Fig. 7.

Kupyaphores are secreted by Mtb for zinc acquisition (A) EIC of kupyaphore species II, V, and VI corresponding to m/z for [M+H]⁺ = 722.55, 834.68, and 848.69 detected from organic extracts of supernatant and cells of WT Mtb grown in Sauton’s medium. (B) Study design and growth kinetics of Δnrps supplemented with supernatant from WT Mtb cultures as compared to Δnrps grown in Sauton’s medium alone. Addition of WT Mtb supernatant rescued the growth defect observed in Δnrps. (C) Radioactive zinc-65 feeding of WT, Δnrps, and Δnrps:nrps Mtb strains. After 4 h, cells were lysed and spotted for estimating intracellular zinc-65 accumulation by autoradiography. (D) Quantitation of autoradiograph signals of three strains as mean pixel intensity in arbitrary units normalized to protein content estimated for each strain. (E) MS1 spectra showing doubly charged peaks corresponding to dimeric kupyaphore-zinc metabolites from WT Mtb extract supplemented with zinc. Putative zinc adducts for both homodimeric and heterodimeric kupyaphore species could be detected. Single charged ions are marked with red asterisks. The corresponding mass peaks were absent from WT Mtb supernatant extract, which was not supplemented with zinc. The calculated masses for metabolites are within 5 ppm mass error tolerance at MS1. Data represents mean ± SEM (n = 3 biological replicates).