Infection metallomics of pulmonary mucormycosis: multi-omics study of siderophore secretion, neutrophil recruitment, and lipid remodeling in rat lungs.

Invasive pulmonary mucormycosis caused by Rhizopus microsporus is often fatal, yet analytical tools to monitor fungal burden and host responses in vivo remain limited. We developed a multimodal infection metallomics workflow combining ⁶⁸Ga-desferrioxamine B PET/CT, high-resolution MALDI mass spectrometry imaging (MALDI-MSI), and targeted LC-MS to map fungal siderophore production and host tissue remodeling in a neutropenic rat model and in human samples. ⁶⁸Ga-desferrioxamine B was rapidly taken up by R. microsporus in vitro and accumulated in infected lungs in vivo. MALDI-MSI visualized rhizoferrin (m/z 435.1259, [M-H]⁻) and homorhizoferrin (m/z 449.1420, [M-H]⁻) confined to hyphal foci and absent from control lungs, whereas heme b was depleted and spatially segregated. Neutrophil α-defensins (RatNP-2/3/4) increased 14-60-fold and formed halos around lesions, showing an inverse trend with siderophore abundance. Lipid MSI revealed remodeling of anionic surfactant lipids, with depletion of short-chain phosphatidylglycerols and phosphatidic acid-derived species and accumulation of long-chain polyunsaturated phosphatidylglycerols and phosphatidylinositols in infected regions. Targeted LC-MS of serial urine showed that rhizoferrin and homorhizoferrin emerged by day 2, peaked on day 4 (37.2 and 15.0 µg/mL), and declined with immune reconstitution; rhizoferrin was also detected in bronchoalveolar lavage from a patient with mucormycosis. Analytically, the workflow links radiotracer uptake, high-mass-accuracy spatial ion mapping of peptides with lipids, and matrix-matched urinary LC-MS quantitation within the same infection model. This integrated platform enables spatially resolved mechanistic readouts and supports non-invasive metallophore-based diagnostics of invasive mucormycosis.