Browsing by Author "Leisching, Gina"

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    The association of OASL and type I interferons in the pathogenesis and survival of intracellular replicating bacterial species
    (Frontiers Media, 2017) Leisching, Gina; Wiid, Ian; Baker, Bienyameen
    The type I IFN response quickly became associated with its role in the innate immune response to viral infection. The past few years have seen the significance of IFNs expand in breadth to include non-viral pathogens. Previous work has identified that following viral infection, type I IFN signaling induces the production of the 2′-5′-oligoadenylate synthetase (OAS) family, which include OAS1, OAS2, OAS3, and OAS-like (OASL) protein. OASL was identified to be strongly induced following viral infection through engaging the RNA sensor RIG-I and increasing signaling through this pathway to enhance the anti-viral type I IFN response. Surprisingly, infection with viral dsDNA revealed an IFN inhibitory role and therefore pro-viral function of OASL through the inhibition of the cGAS cytosolic DNA sensing mechanism. Intracellular bacteria are able to activate the cytosolic DNA sensing pathway, however the role of OASL during bacterial infection is largely unknown. Vacuolar pathogenic microbes such as mycobacteria induce OASL early post infection, where it functions in a prosurvival fashion by inhibiting autophagic mechanisms and antimicrobial peptide expression. This suggests an underestimated role of OASL in the innate immune response to infection with a variety of pathogens and points to OASL-associated modulation of the type I IFN response. OASL may therefore play a critical role in defining the outcome of infection. We provide a brief update on the recent developments of the OAS family of proteins in response to DNA and RNA virus infections, as well as discuss evidence of Oasl expression in response to a number of cytosolic and vacuolar replicating bacterial pathogens.
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    Bcl‑2 confers survival in cisplatin treated cervical cancer cells: circumventing cisplatin dose‑dependent toxicity and resistance
    (BioMed Central, 2015-10) Leisching, Gina; Loos, Benjamin; Botha, Matthys; Engelbrecht, Anna-Mart
    Background: Cisplatin is the main chemotherapeutic drug for the treatment of cervical cancers, however resistance to cisplatin is increasingly common and therefore has limited the efficacy and use of this drug in the clinic. Dosedependent toxicity poses an additional challenge since patients suffer long-term and often permanent side-effects after treatment. Bcl-2 up-regulation has been implicated in the resistance to cisplatin in a variety of cancer cell lines, however its role in cervical cancer is confounding. Methods: A low, non-cytotoxic concentration of cisplatin was used in the treatment of HeLa and CaSki cells. Bcl-2 expression was determined through Western blotting and immunocytochemistry before and after treatment with cisplatin. To assess the reliance of the cervical cancer cells on Bcl-2 in the presence of cisplatin, Bcl-2 knock-down was achieved through RNA interference, where after apoptosis was assessed through PARP cleavage (Western blotting), Caspase activity (Caspase-Glo©) and PI inclusion analysis (Flow cytometry). Finally, pre-malignant and malignant cervical tissue was analysed for the presence of Bcl-2 through Western blotting and immunofluorescence. Results: Cervical cancer cells upregulate Bcl-2 when treated with a non-cytotoxic concentration of cisplatin, which when silenced, effectively enhanced cisplatin sensitivity, and therefore significantly induced apoptosis. Analysis of the expression profile of Bcl-2 in cervical tissue revealed its up-regulation in cervical carcinoma, which agrees with results obtained from the in vitro data. Conclusions: Our data strongly suggest that utilising a lower dose of cisplatin is feasible when combined with Bcl-2 silencing as an adjuvant treatment, thereby improving both the dose-dependent toxicity, as well as cervical cancer resistance.
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    Distinct host-immune response toward species related intracellular mycobacterial killing : a transcriptomic study
    (Taylor & Francis, 2020) Madhvi, Abhilasha; Mishra, Hridesh; Chegoua, Novel N.; Tromp, Gerard; Van Heerden, Carel J.; Pietersen, R. D.; Leisching, Gina; Baker, Bienyameen
    The comparison of the host immune response when challenged with pathogenic and nonpatho- genic species of mycobacteria can provide answers to the unresolved question of how pathogens subvert or inhibit an effective response. We infected human monocyte derived macrophages (hMDMs) with different species of mycobacteria, in increasing order of pathogenicity, i.e. M. smegmatis, M. bovis BCG, and M. tuberculosis R179 that had been cultured in the absence of detergents. RNA was isolated post-infection and transcriptomic analysis using amplicons (Ampliseq) revealed 274 differentially expressed genes (DEGs) across three species, out of which we selected 19 DEGs for further validation. We used qRT-PCR to confirm the differential expression of 19 DEGs. We studied biological network through Ingenuity Pathway Analysis® (IPA) which revealed up-regulated pathways of the interferon and interleukin family related to the killing of M. smegmatis. Apart from interferon and interleukin family, we found one up-regulated (EIF2AK2) and two down-regulated (MT1A and TRIB3) genes as unique potential targets found by Ampliseq and qRT-PCR which may be involved in the intracellular mycobacterial killing. The roles of these genes have not previously been described in tuberculosis. Multiplex ELISA of culture supernatants showed increased host immune response toward M. smegmatis as compared to M. bovis BCG and M.tb R179. These results enhance our understanding of host immune response against M.tb infection.
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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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    OAS1, OAS2 and OAS3 restrict intracellular M. tb replication and enhance cytokine secretion
    (Elsevier, 2019) Leisching, Gina; Cole, Victoria; Ali, Aus T.; Baker, Bienyameen
    The 2′,5′ (OASs) are known as mediators of the antiviral response system through activation of the RNA cleavage pathway. Interestingly, we observe OAS1 , OAS2 and OAS3 upregulation in a number of gene expression signatures which discriminate active TB from latent TB infection, however their biological role during bacterial infection has not yet been elucidated. We observed that the expression of these genes was associated with pathogenicity and virulence of mycobacteria as infection with Mycobacterium bovis BCG failed to significantly induce OAS expression. Further, we observed that after silencing of these genes, M. tb CFU counts increased significantly 96 h post-infection in comparison to the respective controls. Luminex revealed that OAS silencing significantly decreased IL-1β, TNF-α and MCP-1 and had no effect of IL-10 secretion. We show for the first time that OAS1, 2 and 3 restrict intracellular pathogenic mycobacterial replication and enhance pro-inflammatory cytokine secretion.