UC Irvine

Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis, can evade host immune response. Peroxidases, ferritins, and folate aldolases are antioxidant enzymes, protecting Mtb from oxidative damage by reactive oxygen species in activated macrophages. Dye-decolorizing peroxidases (DyPs) are encapsulated by a bacterial nanocompartment, encapsulin (Enc), where packaged DyP interacts with Enc via a unique C-terminal extension. Genes encoding for Enc exist in an operon with DyPs. Mtb harbors an encapsulin homolog (Mt-Enc) adjacent to the gene that encodes for Mt-DyP. Our studies indicate that Mt-Enc self-assembles into a 60-subunit icosahedral shell independently of cargo enzymes. Preliminary characterization suggests that Mt-DyP is a heme-peroxidase with a C-terminal extension important for interaction with Mt-Enc--elimination of the C-terminal extension abolishes the interaction.

Mtb iron-storage ferritin protein (Mt-BfrB) and 7,8-dihydroneopterin aldolase (Mt-FolB) have C-terminal tails that also interact with Mt-Enc. Mt-DyP and Mt-BfrB are predicted to be involved in oxidative stress response. Mt-FolB may mitigate oxidative attack, where Mt-FolB allows 7,8-dihydroneopterin to accumulate acting as an antioxidant. We observed encapsulation of Mt-DyP, Mt-BfrB, and Mt-FolB via co-purification and electron microscopy. Protein encapsulation has not been observed in vivo and its purpose is currently unknown, specifically encapsulation of enzymes involved in oxidative stress response. Functional studies of these three enzymes while encapsulated demonstrate that these enzymes retain enzymatic activity: Mt-DyP exhibits peroxidase activity with ABTS as a substrate, Mt-BfrB exhibits ferroxidase activity, and Mt-FolB exhibits aldolase activity; however, Mt-Enc alone does not appear to have enzymatic activity. This is the first study that illustrates mycobacterial encapsulation, providing structural and biochemical clues about mechanisms by which Mtb can detoxify the local environment to ensure long-term survival. We have also begun structural studies, probing Mt-Enc nanocompartment assembly in order to understand formation and utilize Mt-Enc as a therapeutic delivery platform.

In vivo analyses elucidating the role of Mt-Enc, and its involvement in survival during oxidative stress have started. Here, we aim to: i) characterize Mt-Enc; ii) preliminarily characterize DyP as a peroxidase; iii) begin structural analyses of both Mt-Enc and Mt-DyP; and iv) begin studies to determine the role of Mt-Enc and its implications in mycobacterial survival.