Biology subjects

Mhlanga, M. M.

Publications and source records attributed to Mhlanga, M. M..

3 recordsLinked to original sources

bioRxiv2018

CRISPRi-Seq for the Identification and Characterisation of Essential Mycobacterial Genes and Transcriptional Units

High-throughput essentiality screens have enabled genome-wide assessments of the genetic requirements for growth and survival of a variety of bacteria in different experimental models. The reliance in many of these studies on transposon (Tn)-based gene inactivation has, however, limited the ability to probe essential gene function or design targeted screens. We interrogated the potential of targeted, large-scale, pooled CRISPR interference (CRISPRi)-based screens to extend conventional Tn approaches in mycobacteria through the capacity for positionally regulable gene repression. Here, we report the utility of the \"CRISPRi-Seq\" method for targeted, pooled essentiality screening, confirming strong overlap with Tn-Seq datasets. In addition, we exploit this high-throughput approach to provide insight into CRISPRi functionality. By interrogating polar effects and combining image-based phenotyping with CRISPRi-mediated depletion of selected essential genes, we demonstrate that CRISPRi-Seq can functionally validate Transcriptional Units within operons. Together, these observations suggest the utility of CRISPRi-Seq to provide insights into (myco)bacterial gene regulation and expression on a genome-wide scale.

microbiology↗

bioRxiv2018

Transcribed enhancers in the macrophage immune response to Mycobacterium tuberculosis infection

BackgroundTuberculosis is a life-threatening infectious disease caused by Mycobacterium tuberculosis (M.tb). M.tb subverts host immune responses to build a favourable niche and survive inside of host macrophages. Macrophages can control or eliminate the infection, if appropriate transcriptional programs are activated. The role of transcriptional enhancers in the activation and maintenance of these programs remains unexplored.\n\nResultsWe analysed transcribed enhancers in M.tb-infected mouse bone marrow-derived macrophages. We established a link between known M.tb-responsive transcription factors and transcriptional activation of enhancers and their target genes. Our data suggest that enhancers might drive the macrophage response via transcriptional activation of key immune genes, such as Tnf, Tnfrsf1b, Irg1, Hilpda, Ccl3, and Ccl4. We report enhancers acquiring transcription de novo upon infection. Finally, we link highly transcriptionally induced enhancers to the activation of genes with previously unappreciated roles in M.tb infection, such as Fbxl3, Tapt1, Edn1, and Hivep1.\n\nConclusionsOur findings extend current knowledge of the regulation of macrophage responses to M.tb infection and provide a basis for future functional studies on enhancer-gene interactions in this process.

bioinformatics↗

bioRxiv2017

Viral apoptosis evasion via the MAPK pathway by use of a host long noncoding RNA

An emerging realisation of infectious disease is the high incidence of genetic instability resulting from pathogen-induced DNA lesions, often leading to classical hallmarks of cancer such as evasion of apoptosis. The Human Immunodeficiency Virus type 1 (HIV-1) induces apoptosis in CD4+ T cells but is largely non-cytopathic in macrophages, thereby leading to long-term dissemination of the pathogen specifically by these host cells. Apoptosis is triggered by double-strand breaks (DSBs), such as those induced by integrating retroviruses, and is coordinated by the p53-regulated long noncoding RNA lincRNA-p21, in a complex with its protein binding partners HuR and hnRNP-K. Here, we monitor the cellular response to infection to determine how HIV-1 induces DSBs in macrophages yet evades apoptosis in these cells. We show that the virus does so by securing the pro-survival MAP2K1/ERK2 cascade early upon entry, in a gp120-dependent manner, to orchestrate a complex dysregulation of lincRNA-p21. By sequestering HuR in the nucleus, HIV-1 enables lincRNA-p21 degradation. Simultaneously, the virus permits transcription of pro-survival genes by sequestering hnRNP-K in the cytoplasm via the MAP2K1/ERK2 pathway. Notably, this pro-survival cascade is unavailable for similar viral manipulation in CD4+ T cells. The introduction of MAP2K1, ERK2 or HDM2 inhibitors in HIV-infected macrophages results in apoptosis providing strong evidence that the viral-mediated apoptotic block can be released, specifically by restoring the nuclear interaction of lincRNA-p21 and hnRNP-K. These results reveal pathogenic control of apoptosis and DNA damage via a host long noncoding RNA, and present MAP2K1/ERK2 inhibitors as a novel therapeutic intervention strategy for HIV-1 infection in macrophages.

molecular biology↗