Browsing by Author "Van Heerden, Carel"

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    The host response to a clinical MDR mycobacterial strain cultured in a detergent-free environment : a global transcriptomics approach
    (Public Library of Science, 2016) Leisching, Gina; Pietersen, Ray-Dean; Mpongoshe, Vuyiseka; Van Heerden, Carel; Van Helden, Paul; Wiid, Ian; Baker, Bienyameen
    During Mycobacterium tuberculosis (M.tb) infection, the initial interactions between the pathogen and the host cell determines internalization and innate immune response events. It is established that detergents such as Tween alter the mycobacterial cell wall and solubilize various lipids and proteins. The implication of this is significant since induced changes on the cell wall affect macrophage uptake and the immune response to M.tb. Importantly, during transmission between hosts, aerosolized M.tb enters the host in its native form, i.e. in a detergent-free environment, thus in vitro and in vivo studies should mimic this as closely as possible. To this end, we have optimized a procedure for growing and processing detergent-free M.tb and assessed the response of murine macrophages (BMDM) infected with multi drug-resistant M.tb (R179 Beijing 220 clinical isolate) using RNAseq. We compared the effects of the host response to M.tb cultured under standard laboratory conditions (Tween 80 containing medium -R179T), or in detergent-free medium (R179NT). RNAseq comparisons reveal 2651 differentially expressed genes in BMDMs infected with R179T M.tb vs. BMDMs infected with R179NT M.tb. A range of differentially expressed genes involved in BMDM receptor interaction with M.tb (Mrc1, Ifngr1, Tlr9, Fpr1 and Itgax) and pro-inflammatory cytokines/chemokines (Il6, Il1b, Tnf, Ccl5 and Cxcl14) were selected for analysis through qPCR. BMDMs infected with R179NT stimulate a robust inflammatory response. Interestingly, R179NT M.tb induce transcription of Fpr1, a receptor which detects bacterial formyl peptides and initiates a myriad of immune responses. Additionally we show that the host components Cxcl14, with an unknown role in M.tb infection, and Tlr9, an emerging role player, are only stimulated by infection with R179NT M.tb. Taken together, our results suggest that the host response differs significantly in response to Tween 80 cultured M.tb and should therefore not be used in infection experiments.
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    Rapid sequencing of the mycobacterium tuberculosis pncA gene for detection of pyrazinamide susceptibility
    (American Society for Microbiology, 2014) Streicher, Elizabeth M.; Maharaj, Kashmeel; York, Talita; Van Heerden, Carel; Barnard, Marinus; Diacon, Andreas; Mendel, Carl M.; Bosman, Marlein E.; Hepple, Juli A.; Pym, Alexander S.; Warren, Robin M.; Van Helden, Paul D.
    We developed a pyrazinamidase gene DNA-sequencing method to rapidly identify pyrazinamide resistance-causing mutations in GenoLyse-treated, smear-positive sputum specimens. The sensitivity and specificity were 90.9 and 100%, respectively, compared to those of MGIT drug susceptibility testing, after the exclusion of synonymous mutations and nonsynonymous mutations previously associated with susceptibility to pyrazinamide.
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    Transcriptomic changes associated with husk scald incidence on pomegranate fruit peel during cold storage
    (Elsevier, 2020-05-05) Belay, Zinash A.; Caleb, Oluwafemi J.; Vorster, Alvera; Van Heerden, Carel; Opara, Umezuruike Linus
    Pomegranate fruit is valued for its social, economic, aesthetic and health benefits. The fruit rapidly loses quality after harvest due to continued metabolic responses and physiological disorders under sub-optimal conditions. The incidence of physiological disorder such as husk scald manifests during storage and commercial shipping, which affects the appearance and limits marketability. Despite the importance of pomegranate husk scald, little information is available about the origin and molecular mechanisms. Therefore, the aim of this study was to investigate the scald incidence of pomegranate fruit at molecular level using RNA-Seq (Ion Proton™ Next Generation Sequencing) by analyzing peel transcriptomic changes. The RNA-seq analysis generated 98,441,278 raw reads. 652 Differentially Expressed Genes (DEGs) with a fold change of > |2|, a p value ≤ 0.05 and a false discovery rate (FDR) of <0.05 were identified between healthy and scald fruit peels. An analysis of the gene ontologies of these DEGs revealed the 432 genes were assigned with molecular functions, 272 as cellular components and 205 as part of biological processes. In this analysis, genes (Pgr023188 and Pgr025081) that encode uncharacterized protein and gene (Pgr007593) that encodes glycosyltransferase showed significantly highest fold changes. Genes (Pgr003448, Pgr006024 and Pgr023696) involved in various iron binding and oxidoreductase activities were significantly suppressed. This is the first transcriptome analysis of pomegranate fruit peel related to husk scald development. Results obtained from this study will add valuable information on husk scald related changes on pomegranate fruit at genomic level and provide insight on other related physiological disorders.