Cryptococcus neoformans Promotes Its Transmigration into the Central Nervous System by Inducing Molecular and Cellular Changes in Brain Endothelial Cells

<jats:title>ABSTRACT</jats:title> <jats:p> <jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">Cryptococcus</jats:named-content> spp. cause fungal meningitis, a life-threatening infection that occurs predominately in immunocompromised individuals. In order for <jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">Cryptococcus neoformans</jats:named-content> to invade the central nervous system (CNS), it must first penetrate the brain endothelium, also known as the blood-brain barrier (BBB). Despite the importance of the interrelation between <jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">C. neoformans</jats:named-content> and the brain endothelium in establishing CNS infection, very little is known about this microenvironment. Here we sought to resolve the cellular and molecular basis that defines the fungal-BBB interface during cryptococcal attachment to, and internalization by, the human brain endothelium. In order to accomplish this by a systems-wide approach, the proteomic profile of human brain endothelial cells challenged with <jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">C. neoformans</jats:named-content> was resolved using a label-free differential quantitative mass spectrometry method known as spectral counting (SC). Here, we demonstrate that as brain endothelial cells associate with, and internalize, cryptococci, they upregulate the expression of several proteins involved with cytoskeleton, metabolism, signaling, and inflammation, suggesting that they are actively signaling and undergoing cytoskeleton remodeling via annexin A2, S100A10, transgelin, and myosin. Transmission electronic microscopy (TEM) analysis demonstrates dramatic structural changes in nuclei, mitochondria, the endoplasmic reticulum (ER), and the plasma membrane that are indicative of cell stress and cell damage. The translocation of HMGB1, a marker of cell injury, the downregulation of proteins that function in transcription, energy production, protein processing, and the upregulation of cyclophilin A further support the notion that <jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">C. neoformans</jats:named-content> elicits changes in brain endothelial cells that facilitate the migration of cryptococci across the BBB and ultimately induce endothelial cell necrosis. </jats:p>