Symbiosome interactome profiling via proximity labelling in Anaeramoeba flamelloides

Sammanfattning

Anaeramoeba is a recently identified lineage of anaerobic amoebae within the Metamonada and characterized by an obligate association with intracellular sulphate-reducing bacteria belongingto the Desulfobacteraceae. These organisms possess mitochondrion-related organelles (MROs), which are evolutionarily derived from mitochondria and have been extensively modified for anaerobic metabolism, displaying a broad spectrum of functional states across eukaryotes. This study applies a horseradish peroxidase (HRP)-based wheat germ agglutinin (WGA) proximity labelling approach to characterize the molecular environment of the host–symbiont interface in A. flamelloides. This method exploits enzymatic generation of short-lived reactive intermediates to label proteins within a limited spatial range (~20 – 300 nm) from the HRP-tagged WGA bound to symbiont-associated glycoconjugates. Because this labelling radius is comparable to the physical scale of the symbiosome boundary, the resulting biotinylated proteins are expected to represent components of the host–symbiont interfacial domain. The approach was validated using fluorogenic HRP substrates and streptavidin-based Western blot detection of biotinylated proteins. The results show that labelling efficiency is highly sensitive to hydrogen peroxide exposure, with prolonged incubation resulting incomplete loss of signal, likely due to oxidative inactivation of HRP and/or disruption of WGA-binding sites. Although fluorescence microscopy revealed localized signal enrichment near symbiosome-associated regions, imaging was limited by low signal-to-noise ratio and background fluorescence. Overall, this study demonstrates that WGA–HRP proximity labelling provides a sensitive biochemical strategy to probe the host–symbiont interface in anaerobic eukaryotes and highlights the importance of tightly controlled oxidative conditions when applying peroxidase-based labelling in oxygen-sensitive systems.