Abstract
We previously demonstrated the immunostimulatory efficacy of Pseudomonas aeruginosa flagellar hook protein FlgE on epithelial cells, presumably via ectopic ATP synthases or subunits ATP5B on cell membranes. Here, by using recombinant wild-type FlgE, mutant FlgE (FlgEM; bearing mutations on two postulated critical epitopes B and F), and a FlgE analog in pull-down assay, Western blotting, flow cytometry, and ELISA, actual bindings of FlgE proteins or epitope B/F peptides with ATP5B were all confirmed. Upon treatment with FlgE proteins, human umbilical vein endothelial cells (HUVECs) and SV40-immortalized murine vascular endothelial cells manifested decreased proliferation, migration, tube formation, and surface ATP production and increased apoptosis. FlgE proteins increased the permeability of HUVEC monolayers to soluble large molecules like dextran as well as to neutrophils. Immunofluorescence showed that FlgE induced clustering and conjugation of F-actin in HUVECs. In Balb/c-nude mice bearing transplanted solid tumors, FlgE proteins induced a microvascular hyperpermeability in pinna, lungs, tumor mass, and abdominal cavity. All effects observed in FlgE proteins were partially or completely impaired in FlgEM proteins or blocked by pretreatment with anti-ATP5B antibodies. Upon coculture of bacteria with HUVECs, FlgE was detectable in the membrane and cytosol of HUVECs. It was concluded that FlgE posed a pathogenic ligand of ectopic ATP5B that, upon FlgE–ATP5B coupling on endothelial cells, modulated properties and increased permeability of endothelial layers both in vitro and in vivo. The FlgE-ectopic ATP5B duo might contribute to the pathogenesis of disorders associated with bacterial infection or ectopic ATP5B-positive cells.
Highlights
Pseudomonas aeruginosa (PA) is a common pathogen in sites like the lungs or eyes or exposed cutaneous injuries but can be transmitted via blood circulation to other organs (Bachta et al, 2020)
We first checked whether the hypothetical interactions between FlgE and ATP5B proteins would occur in vascular endothelial cells (VECs)
Flow cytometry showed that bindings of FlgE proteins with human umbilical vein endothelial cells (HUVECs) were inhibited by pretreatment of cells with antiATP5B antibodies (Figure 2C), suggesting that FlgE interacted with ectoATP5B directly
Summary
Pseudomonas aeruginosa (PA) is a common pathogen in sites like the lungs or eyes or exposed cutaneous injuries but can be transmitted via blood circulation to other organs (Bachta et al, 2020). Should the proposed FlgE–ATP synthase/subunit interactions occur in living organisms like in humans, it would advance our understanding of bacteria–host interactions in a few conditions concerning ectopic ATP synthase-expressing cells, such as in vascular endothelium Though it still remains unclear why, how, in what cells, or under what situation would ATP synthase relocate from its traditional destination (e.g., inner membrane of mitochondria) to the outer surface of cellular membrane, ectoATP5B on VECs had been reported to mediate, at least partially, cellular responses to several host-derived molecules like angiostatin (Moser et al, 1999), apolipoprotein (Apo) A-I (Radojkovic et al, 2009; Cavelier et al, 2012), kringle 15 of plasminogen (Veitonmaki et al, 2004), or pigment epithelium-derived factor (Notari et al, 2010). The effect of FlgE on systemic vascular permeability was studied by using an animal model
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