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1.  Phase Variation in the Helicobacter pylori Phospholipase A Gene and Its Role in Acid Adaptation 
Infection and Immunity  2001;69(12):7334-7340.
Previously, we have shown that Helicobacter pylori can spontaneously and reversibly change its membrane lipid composition, producing variants with low or high content of lysophospholipids. The “lyso” variant contains a high percentage of lysophospholipids, adheres better to epithelial cells, and releases more proteins such as urease and VacA, compared to the “normal” variant, which has a low content of lysophospholipids. Prolonged growth of the normal variant at pH 3.5, but not under neutral conditions, leads to enrichment of lyso variant colonies, suggesting that the colony switch is relevant to acid adaptation. In this study we show that the change in membrane lipid composition is due to phase variation in the pldA gene. A change in the (C) tract length of this gene results in reversible frameshifts, translation of a full-length or truncated pldA, and the production of active or inactive outer membrane phospholipase A (OMPLA). The role of OMPLA in determining the colony morphology was confirmed by the construction of an OMPLA-negative mutant. Furthermore, variants with an active OMPLA were able to survive acidic conditions better than variants with the inactive form. This explains why the lyso variant is selected at low pH. Our studies demonstrate that phase variation in the pldA gene, resulting in an active form of OMPLA, is important for survival under acidic conditions. We also demonstrated the active OMPLA genotype in fresh isolates of H. pylori from patients referred to gastroscopy for dyspepsia.
PMCID: PMC98819  PMID: 11705905
2.  Molecular characterization of enterobacterial pldA genes encoding outer membrane phospholipase A. 
Journal of Bacteriology  1994;176(3):861-870.
The pldA gene of Escherichia coli encodes an outer membrane phospholipase A. A strain carrying the most commonly used mutant pldA allele appeared to express a correctly assembled PldA protein in the outer membrane. Nucleotide sequence analysis revealed that the only difference between the wild type and the mutant is the replacement of the serine residue in position 152 by phenylalanine. Since mutants that lack the pldA gene were normally viable under laboratory conditions and had no apparent phenotype except for the lack of outer membrane phospholipase activity, the exact role of the enzyme remains unknown. Nevertheless, the enzyme seems to be important for the bacteria, since Western blotting (immunoblotting) and enzyme assays showed that it is widely spread among species of the family Enterobacteriaceae. To characterize the PldA protein further, the pldA genes of Salmonella typhimurium, Klebsiella pneumoniae, and Proteus vulgaris were cloned and sequenced. The cloned genes were expressed in E. coli, and their gene products were enzymatically active. Comparison of the predicted PldA primary structures with that of E. coli PldA revealed a high degree of homology, with 79% of the amino acid residues being identical in all four proteins. Implications of the sequence comparison for the structure and the structure-function relationship of PldA protein are discussed.
PMCID: PMC205124  PMID: 8300539
3.  Function of Neisserial Outer Membrane Phospholipase A in Autolysis and Assessment of Its Vaccine Potential  
Infection and Immunity  2005;73(4):2222-2231.
Outer membrane phospholipase A (OMPLA) is an outer membrane-localized enzyme, present in many gram-negative bacterial species. It is implicated in the virulence of several pathogens. Here, we investigated the presence, function, and vaccine potential of OMPLA in the human pathogen Neisseria meningitidis. Immunoblot analysis showed the presence of OMPLA in 28 out of 33 meningococcal strains investigated. The OMPLA-negative strains all contained a pldA gene, but these alleles contained premature stop codons. All six Neisseria gonorrhoeae strains tested, but only two out of seven commensal neisserial strains investigated, expressed OMPLA, showing that OMPLA is expressed by, but not limited to, many pathogenic neisserial strains. The function of OMPLA was investigated by assessing the phenotypes of isogenic strains, expressing no OMPLA, expressing wild-type levels of OMPLA, or overexpressing OMPLA. OMPLA exhibited phospholipase activity against endogenous phospholipids. Furthermore, OMPLA was characterized as an autolysin that acted under specific conditions, such as prolonged growth of the bacteria. The vaccine potential of the protein was investigated by immunizing mice with in vitro refolded, recombinant OMPLA. High levels of antibody titers were obtained, but the murine sera were neither bactericidal nor protective. Also, convalescent patients and vaccinee sera did not contain detectable levels of anti-OMPLA antibodies, indicating that OMPLA may not be sufficiently immunogenic to be included in a meningococcal vaccine.
PMCID: PMC1087465  PMID: 15784566
4.  Bacterial phospholipid hydrolysis enhances the destruction of Escherichia coli ingested by rabbit neutrophils. Role of cellular and extracellular phospholipases. 
Journal of Clinical Investigation  1990;85(6):1925-1935.
