Journal: The Journal of Biological Chemistry
Article Title: Mycobacterium tuberculosis EsxL inhibits MHC-II expression by promoting hypermethylation in class-II transactivator loci in macrophages
Figure Lengend Snippet: Genetic organization, growth analysis, bacterial survival, and Mtb Δ esxL mutant construction. A , schematic representation of e sxL in the M. tuberculosis H37Rv genome. RAW 264.7 ( B ) and THP-1 ( C ) were infected with M. smegmatis ( Msm ) pSMT3 and recombinant M. smegmatis esxL strains. The cells were lysed, and intracellular survival was determined 1, 8, and 24 h post-infection by a cfu assay. D , in vitro growth curve of the M. smegmatis WT, M. smegmatis pSMT3, and recombinant M. smegmatis esxL was determined by growing bacteria in 7H9 medium and measuring OD ( O.D 600 nm ) . E , extracellular expression of the esxL transcript was measured by qRT-PCR after growing M. smegmatis esxL in vitro for 4, 12, and 24 h. RNA was isolated at the respective time points. cDNA was synthesized, and the expression of esxL was determined using qRT-PCR. Transcript levels are represented relative to mRNA -fold change of M. smegmatis esxL at 4 h, which is assigned a value of 1. The expression values were normalized with sigA. F , intracellular expression of esxL transcript was measured by qRT-PCR. RNA was isolated from M. smegmatis esxL -infected macrophages at different time points. cDNA was synthesized, and the expression of esxL was determined using qRT PCR. Transcript levels are represented relative to mRNA level of M. smegmatis esxL at 4 h, which is assigned a value of 1. The expression values were normalized with sigA. G , schematic representation of construction of Mtb Δ esxL mutant by homologous recombination. The location of primers used for the confirmation of deletion mutant generation is depicted. H , confirmation of Mtb Δ esxL mutant generation. F1 and R2 primers were designed beyond the flanks, whereas R1 and F2 primers anneal to sacB-hyg r cassette. PCR using F1 and R1 is expected to give no product with the M. tuberculosis ( lane 1 ) and ∼1.3 kb with the Mtb Δ esxL ( lane 2 ); F2-R2 primer sets were expected to give no product with M. tuberculosis and ∼1.5 kb in Mtb Δ esxL mutant. Amplification of udgB with gene-specific primers was performed as a control. The experiments were performed in triplicate ( n = 3). Results are shown as mean ± S.D. ( error bars ). *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ns , not significant.
Article Snippet: Mycobacterium smegmatis mc2 155 was grown in Middlebrook's 7H9 broth medium (Difco) containing 0.05% Tween 80, 0.5% glucose, and 0.5% albumin at 37 °C on a shaker at 120 rpm.
Techniques: Mutagenesis, Infection, Recombinant, Colony-forming Unit Assay, In Vitro, Expressing, Quantitative RT-PCR, Isolation, Synthesized, Homologous Recombination, Polymerase Chain Reaction, Amplification