Functional characterization of the Shigella spp. effector OspB

Abstract

Shigella flexneri is an intracellular bacterial pathogen that uses a Type 3 Secretion System (T3SS) to deliver effector proteins into host epithelial cells, enabling invasion, replication, and intercellular spread. Among these effectors, OspB is a conserved but poorly understood protein previously linked to host cell proliferation. In this work, we sought to fill this gap, defining OspB as a cysteine protease, characterizing its substrate recognition motif, and investigating its function during infection. Transcriptomic profiling reveals that OspB alters host gene expression during infection, but not during ectopic expression, suggesting that its activity is dependent on infection-specific conditions. We found that during S. flexneri infection, OspB upregulates pathways related to vesicle trafficking, chromatin remodeling, and cell proliferation, highlighting its potential to reshape the host cellular environment. Using a combination of motif prediction and N-terminomics mass spectrometry, we identify CHMP2B, a core component of the ESCRT-III membrane repair complex, as a substrate of OspB. We show using functional assays that OspB cleaves CHMP2B in transfected cells and enhances membrane repair during infection. Moreover, ESCRT-III components, including CHMP2B and VPS4, localize to actin tails at sites of Shigella cell-to-cell spread, suggesting that this machinery plays an active role in resolving membrane stress during bacterial dissemination. Taken together, this work establishes host membrane repair – specifically ESCRT-mediated repair – as an important and previously unappreciated target of Shigella manipulation during epithelial cell infection. OspB enhances this process through its protease activity, revealing a novel consequence of OspB during Shigella infection.