Escherichia coli ingested by PMN are promptly growth arrested but undergo limited destruction. We have studied bacterial phospholipid hydrolysis as a possible limiting factor in the disassembly of ingested E. coli, comparing the fates, during phagocytosis by rabbit peritoneal exudate PMN, of three isogenic strains, differing in their content of the pldA gene encoding the principal E. coli phospholipase A (PLA), i.e., pldA-, pldA+, pldA (the latter strain bearing the pldA gene in a multicopy plasmid resulting in a 20-fold increase in PLA content). Ingestion and growth inhibition (greater than 99% within 15 min) were the same for the three strains, but phospholipid degradation differed according to bacterial PLA content: pldA up to 60%, pldA+ up to 30%, and pldA- up to 20%. Since the pldA- strain has no activatable PLA, phospholipid degradation in this strain demonstrates the action of a PMN PLA. Added PLA2-rich ascitic fluid (AF) or purified AF PLA2 increased the rate and extent of degradation of the pldA- strain, provided the enzyme was added before ingestion was complete. 125I-AF-PLA2 binds to both E. coli and PMN and thus can enter the vacuole during phagocytosis. Although up to 50-fold more AF-PLA2 than the PLA2 content of the PMN could be loaded into the PMN in this way, degradation of pldA- E. coli did not exceed 30%. Increased phospholipid degradation had no effect on the degradation of bacterial macromolecules. In contrast, bacterial disassembly manifest as structural disorganization, release of bacterial protein derived material, and inhibition of protein synthesis were markedly enhanced when greater than 50% of prelabelled bacterial phospholipids were degraded. These findings reveal a link between envelope phospholipid degradation and overall bacterial destruction, suggesting therefore that factors limiting PLA action limit the destruction of E. coli ingested by PMN.
PMCID: PMC296660  PMID: 2189895
5.  Absence of the Outer Membrane Phospholipase A Suppresses the Temperature-Sensitive Phenotype of Escherichia coli degP Mutants and Induces the Cpx and ςE Extracytoplasmic Stress Responses 
Journal of Bacteriology  2001;183(18):5230-5238.
DegP is a periplasmic protease that is a member of both the ςE and Cpx extracytoplasmic stress regulons of Escherichia coli and is essential for viability at temperatures above 42°C. [U-14C]acetate labeling experiments demonstrated that phospholipids were degraded in degP mutants at elevated temperatures. In addition, chloramphenicol acetyltransferase, β-lactamase, and β-galactosidase assays as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that large amounts of cellular proteins are released from degP cells at the nonpermissive temperature. A mutation in pldA, which encodes outer membrane phospholipase A (OMPLA), was found to rescue degP cells from the temperature-sensitive phenotype. pldA degP mutants had a normal plating efficiency at 42°C, displayed increased viability at 44°C, showed no degradation of phospholipids, and released far lower amounts of cellular protein to culture supernatants. degP and pldA degP mutants containing chromosomal lacZ fusions to Cpx and ςE regulon promoters indicated that both regulons were activated in the pldA mutants. The overexpression of the envelope lipoprotein, NlpE, which induces the Cpx regulon, was also found to suppress the temperature-sensitive phenotype of degP mutants but did not prevent the degradation of phospholipids. These results suggest that the absence of OMPLA corrects the degP temperature-sensitive phenotype by inducing the Cpx and ςE regulons rather than by inactivating the phospholipase per se.
PMCID: PMC95403  PMID: 11514504
6.  Molecular characterization of pldA, the structural gene for a phospholipase A from Campylobacter coli, and its contribution to cell-associated hemolysis. 
Infection and Immunity  1997;65(4):1172-1180.
A gene (pldA) encoding a 35.0-kDa protein with significant homology to the Escherichia coli outer membrane phospholipase was identified upstream of an operon encoding an enterochelin transport system in Campylobacter coli. The results of this study suggest that this gene encodes an outer membrane phospholipase A in C. coli. First, expression of the pldA gene product in a PldA-deficient mutant of E. coli led to the restoration of phospholipase A activity. The recombinant product also partitioned to the outer membrane, suggesting that it may be similarly located in C. coli. Second, heterologous overexpression in E. coli, followed by in vitro folding and purification of C. coli PldA, resulted in pure protein which displayed calcium-dependent lysophospholipase and phospholipase A activities in vitro. Finally, mutants of C. coli in which the pldA gene had been inactivated by allelic exchange were deficient in phospholipase A activity. Phospholipases are associated with lysis of erythrocytes by a number of bacterial pathogens. The pldA mutant was shown to have a reduced hemolytic activity compared to the wild-type strain, suggesting a role for the phospholipase A in the lysis of erythrocytes by C. coli. Since hemolysins are intimately associated with the disease-causing potential of a number of bacterial pathogens, it is likely that the phospholipase A plays some role in Campylobacter virulence.
PMCID: PMC175114  PMID: 9119448
7.  Topology of the outer membrane phospholipase A of Salmonella typhimurium. 
Journal of Bacteriology  1997;179(11):3443-3450.
