Symmetric division of Gram-negative bacteria depends on the combined action of three proteins that ensure correct positioning of the cell division septum, namely, MinC, MinD, and MinE. To achieve this function, MinC and MinD form a membrane-bound complex that blocks cell division at all potential sites. Opposing this inhibition is MinE, which interacts with MinD via its N-terminal anti-MinCD domain to site-specifically counter the action of the MinCD complex. The anti-MinCD domain has been proposed to bind MinD in a helical conformation; however, little is actually known about the structure of this functionally critical region. To understand how MinE can perform its anti-MinCD function, we have therefore investigated the conformation of the full-length MinE from Neisseria gonorrhoeae by solution NMR. Although solubility considerations required the use of sample conditions that limit the observation of amide resonances to regions that are protected from solvent exchange, backbone chemical shifts from both N- and C-terminal domains could be assigned. In contrast to previous models, secondary chemical shift analysis of these solvent-protected regions shows that parts of the N-terminal anti-MinCD domain are stably folded with many functionally important residues localizing to a beta-structure. In addition, this N-terminal domain may be interacting with the C-terminal topological specificity domain, since mutations made in one domain led to NMR spectral changes in both domains. The nonfunctional MinE mutant L22D showed even larger evidence of structural perturbations in both domains, with significant destabilization of the entire MinE structure. Overall, these results suggest that there is an intimate structural association between the anti-MinCD and topological specificity domains, allowing the functional properties of the two domains to be modulated through this interaction.
Objective Immune cell recruitment into tissues is an essential step in inflammatory responses. This occurs in a highly tissue- and stimulus-specific manner, which presents a significant challenge to modeling disease and testing therapeutics ex vivo. We previously developed an advanced primary human vascularized Colon Intestine-Chip model and showed that it recapitulates physiologic cell composition, morphology and barrier function. The goal of this study was to test the ability of this system to model inflammatory bowel disease (IBD)-like immune cell reactions ex vivo. Methods We perfused primary human peripheral blood mononuclear cells (PBMC) across the vascular channel in untreated ‘resting’ or TNFα/chemokine-treated ‘inflamed’ Colon Intestine-Chips. We analyzed total cell recruitment, inflammatory cytokine secretion and barrier function in the following 24–72 hours. Results We show that the perfused PBMC efficiently adhered and transmigrated to the epithelial channel in an inflammation-specific manner. This was followed by an accumulation of proinflammatory cytokines characteristic of IBD (e.g., INFγ, IL1β, IL18), as well as loss of barrier function, the hallmark feature of IBD. We further showed that 1) the recruited cells were strongly enriched in the ‘gut trophic’ α4β7+/CCR9+ PBMC subsets and 2) this recruitment could be blocked with the IBD therapeutic Entyvio, which targets the α4β7-MAdCAM-1 interaction. Conclusion Our findings indicate that our Colon Intestine-Chip can model inflammatory immune cell recruitment and in situ immune reactions that reflect key clinical correlates of IBD. This model may prove effective for development of new anti-inflammatory therapeutics for human intestinal diseases. Supported by Emulate Bio
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