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Arcanobacterium haemolyticum Phospholipase D Enzymatic Activity Promotes the Hemolytic Activity of the Cholesterol-Dependent Cytolysin Arcanolysin.

ABSTRACT: Arcanolysin, produced by the human pathogen Arcanobacterium haemolyticum, is a cholesterol-dependent cytolysin. To mediate the pore-formation process, arcanolysin is secreted by A. haemolyticum and then must interact with cholesterol embedded within a host membrane. However, arcanolysin must compete with membrane components, such as the phospholipid sphingomyelin, to interact with cholesterol and form pores. Cholesterol forms transient hydrogen bonds with the extracellular portion of sphingomyelin, shielding cholesterol from extracellular factors, including arcanolysin. A. haemolyticum also produces a sphingomyelin-specific phospholipase D, which removes the choline head from sphingomyelin, leaving cyclic-ceramide phosphate and eliminating the potential for cholesterol sequestration. We hypothesized that the enzymatic activity of phospholipase D decreases sphingomyelin-mediated cholesterol sequestration and increases cholesterol accessibility for arcanolysin. Using purified arcanolysin and phospholipase D, we demonstrate that the enzymatic activity of phospholipase D is necessary to promote arcanolysin-mediated hemolysis in both time- and concentration-dependent manners. Phospholipase D promotion of arcanolysin-mediated cytotoxicity was confirmed in Detroit 562 epithelial cells. Furthermore, we determined that incubating phospholipase D with erythrocytes corresponds with an increase in the amount of arcanolysin bound to host membranes. This observation suggests that phospholipase D promotes arcanolysin-mediated cytotoxicity by increasing the ability of arcanolysin to bind to a host membrane.

SUBMITTER: Gellings PS

PROVIDER: S-EPMC6024514 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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Arcanobacterium haemolyticum Phospholipase D Enzymatic Activity Promotes the Hemolytic Activity of the Cholesterol-Dependent Cytolysin Arcanolysin.

Gellings Patrick S PS McGee David J DJ

Toxins 20180523 6

Arcanolysin, produced by the human pathogen Arcanobacterium haemolyticum, is a cholesterol-dependent cytolysin. To mediate the pore-formation process, arcanolysin is secreted by A. haemolyticum and then must interact with cholesterol embedded within a host membrane. However, arcanolysin must compete with membrane components, such as the phospholipid sphingomyelin, to interact with cholesterol and form pores. Cholesterol forms transient hydrogen bonds with the extracellular portion ...[more]

PMID: 29882842

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Project description: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.

Dataset Information

Arcanobacterium haemolyticum Phospholipase D Enzymatic Activity Promotes the Hemolytic Activity of the Cholesterol-Dependent Cytolysin Arcanolysin.

Publications

<i>Arcanobacterium haemolyticum</i> Phospholipase D Enzymatic Activity Promotes the Hemolytic Activity of the Cholesterol-Dependent Cytolysin Arcanolysin.

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