The outer membrane phospholipase A (OMPLA) of Enterobacteriaceae has been proposed to span the membrane 14 times as antiparallel amphipathic beta-strands, thereby exposing seven loops to the cell surface. We have employed the epitope insertion method to probe the topology of OMPLA of Salmonella typhimurium. First, missense mutations were introduced at various positions in the pldA gene, encoding OMPLA, to create unique BamHI sites. These BamHI sites were subsequently used to insert linkers, encoding a 16-amino-acid B-cell epitope. Proper assembly of all mutant proteins was revealed by their heat modifiability in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The accessibility of the inserted epitopes was assessed. Immunofluorescence analysis of intact cells with antibodies against the inserted epitope showed that three of seven predicted loops are indeed cell surface exposed. Trypsin accessibility experiments verified the cell surface exposure of two additional loops and provided support for the proposed periplasmic localization of three predicted turns. For two other predicted exposed loops, the results were not conclusive. These results support to a large extent the proposed topology model of OMPLA. Furthermore, the observation that the substitutions Glu66Pro and Glu247Gly virtually abolished enzymatic activity indicates that these residues might play a major role in catalysis.
PMCID: PMC179134  PMID: 9171386
8.  Genome-wide and molecular evolution analyses of the phospholipase D gene family in Poplar and Grape 
BMC Plant Biology  2010;10:117.
The Phospholipase D (PLD) family plays an important role in the regulation of cellular processes in plants, including abscisic acid signaling, programmed cell death, root hair patterning, root growth, freezing tolerance and other stress responses. PLD genes constitute an important gene family in higher plants. However, until now our knowledge concerning the PLD gene family members and their evolutionary relationship in woody plants such as Poplar and Grape has been limited.
In this study, we have provided a genome-wide analysis of the PLD gene family in Poplar and Grape. Eighteen and eleven members of the PLD gene family were identified in Poplar and Grape respectively. Phylogenetic and gene structure analyses showed that the PLD gene family can be divided into 6 subgroups: α, β/γ, δ, ε, ζ, and φ, and that the 6 PLD subgroups originated from 4 original ancestors through a series of gene duplications. Interestingly, the majority of the PLD genes from both Poplar (76.5%, 13/17) and Grape (90.9%, 10/11) clustered closely together in the phylogenetic tree to the extent that their evolutionary relationship appears more tightly linked to each other, at least in terms of the PLD gene family, than it does to either Arabidopsis or rice. Five pairs of duplicated PLD genes were identified in Poplar, more than those in Grape, suggesting that frequent gene duplications occurred after these species diverged, resulting in a rapid expansion of the PLD gene family in Poplar. The majority of the gene duplications in Poplar were caused by segmental duplication and were distinct from those in Arabidopsis, rice and Grape. Additionally, the gene duplications in Poplar were estimated to have occurred from 11.31 to 13.76 million years ago, which are later than those that occurred in the other three plant species. Adaptive evolution analysis showed that positive selection contributed to the evolution of the PXPH- and SP-PLDs, whereas purifying selection has driven the evolution of C2-PLDs that contain a C2 domain in their N-terminal. Analyses have shown that the C2-PLDs generally contain 23 motifs, more than 17 motifs in PXPH-PLDs that contain PX and PH domains in N-terminal. Among these identified motifs, eight, (6, 8, 5, 4, 3, 14, 1 and 19) were shared by both the C2- and PXPH-PLD subfamilies, implying that they may be necessary for PLD function. Five of these shared motifs are located in the central region of the proteins, thus strongly suggesting that this region containing a HKD domain (named after three conserved H, K and D residues) plays a key role in the lipase activity of the PLDs.
As a first step towards genome wide analyses of the PLD genes in woody plants, our results provide valuable information for increasing our understanding of the function and evolution of the PLD gene family in higher plants.
PMCID: PMC3095279  PMID: 20565843
9.  The Vps/VacJ ABC Transporter Is Required for Intercellular Spread of Shigella flexneri 
Infection and Immunity  2014;82(2):660-669.
The Vps/VacJ ABC transporter system is proposed to function in maintaining the lipid asymmetry of the outer membrane. Mutations in vps or vacJ in Shigella flexneri resulted in increased sensitivity to lysis by the detergent sodium dodecyl sulfate (SDS), and the vpsC mutant showed minor differences in its phospholipid profile compared to the wild type. vpsC mutants were unable to form plaques in cultured epithelial cells, but this was not due to a failure to invade, to replicate intracellularly, or to polymerize actin via IcsA for movement within epithelial cells. The addition of the outer membrane phospholipase gene pldA on a multicopy plasmid in a vpsC or vacJ mutant restored its resistance to SDS, suggesting a restoration of lipid asymmetry to the outer membrane. However, the pldA plasmid did not restore the mutant's ability to form plaques in tissue culture cells. Increased PldA levels also failed to restore the mutant's phospholipid profile to that of the wild type. We propose a dual function of the Vps/VacJ ABC transporter system in S. flexneri in both the maintenance of lipid asymmetry in the outer membrane and the intercellular spread of the bacteria between adjacent epithelial cells.
PMCID: PMC3911398  PMID: 24478081
10.  Determinants of activation by complement of group II phospholipase A2 acting against Escherichia coli. 
Infection and Immunity  1996;64(7):2425-2430.
