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Temperature influences the composition and cytotoxicity of extracellular vesicles in Staphylococcus aureus

ABSTRACT: Staphylococcus aureus is a pathogenic bacterium but also a commensal of skin and anterior nares in humans. As S. aureus transits from skins/nares to inside the human body, it experiences changes in temperature. The production and content of S. aureus extracellular vesicles (EV) have been increasingly studied over the past few years and EVs are increasingly being recognized as important to the infectious process. Nonetheless, the impact of temperature variation on S. aureus EVs has not been studied in detail as most reports that investigate EV cargoes and host cell interactions are performed using vesicles produced at 37˚C. Here we report that S. aureus vesicle production is temperature-dependent with greater vesiculation at 40˚C. We demonstrate that the temperature dependent regulation of vesicle production in S. aureus is mediated by the alpha phenol soluble modulin peptides (αPSMs). Through proteomic analysis, we observed increased packaging of virulence factors at 40˚C whereas more total proteins are present in EVs produced at 34˚C. Similar to the protein content, we perform transcriptomic analysis and demonstrate that the RNA cargo is impacted by temperature also. Finally, we demonstrate greater αPSM- and α-toxin mediated erythrocyte lysis with 40˚C EVs but 34˚C EVs are more cytotoxic toward THP-1 cells. Together our study demonstrates that small temperature variations have great impact on EV biogenesis and shape the interaction with host cells.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Staphylococcus Aureus

SUBMITTER: Ronan Carroll

LAB HEAD: Ronan Carroll

PROVIDER: PXD025236 | Pride | 2021-10-12

REPOSITORIES: pride

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			Action	DRS
	20201125_Staph-Aureus-EVs_34-1.raw	Raw
	20201125_Staph-Aureus-EVs_34-2.raw	Raw
	20201125_Staph-Aureus-EVs_34-3.raw	Raw
	20201125_Staph-Aureus-EVs_37-1.raw	Raw
	20201125_Staph-Aureus-EVs_37-2.raw	Raw

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Project description:Background: Acute coronary syndromes (ACS) are associated with aberrant gene expression and epigenetic mechanisms. In particular, de novo DNA methylation is typically linked to gene silencing, but its role in heart disease remains not fully understood. Extracellular vesicles (EVs) are active components in cellular communication for their ability to carry a plethora of signalling biomolecules, thus representing a promising new diagnostic/therapeutic approach in cardiovascular diseases (CVDs). Indeed, there is the need of novel biomarkers for ACS prediction and timely detection. Purpose: We hypothesized that specific epigenetic signals can be carried by EVs. In this regard, we isolated and characterized circulating EVs from ACS patients and evaluated their potential role to influence DNA methylation in target cells. Methods: Circulating EVs were recovered, by ultracentrifugation, from plasma samples of 19 ACS patients and 50 healthy subjects (HS). Nanoparticle tracking analysis (NTA) and western blot (WB) were used to confirm the EVs integrity and purity. Peripheral blood mononuclear cells (PBMCs) of volunteer donors were treated with both ACS and HS derived EVs and genomic DNA was extracted to perform epigenome wide analysis through Reduced Representation Bisulfite Sequencing. ShinyGO, PANTHER, and STRING tools were interrogated to perform GO and PPI network analyses. Flow Cytometry, qRT-PCR, and WB analysis were also performed to evaluate and validate both intra-vesicular and intra-cellular signals. Results: Plasma ACS-derived EVs showed a significant up-regulation of DNA methyltransferases (DNMTs) gene expression levels as compared to HS (P<0.001). Specifically, de novo methylation transcripts, as DNMT3A and DNMT3B were significantly increased in plasma ACS-EVs. DNA methylation analysis of PBMCs from volunteer donors treated with HS- and ACS-derived EVs showed that RNF166 and CCND3 genes resulted the most hyper- and hypo-methylated, respectively, after by ACS-EV treatment. In addition, PPI network analysis specifically evidenced the subnetwork with SRC, as a hub gene, connecting it to NOTCH1, FOXO3, CDC42, IKBKG, RXRA, DGKG, known as important genes already involved in the onset of CVDs. Surprising, other novel genes, BAIAP2, SYP, CHL1, and SHB, which were hypomethylated, were found significantly overexpressed in PBMCs (P<0.005), underlying the fundamental modulating properties of EV cargo in atherosclerosis. Conclusions: These findings support the significant role of ACS plasma-derived EVs, inducing de novo DNA methylation signals, and modulating specific signaling pathways in target cells.

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Temperature influences the composition and cytotoxicity of extracellular vesicles in Staphylococcus aureus

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