title: The Host Response to a Clinical MDR Mycobacterial Strain Cultured in a Detergent-Free Environment: A Global Transcriptomics Approach.
abstract: 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.
authors.affiliation: SA MRC Centre for TB Research, DST-NRF Centre of Excellence for Biomedical Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Stellenbosch, South Africa.
authors.forename: Gina
authors.initials: G
authors.lastname: Leisching
authors.affiliation: SA MRC Centre for TB Research, DST-NRF Centre of Excellence for Biomedical Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Stellenbosch, South Africa.
authors.forename: Ray-Dean
authors.initials: RD
authors.lastname: Pietersen
authors.affiliation: SA MRC Centre for TB Research, DST-NRF Centre of Excellence for Biomedical Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Stellenbosch, South Africa.
authors.forename: Vuyiseka
authors.initials: V
authors.lastname: Mpongoshe
authors.affiliation: Central Analytical Facility (CAF), DNA sequencing unit, Stellenbosch University, Stellenbosch, South Africa.
authors.forename: Carel
authors.initials: C
authors.lastname: van Heerden
authors.affiliation: SA MRC Centre for TB Research, DST-NRF Centre of Excellence for Biomedical Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Stellenbosch, South Africa.
authors.forename: Paul
authors.initials: P
authors.lastname: van Helden
authors.affiliation: SA MRC Centre for TB Research, DST-NRF Centre of Excellence for Biomedical Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Stellenbosch, South Africa.
authors.forename: Ian
authors.initials: I
authors.lastname: Wiid
authors.affiliation: SA MRC Centre for TB Research, DST-NRF Centre of Excellence for Biomedical Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Stellenbosch, South Africa.
authors.forename: Bienyameen
authors.initials: B
authors.lastname: Baker
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D003470:Culture Media / Q000737:chemistry / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology*; D004195:Disease Models, Animal; D024901:Drug Resistance, Multiple, Bacterial / Q000187:drug effects; D020869:Gene Expression Profiling / Q000379:methods*; D005786:Gene Expression Regulation / Q000187:drug effects; D007113:Immunity, Innate / Q000187:drug effects; D008264:Macrophages / Q000276:immunology / Q000382:microbiology; D051379:Mice; D011136:Polysorbates / Q000494:pharmacology*; D017423:Sequence Analysis, RNA / Q000379:methods; D014376:Tuberculosis / Q000235:genetics* / Q000276:immunology / Q000382:microbiology
chemical_list: D003470:Culture Media; D003902:Detergents; D011136:Polysorbates