Prompt killing of many strains of Escherichia coli during phagocytosis in vitro by isolated polymorphonuclear leukocytes (PMN) requires the presence of nonlethal doses of nonimmune serum (B. A. Mannion, J. Weiss, and P. Elsbach, J. Clin. Invest. 86:631-641, 1990). Because this requirement is bypassed in a phospholipase A (PLA)-rich mutant (pldA ) of E. coli, we have examined the effect of serum on bacteria] phospholipid (PL) degradation during phagocytosis of wild-type (pldA+) and PLA-deficient (pldA) E. coli. In parallel with increased killing, nonlethal doses of serum increased the degradation of prelabeled bacterial PL during phagocytosis by two- to fivefold, to nearly the same levels (ca. 50 to 60%) as those produced during phagocytosis of E. coli pldA in the absence of serum. The effects on the E. coli pldA mutant imply that there is a serum-mediated enhancement of granule-associated group II PMN PLA2 activity. At the same doses, serum promoted action against E. coli in the presence of purified rabbit and human group II PLA2 but did not activate bacterial PLA. Related PLA2s that lack specific structural determinants needed for optimal activity against E. coli treated with the bactericidal/permeability-increasing protein (BPI) of PMN are also less active than wild-type group II PLA2 against serum-treated E. coli. Treatment of E. coli with C7- or C9-depleted serum did not enhance bacterial killing or PL degradation during phagocytosis or the action of purified PLA2. In summary, these findings suggest that (i) nonlethal assemblies of the membrane attack complex promote intracellular killing and destruction of E. coli ingested by PMN, in part by promoting the action of granule-associated PLA2 against ingested bacteria, and (ii) structural determinants first implicated in PLA2 action against BPI-treated E. coli are also important in PLA2 action in concert with other host defense systems, such as complement.
PMCID: PMC174093  PMID: 8698462
11.  Application of High-Density Array-Based Signature-Tagged Mutagenesis To Discover Novel Yersinia Virulence-Associated Genes 
Infection and Immunity  2001;69(12):7810-7819.
Yersinia pestis, the causative agent of plague, and the enteropathogen Yersinia pseudotuberculosis have nearly identical nucleotide similarity yet cause markedly different diseases. To investigate this conundrum and to study Yersinia pathogenicity, we developed a high-density oligonucleotide array-based modification of signature-tagged mutagenesis (STM). Y. pseudotuberculosis YPIII mutants constructed with the tagged transposons were evaluated in the murine yersiniosis infection model. The DNA tags were amplified using biotinylated primers and hybridized to high-density oligonucleotide arrays containing DNA complementary to the tags. Comparison of the hybridization signals from input pools and output pools identified a mutant whose relative abundance was significantly reduced in the output pool. Sequence data from 31 transposon insertion regions was compared to the complete Y. pestis CO92 genome sequence. The 26 genes present in both species were found to be almost identical, but five Y. pseudotuberculosis genes identified through STM did not have counterparts in the Y. pestis genome and may contribute to the different tropisms in these closely related pathogens. Potential virulence genes identified include those involved in lipopolysaccharide biosynthesis, adhesion, phospholipase activity, iron assimilation, and gene regulation. The phospholipase A (PldA) mutant exhibited reduced phospholipase activity compared to the wild-type strain and in vivo attenuation of the mutant was confirmed. The combination of optimized double tag sequences and high-density array hybridization technology offers improved performance, efficiency, and reliability over classical STM and permits quantitative analysis of data.
PMCID: PMC98877  PMID: 11705963
12.  Interleukin-8 is the single most up-regulated gene in whole genome profiling of H. pylori exposed gastric epithelial cells 
BMC Microbiology  2012;12:9.
The association between Helicobacter pylori infection and upper gastrointestinal disease is well established. However, only a small fraction of H. pylori carriers develop disease, and there are great geographical differences in disease penetrance. The explanation to this enigma lies in the interaction between the bacterium and the host. H. pylori Outer Membrane Phospholipase A (OMPLA) has been suggested to play a role in the virulence of this bacterium. The aim of this study was to profile the most significant cellular pathways and biological processes affected in gastric epithelial cells during 24 h of H. pylori exposure, and to study the inflammatory response to OMPLA+ and OMPLA- H. pylori variants.
Interleukin-8 was the most significantly up-regulated gene and appears to play a paramount role in the epithelial cell response to H. pylori infection and in the pathological processes leading to gastric disease. MAPK and NF-kappaB cellular pathways were powerfully activated, but did not seem to explain the impressive IL-8 response. There was marked up-regulation of TP53BP2, whose corresponding protein ASPP2 may interact with H. pylori CagA and cause marked p53 suppression of apoptosis. Other regulators of apoptosis also showed abberant regulation. We also identified up-regulation of several oncogenes and down-regulation of tumor suppressor genes as early as during the first 24 h of infection. H. pylori OMPLA phase variation did not seem to influence the inflammatory epithelial cell gene response in this experiment.
In whole genome analysis of the epithelial response to H. pylori exposure, IL-8 demonstrated the most marked up-regulation, and was involved in many of the most important cellular response processes to the infection. There was dysregulation of apoptosis, tumor suppressor genes and oncogenes as early as in the first 24 h of H. pylori infection, which may represent early signs of gastric tumorigenesis. OMPLA+/-did not affect the acute inflammatory response to H. pylori.
