Ubiquitin and ubiquitin-modified proteins activate the Pseudomonas aeruginosa T3SS cytotoxin, ExoU

Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
Molecular Microbiology (Impact Factor: 4.42). 12/2011; 82(6):1454-67. DOI: 10.1111/j.1365-2958.2011.07904.x
Source: PubMed


Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that possesses a type III secretion system (T3SS) critical for evading innate immunity and establishing acute infections in compromised patients. Our research has focused on the structure-activity relationships of ExoU, the most toxic and destructive type III effector produced by P. aeruginosa. ExoU possesses phospholipase activity, which is detectable in vitro only when a eukaryotic cofactor is provided with membrane substrates. We report here that a subpopulation of ubiquitylated yeast SOD1 and other ubiquitylated mammalian proteins activate ExoU. Phospholipase activity was detected using purified ubiquitin of various chain lengths and linkage types; however, free monoubiquitin is sufficient in a genetically engineered dual expression system. The use of ubiquitin by a bacterial enzyme as an activator is unprecedented and represents a new aspect in the manipulation of the eukaryotic ubiquitin system to facilitate bacterial replication and dissemination.

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    • "ExoU is held inactive inside the bacterium and is unable to exert toxicity from outside the host cells. To cause cytotoxicity, ExoU requires to be translocated into the cytoplasm of the host cell, where it is activated by the contact with three cofactors: the eukaryotic cytoplasmic superoxide dismutase [88], ubiquitin or ubiquitin-modified proteins [89] [90], and PI(4,5)P 2 [91]. Distinct residues of ExoU are critical for its activation by ubiquitin and by PI(4,5),P 2 , indicating that these factors activate ExoU by discrete mechanisms [91]. "
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    ABSTRACT: Bacterial sphingomyelinases and phospholipases constitute a heterogeneous group of surface associated or secreted esterases, produced by a variety of Gram-positive and Gram-negative bacteria. In several cases mutant strains lacking a gene encoding a sphingomyelinase or a phospholipase have reduced pathogenicity in experimental animals demonstrating the role of the corresponding enzyme in pathogenesis. Bacterial sphingomyelinases and phospholipases might favor in different ways the colonization of the infected tissue, the establishment and progression of the infection or the evasion of the immune response by both intracellular and extracellular pathogens. This chapter presents an overview of the classification, structure, and main physiopathological activities of bacterial sphingomyelinases and phospholipases, providing examples of their roles as virulence factors in several human and animal diseases.
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    • "ExoU is a potent phospholipase A2 cytotoxin that rapidly causes eukaryotic cell lysis and stimulates lipid signal transduction cascades [44,45]. ExoU activation is mediated by interactions with eukaryotic mono- and poly-ubiquitin, and ubiquitinylated proteins such as Cu/Zn superoxide dismutase 1 [46–49]. ExoS and ExoT are dual functioning Rho GTPase activating and ADP-ribosyltranferase effectors that disrupt eukaryotic cell signaling, prevent phagocytosis, and mediate the pathogen’s ability to disrupt the epithelial barrier [43,45,50,51]. "
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    ABSTRACT: Herein we describe a pathogenic role for the Pseudomonas aeruginosa type three secretion system (T3SS) needle tip complex protein, PcrV, in causing lung endothelial injury. We first established a model in which P. aeruginosa wild type strain PA103 caused pneumonia-induced sepsis and distal organ dysfunction. Interestingly, a PA103 derivative strain lacking its two known secreted effectors, ExoU and ExoT [denoted PA103 (ΔU/ΔT)], also caused sepsis and modest distal organ injury whereas an isogenic PA103 strain lacking the T3SS needle tip complex assembly protein [denoted PA103 (ΔPcrV)] did not. PA103 (ΔU/ΔT) infection caused neutrophil influx into the lung parenchyma, lung endothelial injury, and distal organ injury (reminiscent of sepsis). In contrast, PA103 (ΔPcrV) infection caused nominal neutrophil infiltration and lung endothelial injury, but no distal organ injury. We further examined pathogenic mechanisms of the T3SS needle tip complex using cultured rat pulmonary microvascular endothelial cells (PMVECs) and revealed a two-phase, temporal nature of infection. At 5-hours post-inoculation (early phase infection), PA103 (ΔU/ΔT) elicited PMVEC barrier disruption via perturbation of the actin cytoskeleton and did so in a cell death-independent manner. Conversely, PA103 (ΔPcrV) infection did not elicit early phase PMVEC barrier disruption. At 24-hours post-inoculation (late phase infection), PA103 (ΔU/ΔT) induced PMVEC damage and death that displayed an apoptotic component. Although PA103 (ΔPcrV) infection induced late phase PMVEC damage and death, it did so to an attenuated extent. The PA103 (ΔU/ΔT) and PA103 (ΔPcrV) mutants grew at similar rates and were able to adhere equally to PMVECs post-inoculation indicating that the observed differences in damage and barrier disruption are likely attributable to T3SS needle tip complex-mediated pathogenic differences post host cell attachment. Together, these infection data suggest that the T3SS needle tip complex and/or another undefined secreted effector(s) are important determinants of P. aeruginosa pneumonia-induced lung endothelial barrier disruption.
    Full-text · Article · Nov 2013 · PLoS ONE
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    • "ExoU has been characterized as a member of the phospholipase family of enzymes and has at least phospholipase A2 activity [19]. Similar to ExoS, ExoT and ExoY, ExoU requires either a eukaryote-specific cofactor for its activity and ubiquitinated proteins, as well as ubiquitin itself, have been suggested as being potential activators of the toxin [20]. In mammalian cells, the direct injection of ExoU causes irreversible damage to cellular membranes and rapid necrotic death. "
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    ABSTRACT: The type III secretion system (T3SS) is a complex nanomachine of many pathogenic gram-negative bacteria. It forms a proteinaceous channel that is inserted into the host eukaryotic cell membrane for injection of bacterial proteins that manipulate host cell signaling. However, few studies have focused on the effector-independent functions of the T3SS. Using a murine model of acute lung infection with Pseudomonas aeruginosa, an important human opportunistic pathogen, we compared the pathogenicity of mutant bacteria that lack all of the known effector toxins ( ΔSTY), with mutant bacteria that also lack the major translocator protein PopB (ΔSTY/ΔPopB) and so cannot form a functional T3SS channel in the host cell membrane. Mortality was higher among mice challenged with ΔSTY compared to mice challenged with ΔSTY/ΔPopB mutant bacteria. In addition, mice infected with ΔSTY showed decreased bacterial clearance from the lungs compared to those infected with ΔSTY/ΔPopB. Infection was in both cases associated with substantial killing of lung infiltrating macrophages. However, macrophages from ΔSTY-infected mice died by pro-inflammatory necrosis characterized by membrane permeabilization and caspase-1 mediated IL-1β production, whereas macrophages from ΔSTY/ΔPopB infected mice died by apoptosis, which is characterized by annexin V positive staining of the cell membrane and caspase-3 activation. This was confirmed in macrophages infected in vitro. These results demonstrate a T3SS effector toxin independent role for the T3SS, in particular the T3SS translocator protein PopB, in the pathogenicity of P. aeruginosa during acute lung infection.
    Full-text · Article · Jul 2012 · PLoS ONE
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