Saturday, May 31, 2025
5/31/2025
In vivo production of Staphylococcus aureus extracellular membrane vesicles - Project Summary Staphylococcus aureus is a bacterial pathogen that provokes a diverse array of human diseases, ranging from mild skin lesions to invasive and life-threatening infections. The success of S. aureus as a pathogen is attributed to its resistance to antibiotic therapy and production of surface adhesins and glycopolymers, as well as secreted proteins such as cytolysins, superantigens, and proteases, many of which play vital roles in immune evasion. S. aureus also produces extracellular membrane vesicles (MVs) that encapsulate cargo that includes lipoproteins, nucleic acids, glycopolymers, adhesins, and exoproteins, including proteases and pore-forming toxins. Numerous studies have characterized MV production from cultures of various S. aureus isolates and demonstrated the multiple biological activities of MVs in vitro and in vivo. However, the generation of MVs in vivo during staphylococcal infection remains unproven, resulting in questions about the biological relevance of MVs and their contribution to microbial virulence. The long-term goal of this project is to understand the biological roles of MVs in the pathogenesis of staphylococcal infections. The objective of this proposal is to characterize the production of S. aureus MVs in vivo to better understand their contributions to disease. The central hypothesis is that release of MVs is a critical virulence mechanism by which S. aureus targets host cells while protecting the MV cargo from destruction by environmental factors. This expectation is based on published findings showing that S. aureus MVs purified from in vitro cultures encapsulate a variety of virulence determinants with pronounced biological activities. Preliminary data in this application show that S. aureus MVs are generated in a murine air pouch infection model. This project will establish and optimize methodology for the purification and analysis of S. aureus MVs recovered from infected animals. The specific aims of the application are to: (1) Characterize the production of S. aureus MVs during infection – murine air pouch and pneumonia models. The pneumonia model will allow a more comprehensive understanding of S. aureus MV production in a model resembling human disease. The protein composition of S. aureus MVs generated in vitro vs. in vivo will be characterized by LC- MS/MS. If the MV protein content differs in vitro vs. in vivo, RNA-Seq analysis will be performed on S. aureus bacteria cultivated in vitro vs. in vivo to correlate gene expression with MV protein cargo. (2) Evaluate the biological effects of “in vivo” MVs on the host. To uncouple the biological responses to MVs from those elicited by bacterial cells, physiologically relevant numbers of MVs purified from air pouch and bronchoalveolar lavage fluids will be used for (a) in vitro cytotoxicity assays and (b) to inoculate naive mice. The animals will be monitored for leukocyte influx, cytokine levels, and tissue pathology. To accomplish these goals, a multidisciplinary approach will be taken that combines microbiology, molecular biology, biochemistry, immunology, and animal models. Knowledge gained from these studies will deepen the currently limited understanding of the role that MVs play in bacterial pathogenesis, especially those produced by Gram-positive pathogens.
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