Cells Responding to Closely Related Cholesterol-Dependent Cytolysins Release Extracellular Vesicles with a Common Proteomic Content Including Membrane Repair Proteins
Abstract:The plasma membrane (PM) protects cells from extracellular threats and supports cellular homeostasis. Some pathogens produce pore-forming toxins (PFTs) that disrupt PM integrity by forming transmembrane pores. High PFT concentrations cause massive damage leading to cell death and facilitating infection. Sub-lytic PFT doses activate repair mechanisms to restore PM integrity, support cell survival and limit disease. Shedding of extracellular vesicles (EVs) has been proposed as a key mechanism to eliminate PFT po… Show more
“…As part of a membrane repair mechanism, host cells infected with S. pneumoniae can release extracellular membrane vesicles through which they expel membrane-bound bacterial products such as PLY [54, 55]. To determine if CD73 was shed in host-derived vesicles, H292 cells were treated with CellTracker Green dye CMFDA, which stains the cell cytoplasm and can be used to track host-derived vesicles.…”
Section: Resultsmentioning
confidence: 99%
“…Each probe comprises a truncated form of the GTPase and the Rho-binding domain (RBD) of effectors, bound to the CFP or YFP, respectively. The binding of GTP to the GTPase (resulting in activation) brings the YFP-bound GTPase domain and the CFP-bound RBD into close proximity, allowing FRET from CFP to YFP [55, 56]. Transfected HeLa cells were challenged with 0.25 nM of PLY and live-cell microscopy was performed to visualize the activation status of RhoA and Cdc42.…”
Section: Resultsmentioning
confidence: 99%
“…Apart from stimulating membrane repair through annexins, injury to plasma membrane by PLY, LLO or SLO also results in a phenomenon called shedding whereby the host cell releases extracellular vesicles to not only remove the injured part of the membrane but also to remove the membrane bound toxin [54, 88]. Blebbing is one of the ways to mediate the shedding of larger vesicular structures whereby the cell undergoes actomyosin-dependent localized contraction in the plasma membrane to create confined spaces or a localized bulge with elevated Ca 2+ levels to prevent leakage to cytoplasmic contents and proteins in the extracellular milieu [13, 65].…”
A return to homeostasis after infection-associated cellular injury can be accelerated by a rapid damage response. S. pneumoniae, a typically asymptomatic colonizer of the host upper respiratory tract, can cause serious and life-threatening infections when it gains access to the lungs and other organs. The cholesterol binding S. pneumoniaepore-forming toxin, pneumolysin (PLY), is central to the induction of host cell damage. Here, we first found that mouse lung infection by S. pneumoniae diminished pulmonary expression of CD73, a glycosylphosphatidylinositol anchored protein (GPI-AP) that modulates inflammation. Infection of the human pulmonary epithelial cell line H292 resulted in a PLY-dependent reduction of not only cell surface CD73, but also the population of surface expressed GPI-APs. The decrease in cell surface GPI-APs was rapid, required pore-forming activity, and could be recapitulated by purified PLY and other cholesterol binding cytolysins. In response to PLY-mediated insult, GPI-APs were not released from the surface of epithelial cells in extracellular vesicles but rather internalized by a mechanism dependent on the Rho GTPases RhoA and Cdc42. Internalization of GPI-APs was associated with lower levels of PLY-induced apoptosis and membrane permeabilization. These findings suggest that internalization of GPI-APs from epithelial cell membranes may constitute a rapid innate repair response to cell damage induced by PLY and other pore forming toxins that could help bacteria evade host defenses as many GPI-APs have roles in immunity.
