Ubiquitin activates patatin-like phospholipases from multiple bacterial species

Abstract

Phospholipase A2 enzymes are ubiquitously distributed throughout the prokaryotic and eukaryotic kingdoms and are utilized in a wide array of cellular processes and physiological and immunological responses. Several patatin-like phospholipase homologs of ExoU from Pseudomonas aeruginosa were selected on the premise that ubiquitin activation of this class of bacterial enzymes was a conserved process. We found that ubiquitin activated all phospholipases tested in both in vitro and in vivo assays via a conserved serine-aspartate catalytic dyad. Ubiquitin chains versus monomeric ubiquitin were superior in inducing catalysis, and ubiquitin-like proteins failed to activate phospholipase activity. Toxicity studies in a prokaryotic dual-expression system grouped the enzymes into high- and low-toxicity classes. Toxicity measured in eukaryotic cells also suggested a two-tiered classification but was not predictive of the severity of cellular damage, suggesting that each enzyme may correspond to unique properties perhaps based on its specific biological function. Additional studies on lipid binding preference suggest that some enzymes in this family may be differentially sensitive to phosphatidyl-4,5-bisphosphate in terms of catalytic activation enhancement and binding affinity. Further analysis of the function and amino acid sequences of this enzyme family may lead to a useful approach to formulating a unifying model of how these phospholipases behave after delivery into the cytoplasmic compartment.

FIG 1

Comparison of three patatin-like homologs to ExoU from P. aeruginosa. (A) Diagram of the relative size and location of important residues in each of the four enzymes. GGG, glycine-rich motif postulated to participate in an oxyanion hole; S and D, catalytic residues serine and aspartate, which form a dyad; blue and black domains, regions characterized in ExoU to possess membrane and/or cofactor interaction activity. (B) Sequence alignment of the C-terminal domains corresponding to the black and blue C-terminal domains in panel A. The alpha-helical secondary structure of ExoU is shown as either black or blue arrows above the alignment for reference. In the alignment, sequence identity or similarity is shown as purple or pink highlights, respectively. Boxed amino acids were shown in previous studies to be important for ExoU activity when monoubiquitin serves as a cofactor. A consensus sequence is shown as calculated by the CLUSTALW algorithm.

FIG 2

Recombinant enzyme-ubiquitin coexpression is differentially toxic to E. coli. (A) Spot plates of 10-fold serially diluted strains containing plasmids pJY2 and pJN105 encoding His-tagged enzyme on agar medium containing either glucose (noninducing conditions) or l-arabinose (inducing conditions for production of the cloned enzyme). (B) Western blot of total bacterial lysates from the same strains as in panel A (lacking an expression construct containing ubiquitin) under inducing conditions. Total lysates were probed for the histidine tag of each enzyme after harvesting a constant volume at 1, 2, or 3 h of growth. The overall replication of bacteria was followed by blotting for DnaK, which increased at each time point. (C) Spot plates of 10-fold serially diluted bacteria containing an inducible monoubiquitin construct with an inducible construct expressing each parental enzyme or catalytic point mutant derivative spotted onto medium containing either glucose or l-arabinose. (D) Graph of cell viability of E. coli strains used in panel C when induced in liquid medium for the time indicated before plating on LB agar with glucose and antibiotics (n = 3 or 4).

FIG 3

Representative microscopic analysis of HeLa cell cultures transfected with enzyme-eGFP fusions expressed from the cytomegalovirus (CMV) promoter. (A) Fluorescence signal from eGFP fused to wild-type sequences of each enzyme or an eGFP control at 24 h posttransfection. (B) Propidium iodide staining of the corresponding fluorescence images in panel A. (C) Bright-field (phase-contrast) images of the corresponding fluorescence images in panel A. Scale bars represent 50 μm.

FIG 4

Quantitative analysis of HeLa cell cultures transfected with enzyme-eGFP fusions expressed from the CMV promoter. (A) Western blot signal of each enzyme-eGFP fusion from an equivalent amount of HeLa cell lysate at 24 h posttransfection. The S96A PFU-eGFP lysate was diluted 1:5 compared to the other samples due to the higher expression levels. Numbers are molecular masses in kilodaltons. (B) LDH release from HeLa cells transfected with equivalent amounts of plasmid DNA at 24 h posttransfection. Results are means from 4 independent experiments. (C) Average percentage of PI-positive cells per field in 3 independent fields from 3 replicate experiments. (D) Average percentage of rounded cell phenotypes in 3 independent microscopic fields from 3 replicate experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.0005; ****, P < 0.0001.

FIG 5

Liposome binding and the effect of PIP₂ on enzyme activity. (A) Apparent affinity constants for each enzyme binding to the PML model liposomes determined by a supernatant depletion assay. (B) Apparent affinity constants of each enzyme binding to the EML model liposomes, plus or minus PIP₂, as determined by a supernatant depletion assay. (C) Observed rate of PED6 hydrolysis by each enzyme under subsaturating enzymatic conditions in the presence of increasing amounts of PIP₂. As a negative control, PAU and PYU enzymes were assayed for PED6 hydrolysis in the absence of ubiquitin. (D) Observed rate of PED6 hydrolysis by each enzyme under subsaturating enzymatic conditions in the presence of POPS. All experimental data are from at least 3 independent experiments. *, P < 0.05; **, P < 0.01.