PMCID: PMC3292955  PMID: 22248188
13.  Molecular diversity of phospholipase D in angiosperms 
BMC Genomics  2002;3:2.
The phospholipase D (PLD) family has been identified in plants by recent molecular studies, fostered by the emerging importance of plant PLDs in stress physiology and signal transduction. However, the presence of multiple isoforms limits the power of conventional biochemical and pharmacological approaches, and calls for a wider application of genetic methodology.
Taking advantage of sequence data available in public databases, we attempted to provide a prerequisite for such an approach. We made a complete inventory of the Arabidopsis thaliana PLD family, which was found to comprise 12 distinct genes. The current nomenclature of Arabidopsis PLDs was refined and expanded to include five newly described genes. To assess the degree of plant PLD diversity beyond Arabidopsis we explored data from rice (including the genome draft by Monsanto) as well as cDNA and EST sequences from several other plants. Our analysis revealed two major PLD subfamilies in plants. The first, designated C2-PLD, is characterised by presence of the C2 domain and comprises previously known plant PLDs as well as new isoforms with possibly unusual features-catalytically inactive or independent on Ca2+. The second subfamily (denoted PXPH-PLD) is novel in plants but is related to animal and fungal enzymes possessing the PX and PH domains.
The evolutionary dynamics, and inter-specific diversity, of plant PLDs inferred from our phylogenetic analysis, call for more plant species to be employed in PLD research. This will enable us to obtain generally valid conclusions.
PMCID: PMC77410  PMID: 11876823
14.  Arcanobacterium haemolyticum phospholipase D is genetically and functionally similar to Corynebacterium pseudotuberculosis phospholipase D. 
Infection and Immunity  1993;61(10):4310-4316.
Arcanobacterium haemolyticum, a pathogen of the human upper respiratory tract and other systems, has been reported to produce soluble toxins, including a phospholipase D (PLD). We confirmed production of PLD by this organism and cloned and sequenced pld. Arcanobacterial PLD (PLD-A) was found to be a protein of approximately 31.5 kDa with a pI of approximately 9.4. Cosmid cloning, followed by subcloning into phagemid pBluescriptIISK+, yielded Escherichia coli(pAh140), a recombinant with a gene product corresponding to PLD-A. Evidence of PLD activity was found by three assays in supernatant fluid of cultures of E. coli(pAh140) and A. haemolyticum, but not in E. coli(pBluescriptIISK+). Experiments to determine if this protein was secreted were not conducted, but previous work with PLD from Corynebacterium pseudotuberculosis suggested that the presence of the enzyme in culture supernatant fluids was due to lysis of E. coli rather than to active transport. Antibodies in polyclonal sera from goats immunized with native or recombinant PLD-A neutralized native and recombinant PLD-A activity, and antibodies against native or recombinant PLD from C. pseudotuberculosis (PLD-P) partially neutralized native and recombinant PLD-A. Antibodies prepared against recombinant PLD-A labelled both recombinant and native PLD-A in Western blots (immunoblots) and dot blots, but antibodies against PLD-P did not. Sequencing of the insert in pAh140 revealed an open reading frame of 930 bp coding for 309 amino acids, including a putative signal sequence of 26 amino acids (3.2 kDa, determined on the basis of homology with the 24-amino-acid signal sequence of pld from C. pseudotuberculosis bv. ovis) and the mature PLD protein (31.5 kDa). Sequence comparisons of coding regions revealed 65% DNA homology with pld genes from C. pseudotuberculosis and Corynebacterium ulcerans. Comparison of amino acid sequences revealed 64% homology of PLD-A both with PLD-P and with PLD produced by C. ulcerans.
PMCID: PMC281159  PMID: 8406819
15.  Localization of Phospholipase D1 to Caveolin-enriched Membrane via Palmitoylation: Implications for Epidermal Growth Factor Signaling 
Molecular Biology of the Cell  2002;13(11):3976-3988.
Phospholipase D (PLD) has been suggested to mediate epidermal growth factor (EGF) signaling. However, the molecular mechanism of EGF-induced PLD activation has not yet been elucidated. We investigated the importance of the phosphorylation and compartmentalization of PLD1 in EGF signaling. EGF treatment of COS-7 cells transiently expressing PLD1 stimulated PLD1 activity and induced PLD1 phosphorylation. The EGF-induced phosphorylation of threonine147 was completely blocked and the activity of PLD1 attenuated by point mutations (S2A/T147A/S561A) of PLD1 phosphorylation sites. The expression of a dominant negative PKCα mutant by adenovirus-mediated gene transfer greatly inhibited the phosphorylation and activation of PLD1 induced by EGF in PLD1-transfected COS-7 cells. EGF-induced PLD1 phosphorylation occurred primarily in the caveolin-enriched membrane (CEM) fraction, and the kinetics of PLD1 phosphorylation in the CEM were strongly correlated with PLD1 phosphorylation in the total membrane. Interestingly, EGF-induced PLD1 phosphorylation and activation and the coimmunoprecipitation of PLD1 with caveolin-1 and the EGF receptor in the CEM were significantly attenuated in the palmitoylation-deficient C240S/C241S mutant, which did not localize to the CEM. Immunocytochemical analysis revealed that wild-type PLD1 colocalized with caveolin-1 and the EGF receptor and that phosphorylated PLD1 was localized exclusively in the plasma membrane, although some PLD1 was also detected in vesicular structures. Transfection of wild-type PLD1 but not of C240S/C241S mutant increased EGF-induced raf-1 translocation to the CEM and ERK phosphorylation. This study shows, for the first time, that EGF-induced PLD1 phosphorylation and activation occur in the CEM and that the correct localization of PLD1 to the CEM via palmitoylation is critical for EGF signaling.