“…As part of a membrane repair mechanism, host cells infected with S. pneumoniae can release extracellular membrane vesicles through which they expel membrane-bound bacterial products such as PLY [54, 55]. To determine if CD73 was shed in host-derived vesicles, H292 cells were treated with CellTracker Green dye CMFDA, which stains the cell cytoplasm and can be used to track host-derived vesicles.…”
Section: Resultsmentioning
confidence: 99%
“…Each probe comprises a truncated form of the GTPase and the Rho-binding domain (RBD) of effectors, bound to the CFP or YFP, respectively. The binding of GTP to the GTPase (resulting in activation) brings the YFP-bound GTPase domain and the CFP-bound RBD into close proximity, allowing FRET from CFP to YFP [55, 56]. Transfected HeLa cells were challenged with 0.25 nM of PLY and live-cell microscopy was performed to visualize the activation status of RhoA and Cdc42.…”
Section: Resultsmentioning
confidence: 99%
“…Apart from stimulating membrane repair through annexins, injury to plasma membrane by PLY, LLO or SLO also results in a phenomenon called shedding whereby the host cell releases extracellular vesicles to not only remove the injured part of the membrane but also to remove the membrane bound toxin [54, 88]. Blebbing is one of the ways to mediate the shedding of larger vesicular structures whereby the cell undergoes actomyosin-dependent localized contraction in the plasma membrane to create confined spaces or a localized bulge with elevated Ca 2+ levels to prevent leakage to cytoplasmic contents and proteins in the extracellular milieu [13, 65].…”
A return to homeostasis after infection-associated cellular injury can be accelerated by a rapid damage response. S. pneumoniae, a typically asymptomatic colonizer of the host upper respiratory tract, can cause serious and life-threatening infections when it gains access to the lungs and other organs. The cholesterol binding S. pneumoniaepore-forming toxin, pneumolysin (PLY), is central to the induction of host cell damage. Here, we first found that mouse lung infection by S. pneumoniae diminished pulmonary expression of CD73, a glycosylphosphatidylinositol anchored protein (GPI-AP) that modulates inflammation. Infection of the human pulmonary epithelial cell line H292 resulted in a PLY-dependent reduction of not only cell surface CD73, but also the population of surface expressed GPI-APs. The decrease in cell surface GPI-APs was rapid, required pore-forming activity, and could be recapitulated by purified PLY and other cholesterol binding cytolysins. In response to PLY-mediated insult, GPI-APs were not released from the surface of epithelial cells in extracellular vesicles but rather internalized by a mechanism dependent on the Rho GTPases RhoA and Cdc42. Internalization of GPI-APs was associated with lower levels of PLY-induced apoptosis and membrane permeabilization. These findings suggest that internalization of GPI-APs from epithelial cell membranes may constitute a rapid innate repair response to cell damage induced by PLY and other pore forming toxins that could help bacteria evade host defenses as many GPI-APs have roles in immunity.
“…4 A). Based on existing literature, cellular damage can stimulate EVs release as a membrane repair mechanism [ 27 ]. Fig.…”
Section: Resultsmentioning
confidence: 99%
“…Extracellular vesicles (EVs) are key in intercellular communication and emerging as therapeutic agents. The disruption of blood vessels post-SCI underscores the therapeutic potential of EVs derived from damaged endothelial cells [ 27 ]. UTX KO in spinal cord microvascular endothelial cells (SCMECs) post-SCI facilitates macrophage polarization to the M2 subtype through EVs [ 16 ].…”
Background
Vascular endothelial cells are pivotal in the pathophysiological progression following spinal cord injury (SCI). The UTX (Ubiquitously Transcribed Tetratripeptide Repeat on Chromosome X) serves as a significant regulator of endothelial cell phenotype. The manipulation of endogenous neural stem cells (NSCs) offers a compelling strategy for the amelioration of SCI.
Methods
Two mouse models were used to investigate SCI: NSCs lineage-traced mice and mice with conditional UTX knockout (UTX KO) in endothelial cells. To study the effects of UTX KO on neural differentiation, we harvested extracellular vesicles (EVs) from both UTX KO spinal cord microvascular endothelial cells (SCMECs) and negative control SCMECs. These EVs were then employed to modulate the differentiation trajectory of endogenous NSCs in the SCI model.
Results
In our NSCs lineage-traced mice model of SCI, a marked decrease in neurogenesis was observed post-injury. Notably, NSCs in UTX KO SCMECs mice showed enhanced neuronal differentiation compared to controls. RNA sequencing and western blot analyses revealed an upregulation of L1 cell adhesion molecule (L1CAM), a gene associated with neurogenesis, in UTX KO SCMECs and their secreted EVs. This aligns with the observed promotion of neurogenesis in UTX KO conditions. In vivo administration of L1CAM-rich EVs from UTX KO SCMECs (KO EVs) to the mice significantly enhanced neural differentiation. Similarly, in vitro exposure of NSCs to KO EVs resulted in increased activation of the Akt signaling pathway, further promoting neural differentiation. Conversely, inhibiting Akt phosphorylation or knocking down L1CAM negated the beneficial effects of KO EVs on NSC neuronal differentiation.
Conclusions
In conclusion, our findings substantiate that EVs derived from UTX KO SCMECs can act as facilitators of neural differentiation following SCI. This study not only elucidates a novel mechanism but also opens new horizons for therapeutic interventions in the treatment of SCI.
Graphical Abstract
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