Fig 1.
Schematic representation of the long-term intracellular M. tuberculosis culture protocol.
For each infection cycle, a frozen stock of THP-1 cells was thawed and cells were cultured for 4 passages. Cells were differentiated by PMA stimulation and infected with M. tuberculosis. After 18 h of infection, medium was replaced, and infected cells were incubated for 6 days. At day 7, THP-1 cells were lysed to release the intracellular bacteria, which were diluted in cell culture medium and subjected to clump disaggregation as described in Methods. Approximately 1/10 to 1/20 of this bacterial suspension was used to infect a new THP-1 cell culture. A total of 10 serial infection cycles were performed, and bacterial CFU and THP-1 cells were counted at 18 h and 7 days of infection for each cycle. An aliquot of the initial bacterial inoculum and of the intracellular bacteria collected at the end of the tenth cycle were diluted and spread onto agar plates for single-colony isolation and whole genome sequencing.
Fig 2.
Parameters of the continuous intracellular culture model.
THP-1 cells were infected with M. tuberculosis H37RvSiena strain for 10 serial infection cycles. The graphs show the parameters evaluated for each cycle. (A) Bacterial CFU count. Bacterial CFU at day 1 and day 7 of infection. (B) Intracellular generations. Number of bacterial generations per cycle, calculated by using the formula (log2 CFU day 7- log2 CFU day 1)/log2 2. (C) Count of infected THP-1 cells. Number of total, viable adherent THP-1 cells (black bar), and dead cells (white bar) at day 7 of infection.
Table 1.
Mutations identified in M. tuberculosis genome and their frequency.
Fig 3.
Growth curves of wild type, cut3 Val223Ala and plcA His39Arg mutant strains in liquid medium, THP-1 cells and murine lung.
(A and C) Growth curves in liquid medium. Growth curves in liquid medium over 16 days of incubation for cut3 Val223Ala mutant (A) and plcA His39Arg mutant (C) strains. (B and D) Growth curves in THP-1 cells. Growth curves of cut3 Val223Ala mutant (B) and plcA His39Arg mutant (D) strains in THP-1 cells over 6 days of infection. Data are expressed as CFU increase relative to day 0. (E) Growth curves in murine lung. Growth curves of wild type, cut3 Val223Ala mutant, and plcA His39Arg mutant strains in the murine lung up to 12 weeks post-infection. Panels A-D show the mean and standard deviation of results obtained from three replicates; panel E shows the mean and standard deviation of results obtained from 3–4 animals per time point.
Fig 4.
Morphometric analysis of mouse lungs infected with wild type and cut3 Val223Ala mutant strains at 12 weeks of infection.
(A) Representative image of mouse lung sections. The image represents 4× magnification of lung sections stained with hematoxylin and eosin. Arrows indicate granulomatous lesions. Scale = 1mm. (B-C) Proportion of involved lung and granuloma size. The graphs show the proportion of lung parenchyma occupied by granulomas (B) and the size of granulomas (C). Results represent the mean and standard deviation of data collected from 3 mice per infecting bacterial strain.
Fig 5.
Phospholipase C activity testing.
Time-course hydrolysis of phosphatidylcholine (PC) with 100μg of wild type (blue circles) and His39Arg mutant (red circles) recombinant PlcA. The release of phosphocholine was measured indirectly by the fluorescence measurement of resorufin (AU) released using the Amplex red phosphatidylcholine kit, and continuously monitored at λexc of 510 nm and λem of 590 nm. Blue and red curves represent the mean of three independent experiments (coefficient of variation was 0.0195 and 0.0293, respectively).