Identification and Verification of Ubiquitin-Activated Bacterial Phospholipases

Abstract

ExoU is a potent type III secretion system effector that is injected directly into mammalian cells by the opportunistic pathogen Pseudomonas aeruginosa. As a ubiquitin-activated phospholipase A2 (PLA2), ExoU exhibits cytotoxicity by cleaving membrane phospholipids, resulting in lysis of the host cells and inhibition of the innate immune response. Recently, ExoU has been established as a model protein for a group of ubiquitin-activated PLA2 enzymes encoded by a variety of bacteria. Bioinformatic analyses of homologous proteins is a powerful approach that can complement and enhance the overall understanding of protein structure and function. To conduct homology studies, it is important to have efficient and effective tools to screen and to validate the putative homologs of interest. Here we make use of an Escherichia coli-based dual expression system to screen putative ubiquitin-activated PLA2 enzymes from a variety of bacteria that are known to colonize humans and to cause human infections. The screen effectively identified multiple ubiquitin-activated phospholipases, which were validated using both biological and biochemical techniques. In this study, two new ExoU orthologs were identified and the ubiquitin activation of the rickettsial enzyme RP534 was verified. Conversely, ubiquitin was not found to regulate the activity of several other tested enzymes. Based on structural homology analyses, functional properties were predicted for AxoU, a unique member of the group expressed by Achromobacter xylosoxidans. IMPORTANCE Bacterial phospholipases act as intracellular and extracellular enzymes promoting the destruction of phospholipid barriers and inflammation during infections. Identifying enzymes with a common mechanism of activation is an initial step in understanding structural and functional properties. These properties serve as critical information for the design of specific inhibitors to reduce enzymatic activity and ameliorate host cell death. In this study, we identify and verify cytotoxic PLA2 enzymes from several bacterial pathogens. Similar to the founding member of the group, ExoU, these enzymes share the property of ubiquitin-mediated activation. The identification and validation of potential toxins from multiple bacterial species provide additional proteins from which to derive structural insights that could lead to paninhibitors useful for treating a variety of infections.

Characterization of the ExoU activation mechanism using EPR and integrative modeling

“…Here we use ExoU, a bacterial phospholipase synthesized by a particularly virulent subset of P. aeruginosa strains, as a model system to map regions of the molecule involved in activation of the enzyme. ExoU homologs and orthologs are encoded by several bacterial pathogens 42 , 43 , suggesting that knowledge of the activation mechanism could translate into inhibitory drugs against multiple Gram-negative pathogens. In support of this notion, experimental evidence is provided that VipD, synthesized by Legionella pneumophila, and ExoU share a conserved activation mechanism despite the use of distinctly separate eukaryotic cofactors, Rab5 and ubiquitin, respectively.…”

“…In all other PLA 2 crystal structures that include coordinates for the catalytic aspartate, this location is occupied by two antiparallel β-strands orienting the catalytic aspartate towards the catalytic serine 31 , 32 , 34 , 45 , 46 . Residues of ExoU analogous to the β-structure preceding the catalytic aspartate are unresolved in the existing published crystal structures, but are predicted to form two β-strands 42 . While the investigation of this region is in progress, we hypothesize residues 180–209 must be displaced from their apo position to allow the ExoU catalytic aspartate and preceding residues to fold into the analogous β structure observed in other PLA 2 enzymes.…”

“…Linear diubiquitin was purified as previously described with slight modifications 42 . Clones encoding linear diubiquitin were induced, suspended in 20 ml of 50 mM sodium acetate buffer (pH 7.3) with 32 µg/ml benzamidine, 64 µg/ml leupeptin, 3.2 µg/ml pepstatin, and 32 µg/ml aprotinin and lysed in a French pressure cell (3–5 passes).…”

“…ExoU activity assays. Activity assays were conducted as previously described 42 . Briefly, 50 μM N-((6-(2,4dinitrophenyl)amino)hexanoyl)-2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphoethanolamine (PED6) was used except for PED6 titrations and 20 μM diubiquitin was used as cofactor.…”

Morphological and physiological effects of a single amino acid substitution in the patatin-like phospholipase CapV inEscherichia coli

“…In mammals, PLPs are mostly involved in lipid metabolism and turnover ( 64 ), with a polymorphism in a patatin phospholipase to provide the genetic predisposition for nonalcoholic fatty liver disease and metabolic syndrome ( 65 ). PLPs are also found in many pathogenic bacterial species and act as toxins in host-pathogen interactions ( 66, 67 ). One of the best characterized PLPs is ExoU, a cytotoxic effector protein of P. aeruginosa secreted through the type III secretion system upon host cell contact ( 68 ).…”