR WGS)” were used to identify relevant articles reporting the molecular epidemiology of drug resistant TB in Africa. Results: Diverse genotypes are associated with drug resistant TB in Africa, with variations in strain predominance within the continent. Lineage 4 predominates across Africa demonstrating the ability of “modern strains” to adapt and spread easily. Most studies under review reported primary drug resistance as the predominant type of transmission. Drug resistant TB strains are associated with community and nosocomial outbreaks involving MDRand XDR-TB strains. The under-use of molecular epidemiological tools is of concern, resulting in gaps in knowledge of the transmission dynamics of drug resistant TB on the continent. Conclusions: Genetic diversity of M. tuberculosis strains has been demonstrated across Africa implying that diverse genotypes are driving the epidemiology of drug resistant TB across the continent.
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    Mycobacterium tuberculosis : genetic and phenotypic comparison
    (Stellenbosch : Stellenbosch University, 2002-03) Sampson, Samantha Leigh; Warren, R. M.; Van Helden, P. D.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Medicine.
    ENGLISH ABSTRACT: This study exploits the Mycobacterium tuberculosis H37Rv genome sequence data in the context of M. tuberculosis clinical isolates, to elucidate genetic variation, and examine the phenotypic and molecular epidemiological implications thereof. The study was initiated by investigation of the insertion sequence IS6110, the primary DNA fingerprinting probe for the molecular epidemiology of tuberculosis. The transposable element is present in variable copy number and chromosomal location in clinical isolates of M. tuberculosis strains, giving rise to extensive genetic diversity. At the inception of this study, little was known about this element in terms of the genetic identity of its surrounding regions, its chromosomal distribution, and the mechanisms contributing to genetic diversity. These shortcomings were therefore addressed by a number of approaches. Firstly, to establish their genetic identity and chromosomal distribution, IS6110 insertion sites from clinical isolates of M. tuberculosis were cloned and sequenced. This data was examined in conjunction with available genome sequence data. The results demonstrated that the majority of insertions occurred within coding regions. Furthermore, the element was shown to have a non-random chromosomal distribution, and a number of preferential integration sites were identified. Secondly, the stability of chromosomal domains flanking IS611 0 elements was investigated by utilizing the insertion site clones as hybridization probes against clinical isolates. This allowed the identification of extensive genetic variation associated with these chromosomal domains, arising from IS6110 transpositions, deletions and point mutations. These events were expressed in terms of a phylogenetic tree which demonstrated ongoing genome evolution associated with IS6110. Thirdly, to investigate the hypothesis that IS6110-mediated deletions occur via homologous recombination between adjacent elements, deletion junctions were mapped and sequenced in clinical isolates representing predecessor and descendant strains. While these results support the involvement of IS6110 as a mediator of genetic deletion, they suggest either alternative mechanisms or the existence of unidentified intermediates. The investigation of IS6110 flanking regions identified the disruption of a number of members of the PPE gene family, leading to the second main area of investigation. The PPE gene family was newly identified as a result of the M. tuberculosis genome sequencing project, and its products are speculated to be of antigenic importance. However, at the commencement of this study very little data was available regarding the biological role of PPE proteins. Therefore, to explore the phenotypic implications of PPE gene disruption, various aspects of the gene family were investigated. Firstly, phylogenetic relationships between members of the PPE family were elucidated, which suggested an evolutionary progression, and highlighted the possibility that there may be functional subdivisions within the gene family. Secondly, the extent and mechanisms of PPE gene variation were analyzed by a combination of hybridization, peR and sequence analysis. This approach revealed extensive variation associated the gene family, although different members of the family exhibit different levels of variation. Of special interest was the discovery that long tandem repeat regions (~69 bp) found within 3 members of the gene family demonstrate variation in the numbers of these tandem repeats. A third avenue of investigation focused on in vitro and in vivo PPE gene expression profiles. RT- , peR was utilized to demonstrate in vitro expression of PPE genes, while RNA:RNA in situ hybridization demonstrated the expression of PPE genes in human tissue samples. Intriguingly, in situ hybridization suggests that there is variable PPE gene expression within the human granuloma. The final approach reported here focused on the subcellular localization of one member of the PPE family, Rv1917c. A combination of cell fractionation and whole-cell antibody binding experiments suggest that the Rv 1917c protein is a cell wallassociated, surface exposed molecule. In summary, the results obtained have potential implications for the interpretation of molecular epidemiological data, support the role of IS6110 as an agent of genome evolution, and emphasize the potential for IS6110 to impact on strain phenotype. Investigation of the PPE family demonstrated that this gene family contributes to genetic variation, is expressed in vitro and in vivo and that at least one protein encoded by the gene family is cell wall associated. Together, the results obtained support the hypothesis that selected members of the PPE gene family may encode products involved in antigenic variation.
