# In vivo production of Staphylococcus aureus extracellular membrane vesicles

> **NIH NIH R21** · BRIGHAM AND WOMEN'S HOSPITAL · 2024 · $257,125

## Abstract

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 mul...

## Key facts

- **NIH application ID:** 10788855
- **Project number:** 1R21AI180326-01
- **Recipient organization:** BRIGHAM AND WOMEN'S HOSPITAL
- **Principal Investigator:** Xiaogang Wang
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $257,125
- **Award type:** 1
- **Project period:** 2024-05-22 → 2024-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10788855

## Citation

> US National Institutes of Health, RePORTER application 10788855, In vivo production of Staphylococcus aureus extracellular membrane vesicles (1R21AI180326-01). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10788855. Licensed CC0.

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