Biochemical Characterization of a <i>Pseudomonas aeruginosa</i> Phospholipase D

Phospholipase D is a ubiquitous protein in eukaryotes that hydrolyzes phospholipids to generate the signaling lipid phosphatidic acid (PtdOH). PldA, a <i>Pseudomonas aeruginosa</i> PLD, is a secreted protein that targets bacterial and eukaryotic cells. Here we have characterized the in vitro factors that modulate enzymatic activity of PldA, including divalent cations and phosphoinositides. We have identified several similarities between the eukaryotic-like PldA and the human PLD isoforms, as well as several properties in which the enzymes diverge. Notable differences include the substrate preference and transphosphatidylation efficiency for PldA. These findings offer new insights into potential regulatory mechanisms of PldA and its role in pathogenesis

Membrane trafficking of the bacterial adhesin GspB and the accessory Sec transport machinery

The serine-rich repeat (SRR) glycoproteins of Gram-positive bacteria are large, cell wall-anchored adhesins that mediate binding to many host cells and proteins and are associated with bacterial virulence. SRR glycoproteins are exported to the cell surface by the accessory Sec (aSec) system comprising SecA2, SecY2, and 3-5 additional proteins (Asp1 to Asp5) that are required for substrate export. These adhesins typically have a 90-amino acid-long signal peptide containing an elongated N-region and a hydrophobic core. Previous studies of GspB (the SRR adhesin of Streptococcus gordonii) have shown that a glycine-rich motif in its hydrophobic core is essential for selective, aSec-mediated transport. However, the role of this extended N-region in transport is poorly understood. Here, using protein-lipid co-flotation assays and site-directed mutagenesis, we report that the N-region of the GspB signal peptide interacts with anionic lipids through electrostatic forces and that this interaction is necessary for GspB preprotein trafficking to lipid membranes. Moreover, we observed that protein-lipid binding is required for engagement of GspB with SecA2 and for aSec-mediated transport. We further found that SecA2 and Asp1 to Asp3 also localize selectively to liposomes that contain anionic lipids. These findings suggest that the GspB signal peptide electrostatically binds anionic lipids at the cell membrane, where it encounters SecA2. After SecA2 engagement with the signal peptide, Asp1 to Asp3 promote SecA2 engagement with the mature domain, which activates GspB translocation

Discovery of Desketoraloxifene Analogues as Inhibitors of Mammalian, <i>Pseudomonas aeruginosa</i>, and NAPE Phospholipase D Enzymes

Phospholipase D (PLD) hydrolyses cellular lipids to produce the important lipid second messenger phosphatidic acid. A PLD enzyme expressed by Pseudomonas aeruginosa (PldA) has been shown to be important in bacterial infection, and NAPE-PLD has emerged as being key in the synthesis of endocannabinoids. In order to better understand the biology and therapeutic potential of these less explored PLD enzymes, small molecule tools are required. Selective estrogen receptor modulators (SERMs) have been previously shown to inhibit mammalian PLD (PLD1 and PLD2). By targeted screening of a library of SERM analogues, additional parallel synthesis, and evaluation in multiple PLD assays, we discovered a novel desketoraloxifene-based scaffold that inhibited not only the two mammalian PLDs but also structurally divergent PldA and NAPE-PLD. This finding represents an important first step toward the development of small molecules possessing universal inhibition of divergent PLD enzymes to advance the field