Journal: Scientific Reports

Article Title: Mycobacterial DNA-binding protein 1 is critical for BCG survival in stressful environments and simultaneously regulates gene expression

doi: 10.1038/s41598-023-40941-9

Figure Lengend Snippet: Survival of BCG MDP1 cKD in macrophages and in vivo ( A ) Intracellular survival of MDP1-cKD BCG compared to VC, over 7 days. After confirmation of MDP1 expression in VC and suppression in MDP1-cKD BCG, THP-1 cells were infected with indicated strains at a multiplicity of infection (MOI) of 1:1 for 4 hrs. The cultures were subsequently washed twice with warm serum-free DMEM to remove uninfected BCG and finally cultured in DMEM supplemented with 200ng/ml ATc every 48 hrs. The cells were incubated at 37 °C in a humidified 5% CO 2 incubator. BCG CFU was determined at indicated timepoints. Data represent mean ± SE from three biological replicates. Statistical differences between VC ATc and MDP1-cKD BCG were assessed by unpaired Welch’s T test, *P < 0.05 **P < 0.01 ***P < 0.001; # p < 0.05 indicate differences between MDP1_#1-cKD and MDP1_#2-cKD. ( B ) In vivo survival of indicated strains over 28 days. Female C57BL/6J mice (n = 2–8) were intraperitoneally infected with 5 × 10 6 CFU of indicated BCG strains, suspended in 200 µl PBS. MDP1 expression was maintained by supplementing mice drinking water with 20 µg/ml Doxycycline (Doxy). BCG CFU was quantified from harvested mice organ homogenates plated on 7H10/OADC agar at indicated time points. Data represent mean ± SE from 2 to 8 mice per group. Statistical differences were analyzed using Mann-Whitney U test, *P < 0.05

Article Snippet: At indicated time points 100 µl of each culture was harvested, and 10-fold serial dilutions were made with Saline Tyloxapol Buffer (STB) to disrupt bacteria clots before plating 10 µl on Middlebrook 7H10/OADC agar supplemented with appropriate antibiotics.

Techniques: In Vivo, Expressing, Infection, Cell Culture, Incubation, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Mycobacterial DNA-binding protein 1 is critical for BCG survival in stressful environments and simultaneously regulates gene expression

doi: 10.1038/s41598-023-40941-9

Figure Lengend Snippet: Growth kinetics of BCG MDP1 cKD ( A ) Position of hupB target sequence start site shown by purple vertical lines for MDP1_ #1 and MDP1 #2 cKD BCG. Numbering indicates first nucleotide on the target sequence relative to annotated start coding sequence. Arrow indicates position of transcription start site. BCG growth kinetics depicted by ( B ) optical density (OD 600 nm) and ( C ) CFU over 21 days. Bacteria were cultured in 7H9/ADC medium supplemented with 200ng/ml ATc every 48 hours to induce sgRNA expression. At indicated time points culture aliquots were harvested to determine the OD and enumerate CFU after culturing on 7H10/OADC agar. Data represent mean ± SE from three biological replicates. Statistical differences between VC ATc and MDP1-cKD BCG were assessed by unpaired Welch’s T test, *P<0.05 **P<0.01 ***P<0.001; # p<0.05 indicate differences between MDP1_#1-cKD and MDP1_#2-cKD. ( D ) Representative western blot images cropped from different gels delineated with black border lines confirming the expression of MDP1: InhA serves as a loading control. Full-length blot images in Supplementary Fig. . 1-VC ATc, 2-MDP1_#1-cKD, 3-MDP1_#2-cKD. Image is representative of three experiments.

Article Snippet: At indicated time points 100 µl of each culture was harvested, and 10-fold serial dilutions were made with Saline Tyloxapol Buffer (STB) to disrupt bacteria clots before plating 10 µl on Middlebrook 7H10/OADC agar supplemented with appropriate antibiotics.

Techniques: Sequencing, Bacteria, Cell Culture, Expressing, Western Blot