Research Projects

Cell-autonomous immunity consists of a set of evolutionarily conserved, robust self-defense strategies that metazoans employ to guard their cytosol from pathogenic bacterial invasion. We have recently established ubiquitin-VCP/p97 axis as a crucial cellular innate immune mechanism. p97 extracts surface bound ubiquitinated proteins to cause extensive damage to bacterial membrane, leading to bacterial killing. This newfound immune pathway efficiently tackles diverse pathogens, irrespective of their shape, size or origin. But, p97 mediated extensive membrane disintegration is possible if large number of surface proteins are extracted simultaneously, implying that p97 must form organized clusters on bacterial membrane. Indeed, we found that p97, following phosphorylation specifically by GSK3β, forms supramolecular assembly on the bacterial surface (preliminary data). The resultant extraction of multiple surface proteins overwhelms bacterial membrane repair machinery and cause widespread disruption of the membrane integrity, leading to pathogen clearance. However, intracellular pathogens, such as Shigella flexneri, must evade p97 mediated killing. Since p97 is not degraded during Shigella infection (preliminary data), we hypothesize that Shigella secreted effector, OspF, a unique phospho-threonine lyase, dephosphorylates p97, inhibiting it’s supramolecular assembly formation on Shigella surface. This may protect Shigella from p97 mediated killing and preserves it’s cytosolic niche. However, only occasionally Shigella leads to productive infections, implying that host possesses countermeasures to protect itself from Shigella infection. These may include preserving the active p97 pool and degradation of OspF. Promiscuity and kinetic properties of OspF suggests that it could rapidly and irreversibly inactivate numerous key proteins in the host cell. We speculate that host employs either ubiquitination or FAT10ylation (conjugation of FAT10 as degradative signal) which has 7-8-fold higher degradation kinetics compared to ubiquitination, to dispose OspF and protect itself from deleterious effects of OspF. We also hypothesize that ISGylation of p97 increases it’s stability and ability to form supramolecular assembly which is necessary for it’s bacteriolytic function. This may provide the host a route to preserve a critical immune executioner like p97. Using Shigella mutants, CRISPR based host gene knock-outs, immunoprecipitation, site-specific phospho-Antibody, TurboID based proximity biotinylation followed by mass spectrometry analysis, and in-vitro reconstitution assays with purified proteins, we aim to explore the mechanistic details of this post-translational modification mediated regulation of p97. This promises to uncover novel strategies that host uses for protection of its immune arsenal and their precise modulation which in-turn will allow us to engineer cell intrinsic immunity for therapeutic applications.