Cultures were inoculated to an initial OD600 of 0.02 to 0.03 and allowed to grow for two weeks. Three cultures per strain were inoculated. Growth of cultures was determined by measurement of OD600 of cultures and also by quantification

of ATP with the luminescence-based Kit BacTiter-GloTM Microbial Cell Viability Assay (Promega). The luminescence was recorded as relative light units (RLU) with the microplate luminometer LB96V (EG & G Berthold). check details Mutants showing differences of growth pattern compared to the WT in both neutral medium and under pH stress conditions were considered for further molecular characterisation. Congo Red plating 100 μl of 1:105 and 1:106 dilutions in sterile water of mutants, complemented strain and WT were spread in triplicate on MB agar plates supplemented with OADC and 100 μg ml-1 Congo Red. Plates were incubated for 2–3 weeks and observed for colony morphology. Mutants showing differences in colony morphology (white vs. red staining, transparent vs. PI3K inhibitor opaque colonies, smooth vs. rough colonies) compared to the WT were considered for further molecular characterisation. Induction

of cytokine expression in THP-1 cells Infection of the cell line THP-1 was performed in 24-well cell culture plates (TPP) with three to five wells per sample. A total of 200,000 cells per well of THP-1 were selleck compound grown along with addition of phorbol-12-myristate-13-acetate (PMA, Sigma, Taufkirchen, Germany) (10 ng ml-1) and allowed to adhere to the surface of the plate well overnight at 37°C and in 5% CO2. Cells were then infected with mutants and WT at a multiplicity of infection (MOI) of 50 colony forming units (CFU). The supernatants were removed after 24 h and cytokines were quantified in appropriate dilutions of the supernatants by ELISA using the Human ELISA Ready to go Kits (Natutec, Frankfurt, Germany). Intracellular survival in THP-1 cells THP-1 cells were seeded, treated with PMA and infected as described above. The supernatants were removed after 4 h infection period and adherent cells were washed twice with RPMI 1640. The cells were then

treated with 200 μg ml-1 of Amikacin (Sigma) for 2 h to kill the mycobacteria in the supernatant. After washing twice with PBS buffer (10 mM sodium phosphate, 126 mM sodium chloride, pH 7.2), 1 ml of medium Demeclocycline supplemented with 5 μg ml-1 of Amikacin was added to each well. Samples for quantification of intracellular bacteria were taken at the end of the infection time after removal and killing of extracellular bacteria and then after 1, 2, and 4 days. For this, the cells were lysed in 1 ml of water at 37°C for 20 min and the mycobacterial DNA in the lysates was quantified by real-time PCR as described in Lewin et al.[41]. Additionally, 100 μl of 1:103 dilution in sterile water of samples were plated in triplicate on agar plates supplemented with ADC for counting of CFU.