PMCID: PMC133608  PMID: 12429840
16.  Cell-Associated Hemolysis Induced by Helicobacter pylori Is Mediated by Phospholipases with Mitogen-Activated Protein Kinase-Activating Properties 
Journal of Clinical Microbiology  2012;50(3):1014-1018.
Pathogenic Helicobacter pylori strains can selectively activate epithelial mitogen-activated protein kinase (MAPK) signaling pathways linked with disease. We now demonstrate that H. pylori-induced hemolysis is strain specific and is mediated by phospholipases PldA1 and PldD. Inactivation of PldD inhibited activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), indicating that H. pylori hemolytic phospholipases also harbor MAPK-activating properties.
PMCID: PMC3295145  PMID: 22205825
17.  Lipoprotein nature of the colicin A lysis protein: effect of amino acid substitutions at the site of modification and processing. 
Journal of Bacteriology  1987;169(5):2187-2194.
The colicin A lysis protein (Cal) is required for the release of colicin A to the medium by producing bacteria. This protein is produced in a precursor form that contains a cysteine at the cleavage site (-Leu-Ala-Ala-Cys). The precursor must be modified by the addition of lipid before it can be processed. The maturation is prevented by globomycin, an inhibitor of signal peptidase II. Using oligonucleotide-directed mutagenesis, the alanine and cystein residues in the -1 and +1 positions of the cleavage site were replaced by proline and threonine residues, respectively, in two different constructs. Both substitutions prevented the normal modification and cleavage of the protein. The marked activation of the outer membrane detergent-resistant phospholipase A observed with wild-type Cal was not observed with the Cal mutants. Both Cal mutants were also defective for the secretion of colicin A. In one mutant, the signal peptide appeared to be cleaved off by an alternative pathway involving signal peptidase I. Electron microscope studies with immunogold labeling of colicin A on cryosections of pldA and cal mutant cells indicated that the colicin remains in the cytoplasm and is not transferred to the periplasmic space. These results demonstrate that Cal must be modified and processed to activate the detergent-resistant phospholipase A and to promote release of colicin A.
PMCID: PMC212125  PMID: 3571165
18.  Discovery of Desketoraloxifene Analogues as Inhibitors of Mammalian, Pseudomonas aeruginosa, and NAPE Phospholipase D Enzymes 
ACS chemical biology  2014;10(2):421-432.
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.
PMCID: PMC4336625  PMID: 25384256
19.  Diverse type VI secretion phospholipases are functionally plastic antibacterial effectors 
Nature  2013;496(7446):508-512.
Membranes allow the compartmentalization of biochemical processes and are therefore fundamental to life. The conservation of the cellular membrane, combined with its accessibility to secreted proteins, has made it a common target of factors mediating antagonistic interactions between diverse organisms. Here we report the discovery of a diverse superfamily of bacterial phospholipase enzymes. Within this superfamily, we defined enzymes with phospholipase A1 (PLA1) and A2 (PLA2) activity, which are common in host cell-targeting bacterial toxins and the venoms of certain insects and reptiles1,2. However, we find that the fundamental role of the superfamily is to mediate antagonistic bacterial interactions as effectors of the type VI secretion system (T6SS) translocation apparatus; accordingly, we name these proteins type VI lipase effectors (Tle). Our analyses indicate that PldA of Pseudomonas aeruginosa, a eukaryotic-like phospholipase D (PLD)3, is a member of the Tle superfamily and the founding substrate of the haemolysin co-regulated protein secretion island II T6SS (H2-T6SS). While prior studies have specifically implicated PldA and the H2-T6SS in pathogenesis3–5, we uncovered a specific role for the effector and its secretory machinery in intra- and inter-species bacterial interactions. Furthermore we find that this effector achieves its antibacterial activity by degrading phosphatidylethanolamine (PE), the major component of bacterial membranes. The surprising finding that virulence-associated phospholipases can serve as specific antibacterial effectors suggests that interbacterial interactions are a relevant factor driving the ongoing evolution of pathogenesis.
PMCID: PMC3652678  PMID: 23552891
20.  Genetic Mapping of the Locus for Detergent-Resistant Phospholipase A (pldA) in Escherichia coli K-12 
Journal of Bacteriology  1974;119(2):543-546.
