Identification of type VI secretion system effector-immunity pairs using structural bioinformatics.

The type VI secretion system (T6SS) is an important mediator of microbe-microbe and microbe-host interactions. Gram-negative bacteria use the T6SS to inject T6SS effectors (T6Es), which are usually proteins with toxic activity, into neighboring cells. Antibacterial effectors have cognate immunity proteins that neutralize self-intoxication. Here, we applied novel structural bioinformatic tools to perform systematic discovery and functional annotation of T6Es and their cognate immunity proteins from a dataset of 17,920 T6SS-encoding bacterial genomes. Using structural clustering, we identified 517 putative T6E families, outperforming sequence-based clustering. We developed a logistic regression model to reliably quantify protein-protein interaction of new T6E-immunity pairs, yielding candidate immunity proteins for 231 out of the 517 T6E families. We used sensitive structure-based annotation which yielded functional annotations for 51% of the T6E families, again outperforming sequence-based annotation. Next, we validated four novel T6E-immunity pairs using basic experiments in E. coli. In particular, we showed that the Pfam domain DUF3289 is a homolog of Colicin M and that DUF943 acts as its cognate immunity protein. Furthermore, we discovered a novel T6E that is a structural homolog of SleB, a lytic transglycosylase, and identified a specific glutamate that acts as its putative catalytic residue. Overall, this study applies novel structural bioinformatic tools to T6E-immunity pair discovery, and provides an extensive database of annotated T6E-immunity pairs.

The biophysical property of the limbal niche maintains stemness through YAP.

The cell fate decisions of stem cells (SCs) largely depend on signals from their microenvironment (niche). However, very little is known about how biochemical niche cues control cell behavior in vivo. To address this question, we focused on the corneal epithelial SC model in which the SC niche, known as the limbus, is spatially segregated from the differentiation compartment. We report that the unique biomechanical property of the limbus supports the nuclear localization and function of Yes-associated protein (YAP), a putative mediator of the mechanotransduction pathway. Perturbation of tissue stiffness or YAP activity affects SC function as well as tissue integrity under homeostasis and significantly inhibited the regeneration of the SC population following SC depletion. In vitro experiments revealed that substrates with the rigidity of the corneal differentiation compartment inhibit nuclear YAP localization and induce differentiation, a mechanism that is mediated by the TGFß-SMAD2/3 pathway. Taken together, these results indicate that SC sense biomechanical niche signals and that manipulation of mechano-sensory machinery or its downstream biochemical output may bear fruits in SC expansion for regenerative therapy.