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    The plasmid-mediated evolution of the mycobacterial ESX (Type VII) secretion systems
    (BioMed Central, 2016-03) Newton-Foot, Mae; Warren, Robin Mark; Sampson, Samantha Leigh; Van Helden, Paul David; Gey van Pittius, Nicolaas Claudius
    BACKGROUND: The genome of Mycobacterium tuberculosis contains five copies of the ESX gene cluster, each encoding a dedicated protein secretion system. These ESX secretion systems have been defined as a novel Type VII secretion machinery, responsible for the secretion of proteins across the characteristic outer mycomembrane of the mycobacteria. Some of these secretion systems are involved in virulence and survival in M. tuberculosis; however they are also present in other non-pathogenic mycobacteria, and have been identified in some non-mycobacterial actinomycetes. Three components of the ESX gene cluster have also been found clustered in some gram positive monoderm organisms and are predicted to have preceded the ESX gene cluster. RESULTS: This study used in silico and phylogenetic analyses to describe the evolution of the ESX gene cluster from the WXG-FtsK cluster of monoderm bacteria to the five ESX clusters present in M. tuberculosis and other slow-growing mycobacteria. The ancestral gene cluster, ESX-4, was identified in several nonmycomembrane producing actinobacteria as well as the mycomembrane-containing Corynebacteriales in which the ESX cluster began to evolve and diversify. A novel ESX gene cluster, ESX-4EVOL, was identified in some non-mycobacterial actinomycetes and M. abscessus subsp. bolletii. ESX-4EVOL contains all of the conserved components of the ESX gene cluster and appears to be a precursor of the mycobacterial ESX duplications. Between two and seven ESX gene clusters were identified in each mycobacterial species, with ESX-2 and ESX-5 specifically associated with the slow growers. The order of ESX duplication in the mycobacteria is redefined as ESX-4, ESX-3, ESX-1 and then ESX-2 and ESX-5. Plasmid-encoded precursor ESX gene clusters were identified for each of the genomic ESX-3, -1, -2 and -5 gene clusters, suggesting a novel plasmid-mediated mechanism of ESX duplication and evolution. CONCLUSIONS: The influence of the various ESX gene clusters on vital biological and virulence-related functions has clearly influenced the diversification and success of the various mycobacterial species, and their evolution from the non-pathogenic fast-growing saprophytic to the slow-growing pathogenic organisms.
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    ProVision: A web based platform for rapid analysis of proteomics data processed by MaxQuant
    (2020-07) Gallant, James; Heunis, Tiaan; Sampson, Samantha Leigh; Bitter, Wilbert
    Proteomics is a powerful tool for protein expression analysis and is becoming more readily available to researchers through core facilities or specialised collaborations. However, one major bottleneck for routine implementation and accessibility of this technology to the wider scientific community is the complexity of data analysis. To this end, we have created ProVision, a free open-source web-based analytics platform that allows users to analyse data from two common proteomics relative quantification workflows, namely label-free and tandem mass tag-based experiments. Furthermore, ProVision allows the freedom to interface with the data analysis pipeline while maintaining a user-friendly environment and providing default parameters for fast statistical and exploratory data analysis. Finally, multiple customisable quality control, differential expression plots as well as enrichments and protein-protein interaction prediction can be generated online in one platform. Availability and implementation: Quick start and step-by-step tutorials as well as tutorial data is fully incorporated in the web application. This application is available online at https://provision.shinyapps.io/provision/ for free use. The source code is available at https://github.com/JamesGallant/ProVision under the GPL version 3.0 license.
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    Two promoters in the esx-3 gene cluster of Mycobacterium smegmatis respond inversely to different iron concentrations in vitro
    (Biomed Central, 2017-08) Fang, Zhuo; Newton-Foot, Mae; Sampson, Samantha Leigh; Gey Van Pittius, Nicolaas Claudius
    Background The ESX secretion system, also known as the Type VII secretion system, is mostly found in mycobacteria and plays important roles in nutrient acquisition and host pathogenicity. One of the five ESXs, ESX-3, is associated with mycobactin-mediated iron acquisition. Although the functions of some of the membrane-associated components of the ESX systems have been described, the role of by mycosin-3 remains elusive. The esx-3 gene cluster encoding ESX-3 in both Mycobacterium tuberculosis and Mycobacterium smegmatis has two promoters, suggesting the presence of two transcriptional units. Previous studies indicated that the two promoters only showed a difference in response under acid stress (pH 4.2). This study aimed to study the effect of a mycosin-3 deletion on the physiology of M. smegmatis and to assess the promoter activities in wildtype, mycosin-3 mutant and complementation strains. Results The gene mycP 3 was deleted from wildtype M. smegmatis via homologous recombination. The mycP 3 gene was complemented in the deletion mutant using each of the two intrinsic promoters from the M. smegmatis esx-3 gene cluster. The four strains were compared in term of bacterial growth and intracellular iron content. The two promoter activities were assessed under iron-rich, iron-deprived and iron-rescued conditions by assessing the mycP 3 expression level. Although the mycP 3 gene deletion did not significantly impact bacterial growth or intracellular iron levels in comparison to the wild-type and complemented strains, the two esx-3 promoters were shown to respond inversely to iron deprivation and iron rescue. Conclusion This finding correlates with the previously published data that the first promoter upstream of msmeg0615, is upregulated under low iron levels but downregulated under high iron levels. In addition, the second promoter, upstream of msmeg0620, behaves in an inverse fashion to the first promoter implying that the genes downstream may have additional roles when the iron levels are high.