A104-18 YAP/Hippo Signaling Modulates Lung Endothelial Responses to Methicillin-Resistant Staphylococcus Aureus (MRSA)-Induced Injury

Abstract Rationale Methicillin-resistant Staphylococcus aureus (MRSA) is a Gram-positive pathogen capable of causing severe pneumonia that can progress to acute respiratory distress syndrome (ARDS). A hallmark of MRSA-induced ARDS is disruption of the alveolar-capillary barrier, resulting in pulmonary edema and respiratory failure. Despite its clinical significance, the molecular mechanisms that regulate endothelial barrier integrity during MRSA infection remain poorly understood. To address this, we performed RNA sequencing (RNA-seq) on human lung endothelial cells (ECs) following MRSA exposure. Differential gene expression and KEGG pathway analyses were performed to identify signaling pathways that may regulate endothelial responses to MRSA-induced lung injury. Methods ECs were exposed to heat-killed (HK) MRSA (2.5*108 CFU) or control media. RNA was extracted using Zymol Direct-zol RNA Kit, and libraries were prepared with the CORALL V2 Total RNA-Seq Kit, followed by sequencing on the NovaSeq X 25B flow cell. Reads were quality-checked, trimmed, and aligned to the human reference genome. Differential expression was analyzed using DESeq2, and KEGG pathway analysis was performed. Protein expression was assessed via western blotting. Functional assays included ECIS for barrier integrity measurements and immunofluorescence microscopy. Pharmacologic modulation of YAP activity was achieved using either Verteporfin (inhibitor) or PY-60 (activator). Results RNA-seq identified more than 5000 differentially expressed genes following MRSA exposure, with KEGG analysis implicating the YAP/Hippo pathway. Western blot studies demonstrated that MRSA modulates nuclear YAP expression in lung ECs: nuclear YAP increased at 4 hours post-exposure, followed by a decline at 8 and 18 hours. MRSA significantly increased endothelial permeability as measured by ECIS, which was further exacerbated by YAP inhibition with Verteporfin, suggesting a protective role for YAP. Conversely, YAP activation with PY-60 attenuated MRSA-induced barrier disruption. VE-cadherin, a key adherens junction protein, was reduced following MRSA exposure, and its level was further reduced by YAP inhibition. Conclusion MRSA induces transient activation of YAP signaling during early lung endothelial injury. Pharmacologic inhibition of YAP worsens barrier dysfunction and junctional protein loss, while activation of YAP preserves barrier function. These findings suggest prolonged YAP signaling is essential for maintaining endothelial barrier function in MRSA-induced lung injury. This abstract is funded by: 1F32HL182249-01 and 1R01HL167518-01