Secretion and delivery of bacteria-derived immunomodulatory RNA and DNA via extracellular membrane vesicles

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2021

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Abstract

Nucleic acids produced by the human pathogen, Staphylococcus aureus, have become increasingly recognized as important pathogen-associated molecular patterns capable of eliciting Type I Interferon (IFN) signaling in host cells. Intracellular Toll-like receptors (TLRs) have been identified as sensors of S. aureus-derived RNA and DNA. However, there is a lack of mechanistic insight into how S. aureus secretes nucleic acids and how nucleic acids derived from S. aureus—which is classified as an extracellular pathogen—are transported into host cells to activate TLRs to modulate host cell functions. Recent studies have uncovered extracellular membrane vesicles (MVs) produced by bacteria as a novel secretion mechanism for biologically active RNA and DNA. These findings raised the intriguing possibility of a new paradigm in bacterial secretion whereby MVs act as a vehicle for both local and distant delivery of regulatory and immunomodulatory RNA and DNA to target cells. As such, we hypothesized that MVs secreted by S. aureus could represent an important source of functional and structurally relevant bacterial RNA and DNA molecules that modulate the host immune response. Through biochemical analysis of S. aureus MVs and their nucleic acid cargo, we found that subpopulations of MV-associated RNA and DNA can withstand nuclease degradation. We report that in cultured mouse macrophages, IFN-β mRNA is induced by S. aureus MVs and that MV-induced IFN-β expression is specifically promoted by activation of endosomal TLR3, TLR7, and TLR9 receptors—pointing to the likelihood that MV-associated, protected RNA and DNA are inducing such a response. By metabolically labeling S. aureus RNA in culture and imaging the resultant MVs using super-resolution microscopy, we provide visual evidence that MVs and their stably associated RNA cargo are internalized by macrophages. Our work suggests that both unprotected and protected S. aureus MV-associated RNA and DNA molecules induce significant responses in host cells, but do so by different pathways, and that these differences could have important functional consequences. Altogether, we provide the first piece of evidence that S. aureus releases MVs laden with RNA and DNA molecules that are internalized by host immune cells to induce IFN-β via TLR-dependent signaling. These novel insights could promote the development of novel therapeutic strategies for the treatment of staphylococcal disease.

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Rodriguez, Blanca Victoria (2021). Secretion and delivery of bacteria-derived immunomodulatory RNA and DNA via extracellular membrane vesicles. Dissertation, Duke University. Retrieved from https://hdl.handle.net/10161/24480.

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