An Escherichia coli K-12 mutant deficient for detergent-resistant (DR) phospholipase A, a principal enzyme catalyzing the first step in phospholipid degradation, was characterized genetically. The mutation was found to affect the locus pldA (phospholipid degradation), which is cotransducible both with ilv and metE at a frequencies of 13 and 78%, respectively, and shown to lie between the ilv and metE loci on the E. coli chromosome. DR phospholipase A1 and A2 activities were simultaneously transduced with pldA+ by phage P1; therefore it is proposed that DR phospholipase A has both activities.
PMCID: PMC245639  PMID: 4604508
21.  The acylated precursor form of the colicin A lysis protein is a natural substrate of the DegP protease. 
Journal of Bacteriology  1989;171(11):6316-6322.
The acylated precursor form of the colicin A lysis protein (pCalm) is specifically cleaved by the DegP protease into two acylated fragments of 6 and 4.5 kilodaltons (kDa). This cleavage was observed after globomycin treatment, which inhibits the processing of pCalm into mature colicin A lysis protein (Cal) and the signal peptide. The cleavage took place in lpp, pldA, and wild-type strans carrying plasmids which express the lysis protein following SOS induction and also in cells containing a plasmid which expresses it under the control of the tac promoter. Furthermore, the DegP protease was responsible for the production of two acylated Cal fragments of 3 and 2.5 kDa in cells carrying plasmids which overproduce the Cal protein, without treatment with globomycin. DegP could also cleave the acylated precursor form of a mutant Cal protein containing a substitution in he amino-terminal portion of the protein, but not that of a mutant Cal containing a frameshift mutation in its carboxyl-terminal end. The functions of Cal in causing protein release, quasi-lysis, and lethality were increased in degP41 cells, suggesting that mature Cal was produced in higher amounts in the mutant than in the wild type. These effects were limited in cells deficient in phospholipase A. Interactions between the DegP protease and phospholipase A were suggested by the characteristics of degP pldA double mutants.
PMCID: PMC210505  PMID: 2681163
22.  Epidemiological association of Campylobacter jejuni groups with pathogenicity-associated genetic markers 
BMC Microbiology  2012;12:171.
Campylobacter jejuni, the most leading cause for bacterial gastroenteritis worldwide, shows a high genetic diversity among its isolates. Recently, we demonstrated the existence of six C. jejuni-groups by combining MLST with six genetic markers. These groups were further characterized by the detection of cj1321-cj1326, fucP, cj0178, cj0755/cfrA, ceuE, pldA, cstII, and cstIII in order (I.) to show further associations between these different genetic markers and MLST CCs. Moreover, different studies were able to associate several of these markers: a sialylated lipoologosaccharide (cstII/III+), the gamma-glytamyl-transpeptidase (ggt+), and the absence of a certain allele of the enterochelin-uptake-binding-protein (ceuE11168-) with severe campylobacteriosis, bloody diarrhea and unpleasant outcome. Additionally more than half of human Campylobacter-isolates were assigned to a non-livestock clade associated with the absence of cj1321-cj1326. These isolates were considered as mere colonizers.
From the combination of marker genes, the ratio of human isolates in a specific group, and clinical data (II.) it should be demonstrated to which of the previous defined groups these Campylobacter-subpopulations, associated with higher virulence, correspond.
Besides the marker gene pldA, all new estimated genetic markers show significant differences in their distribution among the various MLST-based groups. Especially the genes for cj1321-cj1326, fucP, cj0178, cj0755/cfrA are widely associated with each other and split the study population into two major and seven intermediate groups substantiating the previous group-definition, whereas cstII and cstIII indicate at least three groups following an independent distribution pattern.
Based on these data a group of C. jejuni-isolates characterized by the presence of ansB, dmsA, ggt, and the absence of cj1365c, cj1585c, cj1321-cj1326, fucP, cj0178, cj0755/cfrA, and cstII/III was associated with a higher prevalence in human campylobacteriosis, bloody diarrhea as well as hospitalization and bears obviously a higher virulence for humans. In contrast to that better livestock-adapted groups characterized by the ability to utilize L-fucose and the presence of all of the five identified putative C. jejuni iron-uptake systems as well as cj1321-cj1326, cj1365c, cj1585c, and cstII and/or cstIII (sialylated lipoologosaccharide) is more prevalent in animal hosts and was secondary associated with less severe campylobacteriosis.
PMCID: PMC3487957  PMID: 22873291
Campylobacter jejuni; Subgroups; fucP; cj0178; cj0755/cfrA; LOS class; Flagellin glycosylation; Virulence
23.  PLD3 is accumulated on neuritic plaques in Alzheimer’s disease brains 
Recently, a whole-exome sequencing (WES) study showed that a rare variant rs145999145 composed of p.Val232Met located in exon 7 of the phospholipase D3 (PLD3) gene confers a doubled risk for late-onset Alzheimer’s disease (AD). Knockdown of PLD3 elevates the levels of extracellular amyloid-beta (Aβ), suggesting that PLD3 acts as a negative regulator of Aβ precursor protein (APP) processing. However, the precise cellular location and distribution of PLD3 in AD brains remain largely unknown.
By quantitative RT-PCR (qPCR), western blot, immunohistochemistry, and bioinformatics analysis, we studied PLD3 expression patterns and levels in a series of AD and control brains, including amyotrophic lateral sclerosis, Parkinson’s disease, multiple system atrophy, and non-neurological cases.
