Abstract
Pseudomonas aeruginosa is an opportunistic pathogen that is associated with hospital-acquired infections, ventilator-associated pneumonia, and morbidity of immunocompromised individuals. A subpopulation of P. aeruginosa encodes a protein, ExoU, which exhibits acute cytotoxicity. Toxicity is directly related to the phospholipase A2 activity of the protein after injection into the host cytoplasm via a type III secretion system. ExoU enzymatic activity requires eukaryotic cofactors, ubiquitin or ubiquitin-modified proteins. When administered extracellularly, ExoU is unable to intoxicate epithelial cells in culture, even in the presence of the cofactor. Injection or transfection of ExoU is necessary to observe the acute cytotoxic response. Biochemical approaches indicate that ExoU possesses high affinity to a multifunctional phosphoinositide, phosphatidylinositol 4,5-bisphosphate or PI(4,5)P2 and that it is capable of utilizing this phospholipid as a substrate. In eukaryotic cells, PI(4,5)P2 is mainly located in the cytoplasmic side of the plasma membrane and anchors adaptor proteins that are involved in cytoskeletal structures, focal adhesions, and plasma membranes. Time-lapse fluorescent microscopy analyses of infected live cells demonstrate that ExoU intoxication correlates with intracellular damage in the early phases of infection, such as disruption of focal adhesions, cytoskeletal collapse, actin depolymerization, and cell rounding. At later time points, a membrane blebbing phenotype was prominent prior to the loss of the plasma membrane integrity and barrier function. Membrane blebbing appears to accelerate membrane rupture and the release of intracellular markers. Our data suggest that in eukaryotic host cells, intracellular ExoU targets and hydrolyzes PI(4,5)P2 on the plasma membrane, causing a subsequent disruption of cellular structures and membrane integrity.
Highlights
Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen and a causative agent for hospital-acquired infection, ventilator-associated pneumonia (VAP), and infection in immunocompromised individuals [1,2,3] including patients with burns, AIDS, neutropenia, leukemia, cystic fibrosis, or transplantation patients treated with immunosuppressive therapy [4,5,6,7,8,9]
ExoU translocation and cytotoxicity as assessed in human epithelial cells During infection, ExoU is delivered into host cells through the T3S injectisome
To characterize the translocation pattern of this protein, we examined the timing and delivery of non-cytotoxic ExoUS142A into HeLa cells during infection at a multiplicity of infection (MOI) of 2.5
Summary
Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen and a causative agent for hospital-acquired infection, ventilator-associated pneumonia (VAP), and infection in immunocompromised individuals [1,2,3] including patients with burns, AIDS, neutropenia, leukemia, cystic fibrosis, or transplantation patients treated with immunosuppressive therapy [4,5,6,7,8,9]. P. aeruginosa virulence is highly correlated to the expression of a specialized type III secretion system (T3SS) [12,13]. This system is responsible for intoxicating host cells with up to four effector proteins; ExoS, ExoT, ExoY and ExoU [14,15,16,17,18,19,20]. ExoU expression induces lung injury and sepsis [22,31]
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