Figure 6. Δlmxgt1–3^s2 amastigotes are dependent on a functional TCA cycle and susceptible to nitric oxide.

A. Bone marrow derived macrophages infected (MOI 5:1) with either wild type or Δlmxgt1–3^s2 amastigotes, were treated with 0, 1 or 10 mg/ml sodium fluoroacetate (NaFAc) and intracellular survival, expressed as percent infected macrophage, determined after 1, 4 and 6 days (representative experiment; n=4, 100 macrophages counted per replicate, average +/− standard deviation).

B. Levels of reactive oxygen species (ROS) in wild type, Δlmxgt1–3 and Δlmxgt1–3^s2 promastigotes. Promastigotes were suspended in glucose-free RPMI supplemented with 10 % iFCS for 2.5 hours at 27 °C prior to the addition of 2.5 μM menadione (MEN) to increase mitochondrial metabolism or mitochondrial ROS production, respectively. Parasites were labeled with H2DCFDA (30 min, 27 °C) to detect ROS and fluorescence normalised to unlabeled parasites (representative experiment; n=3; average, +/− standard deviation; * p-value<0.07, ** p-value <0.05, *** p-value <0.005).

C. Wild type and Δlmxgt1–3^s2 Ama^axenic were suspended in glucose-free RPMI with 20% iFCS (pH 4.5) containing 0–2 mM sodium nitrite with or without glucose supplementation, and incubated at 35 °C overnight. Amastigote viability was determined after eFluor staining and FACS analysis and normalization to untreated controls.

D. Wild type and Δlmxgt1–3^s2 Ama^axenic were cultured in CDM containing 1 mM sodium nitrite and either ¹³C-U-glutamate or mixed ¹³C-U-free fatty acids. Parasite metabolism was quenched after 3 hours and the ¹³C-enrichment (presented as fractional labelling) of selected carbon cycle intermediates was determined by GC-MS.

E. Wild type and Δlmxgt1–3^s2 Ama^axenic were cultured in RPMI supplemented with 20 % iFCS (pH 4.5) containing varying concentrations of sodium nitrite (0 to 6 mM) for 2 hours. Parasites were labeled with H2DCFDA (30 min, 33–34 °C) to detect ROS and fluorescence normalized to unlabeled parasites (representative experiment; n=3; average, +/− standard deviation; * p-value<0.005, ** p-value <0.0005).

F. Bone marrow derived macrophages infected (MOI 5:1) with either wild type, Δlmxgt1-² or Δlmxgt1–3^s2 promastigotes, were stimulated with lipopolysaccharide (LPS 20 ng/ml) or LPS and interferon gamma (LPS/IFNγ, 20 and 10 ng/ml, respectively) and intracellular survival (expressed as percent infected macrophage) determined after 4 and 7 days (representative experiment; n=3, 100 macrophages counted per replicate; average, +/− standard deviation).