The levels of PLD3 mRNA and protein expression were reduced modestly in AD brains, compared with those in non-AD brains. In all brains, PLD3 was expressed constitutively in cortical neurons, hippocampal pyramidal and granular neurons but not in glial cells. Notably, PLD3 immunoreactivity was accumulated on neuritic plaques in AD brains. We identified the human granulin (GRN) gene encoding progranulin (PRGN) as one of most significant genes coexpressed with PLD3 by bioinformatics database search. PLD3 was actually coexpressed and interacted with PGRN both in cultured cells in vitro and in AD brains in vivo.
We identified an intense accumulation of PLD3 on neuritic plaques coexpressed with PGRN in AD brains, suggesting that PLD3 plays a key role in the pathological processes of AD.
PMCID: PMC4255636  PMID: 25478031
24.  Gene Cloning and Molecular Characterization of an Extracellular Poly(l-Lactic Acid) Depolymerase from Amycolatopsis sp. Strain K104-1 
Journal of Bacteriology  2005;187(21):7333-7340.
We have isolated a polylactide or poly(l-lactic acid) (PLA)-degrading bacterium, Amycolatopsis sp. strain K104-1, and purified PLA depolymerase (PLD) from the culture fluid of the bacterium. Here, we cloned and expressed the pld gene encoding PLD in Streptomyces lividans 1326 and characterized a recombinant PLD (rPLD) preparation. We also describe the processing mechanism from nascent PLD to mature PLD. The pld gene encodes PLD as a 24,225-Da polypeptide consisting of 238 amino acids. Biochemical and Western immunoblot analyses of PLD and its precursors revealed that PLD is synthesized as a precursor (prepro-type), requiring proteolytic cleavage of the N-terminal 35-amino-acid extension including the 26-amino-acid signal sequence and 9-residue prosequence to generate the mature enzyme of 20,904 Da. The cleavage of the prosequence was found to be autocatalytic. PLD showed about 45% similarity to many eukaryotic serine proteases. In addition, three amino acid residues, H57, D102, and S195 (chymotrypsin numbering), which are implicated in forming the catalytic triad necessary for cleavage of amide bond of substrates in eukaryotic serine proteases, were conserved in PLD as residues H74, D111, and S197. The G193 residue (chymotrypsin numbering), which is implicated in forming an oxyanion hole with residue S195 and forms an important hydrogen bond for interaction with the carbonyl group of the scissile peptide bond, was also conserved in PLD. The functional analysis of the PLD mutants H74A, D111A, and S197A revealed that residues H74, D111, and S197 are important for the depolymerase and caseinolytic activities of PLD and for cleavage of the prosequence from pro-type PLD to form the mature one. The PLD preparation had elastase activity which was not inhibited by 1 mM elastatinal, which is 10 times higher than needed for complete inhibition of porcine pancreatic elastase. The rPLD preparation degraded PLA with an average molecular mass of 220 kDa into lactic acid dimers through lactic acid oligomers and finally into lactic acid. The PLD preparation bound to high polymers of 3-hydoxybutyrate, ɛ-caprolacton, and butylene succinate as well as PLA, but it degraded only PLA.
PMCID: PMC1272983  PMID: 16237016
25.  Comparative analysis of Klebsiella pneumoniae genomes identifies a phospholipase D family protein as a novel virulence factor 
BMC Biology  2014;12:41.
Klebsiella pneumoniae strains are pathogenic to animals and humans, in which they are both a frequent cause of nosocomial infections and a re-emerging cause of severe community-acquired infections. K. pneumoniae isolates of the capsular serotype K2 are among the most virulent. In order to identify novel putative virulence factors that may account for the severity of K2 infections, the genome sequence of the K2 reference strain Kp52.145 was determined and compared to two K1 and K2 strains of low virulence and to the reference strains MGH 78578 and NTUH-K2044.
In addition to diverse functions related to host colonization and virulence encoded in genomic regions common to the four strains, four genomic islands specific for Kp52.145 were identified. These regions encoded genes for the synthesis of colibactin toxin, a putative cytotoxin outer membrane protein, secretion systems, nucleases and eukaryotic-like proteins. In addition, an insertion within a type VI secretion system locus included sel1 domain containing proteins and a phospholipase D family protein (PLD1). The pld1 mutant was avirulent in a pneumonia model in mouse. The pld1 mRNA was expressed in vivo and the pld1 gene was associated with K. pneumoniae isolates from severe infections. Analysis of lipid composition of a defective E. coli strain complemented with pld1 suggests an involvement of PLD1 in cardiolipin metabolism.
Determination of the complete genome of the K2 reference strain identified several genomic islands comprising putative elements of pathogenicity. The role of PLD1 in pathogenesis was demonstrated for the first time and suggests that lipid metabolism is a novel virulence mechanism of K. pneumoniae.
PMCID: PMC4068068  PMID: 24885329
Genome sequencing; Host-microbe interactions; Bacterial pathogenesis; Lipid metabolism

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