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 A 2 (PLA 2 ), 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 PLA 2 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 PLA 2 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 PLA 2 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.