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. Author manuscript; available in PMC: 2009 Mar 13.
Published in final edited form as: Cell Host Microbe. 2008 Mar 13;3(3):137–145. doi: 10.1016/j.chom.2008.02.003

Figure 5. D157070 Kills Nonreplicating Mycobacteria.

Figure 5

(A) The yellow compounds D155931 (25 μM) and D157070 (25 μM) partitioned from the medium into BCG over 24 hr at pH 6.6, as evidenced by the color of the bacteria collected by centrifugation as compared to BCG exposed to vehicle alone.

(B) Killing of BCG by D157070 was concentration dependent and synergistic with nitrite. BCG was treated at pH 5.5 (which prevents its replication) without (black symbols) or with 0.5 mM nitrite (white symbols) for 4 days. Nitrite alone at this concentration caused no killing. Dashed line denotes inoculum.

(C) D157070 killed BCG at pH 6.6 in the presence of 50 μM DETANO (white symbols), which generates sublethal NO, but not DETA (black symbols). Dashed line denotes inoculum. DETANO or DETA were added at 0, 24, 48, and 72 hr.

(D) D157070 killed Mtb synergistically with nitrite. Mtb was treated with D157070 at pH 5.5 (black circles), pH 5.5 + 0.5 mM nitrite (white circles), or at pH 6.6 (black squares) for 4 days. Inocula were 6.6 × 107 (pH 5.5) and 5.9 × 107 (pH 6.6) CFU/ml.

(E) D157070 killed mycobacteria in Dulbecc's modified Eagle's medium with 10% FBS (“complete medium”), which did not support mycobacterial replication. BCG (black symbols) or Mtb (white symbols) were inoculated at 8 × 105 (BCG) or 1.5 × 105 (Mtb) CFU/ml into complete medium and treated with D157070 for 4 days.

(F) D157070 killed hypoxic Mtb. Mtb was treated with rifampin (1 μg/ml), isoniazid (INH) (0.4 μg/ml), D157070 (12.5 μg/ml), or DMSO for 4 days after reaching stage 2 nonreplicative persistence (Wayne and Hayes, 1996). Results are means ± SD of triplicates in single experiments, each of which was repeated at least two times.

(G) D157070, but not isoniazid, killed hypoxic Mtb with butyrate instead of carbohydrate as a carbon source. Mtb was treated with D157070 (12.5 μg/ml) (white circles), isoniazid (0.1 μg/ml) (black circles), or DMSO (white squares) for 4 or 6 days in environmental chambers at 1% O2, pH 5.5 with 0.3 mM NO2. Results are mean ± SD of triplicates in single experiments, each of which was repeated at least two times.