Fig. 5: Host fumarate hydratase and S. aureus FumC both regulate fumarate and pathogenesis.

A Relative level of fumarate (left panel) and succinate (right panel) in the BALF of uninfected BL/6 and Ptenl^−/− mice. Fumarate levels differed between BL/6 and Ptenl^−/− mice, *p = 0.0190. B Bacterial burden from the BALF (left panel) and lung (right panel) of BL/6 and Ptenl^−/− mice infected with WT LAC, the ΔfumC mutant and complemented strain; BL/6 infected with WT LAC (n = 11), ΔfumC mutant (n = 13), and ΔfumC::fumC (n = 7), Ptenl^−/− infected with WT LAC (n = 6), ΔfumC mutant (n = 5). The above-mentioned total number of mice per group were from at least 2 independent experiments. The dotted line indicates the limit of detection. The bacterial load of WT LAC and the ΔfumC mutant differed significantly in the BALF and lung of BL/6 mice (p = 0.0009 and 0.0338, respectively) and Ptenl^-/- mice (p = 0.0152 and 0.0173, respectively). Similarly, the burden of the ΔfumC mutant and complemented strain differed significantly in the BALF and lung of BL/6 mice (p = 0.0009 and <0.0001, respectively). WT LAC burden also differed between BL/6 and Ptenl^−/− mice in the BALF (p = 0.0127) and lung (p = 0.0046). Innate immune cells (monocytes left panel, neutrophils middle panel and alveolar macrophages right panel) from the C BALF (*p = 0.0357) and D lungs of uninfected and infected mice from (A). E Cytokine measurements from the BALF of uninfected and infected mice from (A). Statistically significant differences were observed between groups in IL-1β (*p = 0.0472, **p = 0.0087), MIP2 (**p = 0.0087), IL-6 (*p = 0.0062), KC (*p = 0.0140, **p = 0.0016), M-CSF (*p = 0.0140), MIG (*p = 0.0062) and IP-10 (*p = 0.0350). F Expression of murine Fh1, Ass1 and Hk2 from uninfected and infected mouse lungs (n = 5 per condition) by qRT-PCR (left panel) and schematic diagram showing the metabolic reactions catalyzed by the encoded enzymes fumarate hydratase, argininosuccinate synthase and hexokinase (right panel). A statistically significant difference in Hk2 expression was observed in the lungs of uninfected (PBS) and ΔfumC-infected mice (p = 0.0480). G Absolute level of malate in the BALF of uninfected (PBS) BL/6 mice and mice infected with WT LAC or the ΔfumC mutant. H Schematic diagram of reaction catalyzed by FumC. I FumC activity assessed by the absorbance of fumarate (left panel). Fumarate was supplied as a substrate for FumC, preincubated with and without 1 mM itaconate (chemical structure, right panel). For (A–G, I), data represent mean values ± SEM and statistical analyses were performed by Mann–Whitney non-parametric U test (two-tailed), *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.