Gasdermin A is activated by Staphylococcus aureus cysteine protease Staphopain A Restricted; Files Only

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA), is a gram-positive bacterium that is part of the human nasal microbiota. It is also a common opportunistic pathogen associated with community and hospital-acquired infections which causes sepsis and death. MRSA releases a cysteine protease, staphopain A (ScpA) during host epithelial cell cytoplasm invasion that induces cell death by an unknown mechanism. Pyroptosis, or inflammatory cell death, is important for initial host cell survival and signaling during infection. Gasdermin A (GSDMA), a pyroptosis-inducing protein expressed in human mucosa and epithelial cells is only known to be activated by the cysteine protease SpeB from Group A Streptococcus (GAS). Since GAS and MRSA clinical manifestations are similar, and SpeB and ScpA are both secreted cysteine proteases, it was hypothesized that GSDMA is activated by ScpA and that this is responsible for the cell death of keratinocytes observed during MRSA invasion. The data from biochemical assays and in vivo murine studies showed that ScpA affected the GSDMA pathway and induced other inflammatory signals. ScpA was able to cleave GSDMA in vitro to a form consistent with its activation. Murine GSDMA knockout and wild type C57Bl/ 6 mice, infected intradermally with wild type (WT) and ScpA deletion mutant (ΔScpA) MRSA strains had similar CFU burden and lesion sizes, but differences in redness and swelling consistent with a role for ScpA and GSDMA in the inflammatory response to infection. Pro-inflammatory cytokines like IL-1β were induced at higher rates with the WT MRSA injection rather than the ΔScpA MRSA injection. The cytokine concentration difference was more pronounced in GSDMA-KO mice than the WT mice. Overall, it was shown that ScpA plays a role for inflammation in MRSA infection and can cleave GSDMA. ScpA is not required for MRSA virulence as SpeB is required for GAS virulence. This data will inform further studies on ScpA and GSDMA interaction with keratinocytes using cell culture models, biochemical assays, and additional murine models of infection.

Table of Contents

Chapter 1: Introduction................................................................................................................ 8

Chapter 2: ScpA activates hGSDMA and inflammation in vitro........................................... 12

Figure 1.................................................................................................................................. 12

Methods..................................................................................................................................... 13

Results........................................................................................................................................ 15

Figure 2.................................................................................................................................. 15

Chapter 3: ScpA activates GSDMA and inflammation in vivo.............................................. 15

Figure 3.................................................................................................................................. 16

Methods..................................................................................................................................... 18

Results........................................................................................................................................ 22

Figure 4.................................................................................................................................. 22

Figure 5.................................................................................................................................. 23

Figure 6.................................................................................................................................. 24

Figure 7.................................................................................................................................. 25

Figure 8.................................................................................................................................. 27

Figure 9.................................................................................................................................. 29

Chapter 4: Discussion and Conclusion...................................................................................... 31

Discussion.................................................................................................................................. 31

Future Directions and Conclusion............................................................................................. 34

References.................................................................................................................................... 37

About this Honors Thesis

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School	Emory College
Department	Biology
Degree	B.S.
Submission	Honors Thesis
Language	English
Keyword	invasion keratinocytes MRSA inflammasome skin infection Staphylococcus aureus gasdermin A pyroptosis virulence
Committee Chair / Thesis Advisor	Christopher LaRock, Emory University
Committee Members	Nic Vega, Emory University Lindsey Seldin, Emory University

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