Fig 6. Growth of S. pneumoniae under defined growth conditions in vitro.

To achieve defined growth conditions, THB medium was treated with Chelex 100 resin. (A-G) Pneumococci were not able to grow in depleted THB medium + 1% FCS (A-G, green line). (A-C) Depleted THB medium was supplemented with increasing concentrations of certain divalent cations. (A) Addition of MgCl₂ to depleted THB medium + 1% FCS restored growth of S. pneumoniae in a dose dependent manner. Treatment of depleted THB + 1% FCS with different concentrations of either MnSO₄ (B) or ZnCl₂ (C) alone was not able to retrieve pneumococcal growth in vitro. (D) Pneumococcal growth in medium supplemented with 200 μM Mg²⁺ + 50 μM Mn²⁺ or 200 μM Mg²⁺ + 50 μM Zn²⁺ was nearly identical to pneumococcal growth in normal THB + 1% FCS. Additionally, depleted THB + 1% FCS medium supplemented with either Mg²⁺ alone (E), Mg²⁺ + Mn²⁺ (F) or Mg²⁺ + Zn²⁺ (G) was further supplemented with recombinant S100A8/A9 (50 μg/ml). S. pneumoniae was then added and bacterial growth was monitored hourly over a time period of six hours. Untreated THB medium supplemented with 1% FCS served as positive control in all growth experiments (A-G). The Data are representative of two independently performed experiments.