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Dissecting the Synergistic Role of Unfolded Protein Response in Wound Infections
Stella Lee Yue Ting1, Cenk Celik1, Aaron Tan Ming Zhi1,2, Mark Veleba2, Kimberly Kline2,3& Guillaume Thibault1,4,5
1School of Biological Sciences, 2Singapore Center for Life Sciences Engineering, Nanyang Technological University, Singapore
3Department of Microbiology and Molecular Medicine, Faculty of Medicine, University of Geneva, Genève, Switzerland
4Mechanobiology Institute, National University of Singapore & 5Institute of Molecular and Cell Biology, A*STAR, Singapore
Background: Infected wounds affect 7-15% of hospitalized patients and significantly increase the duration of hospitalization and treatment cost per day. Various bacterial species, including biofilm-forming Enterococcus faecalis, have been isolated from wounds associated with diabetic foot ulcers, burns, and surgical sites. Therapeutic efforts have been concentrated on addressing microbial factors. However, this is inadequate in the presence of biofilm-forming microorganisms which renders antimicrobials sub-effective. The arrest in the inflammatory phase during wound healing, due to the incomplete elimination of pathogens, has been underpinning the basis for wound chronicity. Recent evidence from us and others has shown that the mammalian unfolded protein response (UPR) promotes the development of bacterial skin and soft tissue infection and impairs wound healing.
Aims: We want to identify the synergistic nodes between host and pathogen interaction in delayed wound healing. To understand this, we aim to establish the role of the UPR in wound healing by attenuating or hyperactivating the UPR in cell culture and mice and examine the global transcriptomic changes in infected wound healing. We also aim to elucidate the mechanism of E. faecalis-induced UPR by identifying bacterial genes that contribute to host UPR activation.
Methods: We employ single-cell RNAseq to observe transcriptomic changes during infected wound healing in C57BL/6 mice. To validate what we observed in mice, we established an in vitro infection assay to monitor and manipulate UPR to observe cell viability and migration outcomes. We use a UPR reporter cell line to screen an E. faecalis transposon library to reveal bacterial genes involved in UPR activation.
Results: Global transcriptomic changes in the UPR in fibroblasts and keratinocytes were identified using single-cell RNAseq during E.faecalis infection of the wound in C57BL/6 mice. We observed significant UPR activation and diminished wound healing in our in vivo and in vitro infection and wound healing models, especially in the IRE1 and PERK branches. Drug-based attenuation revealed that PERK contributes to delayed cell migration. A high throughput screen with an E. faecalis transposon library consisting of 14976 mutants has so far revealed 108 genes that are potentially involved in host UPR activation. Various hits are in the process of being validated.
Conclusion: These findings highlight the potential interplay between E. faecalis and the PERK signaling pathway of the UPR. Further studies are required to understand the delicate balance of this pathway to manipulate its potential as an alternative therapeutic target for treating infected wounds.
This research was funded by the National Medical Research Council.