Disruption of the host deubiquitinating enzyme, USP14, as a novel therapeutic strategy to combat Listeria monocytogenes infection

Kevin M. Claunch, M.S.

 Introduction. The Unfolded Protein Response (UPR) is a critical homeostatic pathway in mammalian cells that is triggered by ER dysfunction and subsequent accumulation of misfolded and damaged proteins1,7. Activation of the UPR is achieved through three transmembrane proteins, one of which is IRE-1, which catalyzes the splicing of X-box binding protein-1 (Xbp-1), a transcription factor of UPR-specific genes7. Activation of the UPR is modulated in part by the activity of deubiquitinating enzymes, which function both as antagonists of the ubiquitin-proteosome pathway involved in protein turnover and as inhibitors of the UPR7. In addition to its role in cellular homeostasis, the UPR has been implicated in host defense to a number of pathogens, such as    Listeria monocytogenes5, a leading cause of food-borne illness among the elderly, immunocompromised, and pregnant women3. The UPR has thus been investigated as a potential drug target for the treatment of L. monocytogenes infection2,6. Two derivates of the compound WP1130, compounds 9 and 6, were found to activate the UPR via inactivation of the deubiquitinating enzyme USP142,6. Inactivation of USP14 by these compounds frees IRE-1, allowing it to splice Xbp-1 mRNA and induce the UPR4,7. Methods. RAW264.7 mouse macrophages were infected with L. monocytogenes and incubated with compounds 9 and 6 to determine if these compounds reduce bacterial replication in cell culture2,6. Inside-out immunofluorescence microscopy was also performed to determine if compound 6 limited bacterial internalization. In addition, both compounds were tested for direct antimicrobial activity by performing bacterial growth assays in the presence or absence of   each compound6. Results. Both compounds 9 and 6 were found to significantly reduce replication of L. monocytogenes in RAW264.7 macrophages with no detectable toxicity to the macrophages2,6. In addition, compound 6 was found to limit internalization of the bacteria into RAW264.7 cells with roughly one bacterial   cell internalized for every three that remained extracellular6. While these compounds act by inducing the UPR, they also may have some moderate direct antimicrobial effects as well, as evidenced by a modest decrease in bacterial growth rate when L. monocytogenes was grown in the presence of each compound in broth alone2,6. Conclusions. These findings indicate that both compounds 9 and 6 are promising candidates for further research and development as novel therapeutics for L. monocytogenes infection. Although these studies were performed in cell culture only, further work to verify or enhance efficacy of these drugs in animal models of infection is warranted.

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