Purification and Further Characterization of a Haemolysin of Actinomyces pyogenes

Ding, H.; Lämmler, Christoph

doi:10.1111/j.1439-0450.1996.tb00303.x

Cited by 25 publications

(17 citation statements)

References 31 publications

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“…The predicted mature form of PLO would be 55.1-kDa with an isoelectric point (pI) of 9.3. These values are in good agreement with molecular mass and pI values obtained for purified native PLO (13). The TACY family is characterized by a highly conserved undecapeptide located near the C terminus of the protein.…”

Section: Resultssupporting

confidence: 75%

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The Arcanobacterium (Actinomyces) pyogenes hemolysin, pyolysin, is a novel member of the thiol-activated cytolysin family

et al. 1997

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Arcanobacterium (Actinomyces) pyogenes, an animal pathogen, produces a hemolytic exotoxin, pyolysin (PLO). The gene encoding PLO was cloned, and sequence analysis revealed an open reading frame of 1,605 bp encoding a protein of 57.9 kDa. PLO has 30 to 40% identity with the thiol-activated cytolysins (TACYs) of a number of gram-positive bacteria. The activity of PLO was found to be very similar to those of other TACYs, except that it was not thiol activated. The highly conserved TACY undecapeptide is divergent in PLO; in particular, the cysteine residue required for thiol activation has been replaced with alanine. However, mutagenesis of the alanine residue to cysteine did not confer thiol activation on PLO, suggesting a conformational difference in the undecapeptide region of this toxin. Specific antibodies against purified, recombinant PLO completely neutralized the hemolytic activity of A. pyogenes, suggesting that this organism produces a single hemolysin. Furthermore, these antibodies could passively protect mice against lethal challenge with A. pyogenes, suggesting that like other TACYs PLO is an important virulence factor in the pathogenesis of this organism.Arcanobacterium (Actinomyces) pyogenes (36), a gram-positive, normally commensal bacterium, resides on the mucous membranes of cattle, sheep, swine, and other economically important animals (10). It can, through an as yet unknown mechanism, disseminate to cause a wide variety of nonspecific purulent infections involving the visceral organs (19,25,45) and joints (18), as well as acute purulent mastitis (20), chronic abscessing mastitis (37), and abortion (42).Despite the versatility of A. pyogenes as an agent of disease in domestic animals, specific determinants of its virulence have not been characterized. A. pyogenes produces several potential virulence factors including a DNase (24) and several proteases (40,44). In addition to these factors, A. pyogenes produces hemolytic exotoxin pyolysin (PLO) (13), which is cytolytic for the erythrocytes of a number of animal species (14), as well as dermonecrotic and lethal for laboratory animals (27). PLO also exhibits cytotoxic effects on bovine polymorphonuclear leukocytes (PMN) and kangaroo kidney cells (13). PLO was reported to be oxygen stable, and its activity was reported to be unaffected by cholesterol (14). The role of this toxin in pathogenesis is unclear. However, it is expressed in vivo and is immunogenic, as antihemolysin antibodies have been found in the sera of naturally (26) and experimentally (28) infected animals.In order to investigate the role of this toxin in the virulence of A. pyogenes, the gene encoding PLO was cloned and sequenced. The work reported here examines the activity of PLO, its similarity to a family of thiol-activated cytolysins (TACYs) produced by a number of gram-positive bacteria (1,8,12), and its potential role in virulence. MATERIALS AND METHODS Bacterial strains and growth conditions. Escherichia coli DH5␣ (Bethesda Research Laboratories [BRL]), DH5␣FЈlacIq (BRL), and L...

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Section: Resultssupporting

confidence: 75%

“…PLO may mediate macrophage or PMN lysis and, hence, escape from the phagocytic process, contributing to the virulence of A. pyogenes. This hypothesis is supported by studies of purified native PLO in which it displayed time-and dose-dependent cytotoxic effects on bovine PMN (13).…”

Section: Discussionsupporting

confidence: 58%

The Arcanobacterium (Actinomyces) pyogenes hemolysin, pyolysin, is a novel member of the thiol-activated cytolysin family

et al. 1997

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“…Despite its initial identification as a haemolysin, PLO is cytolytic for a number of cell types, including PMNs and macrophages (Jost et al 1999), as well as being dermonecrotic and lethal to laboratory animals via the intravenous and intraperitoneal routes (Lovell 1944;Jost et al 1999). PLO is produced by all A. pyogenes strains examined to date (Billington et al 1997;Ding and La¨mmler 1996;Jost et al 1999), and is expressed primarily in stationary phase cultures (Ding and La¨mmler 1996;Gilbert 2002), where it is detected in culture supernatant as a 55 kD protein (Billington et al 1997;Jost et al 1999). PLO belongs to the cholesterol-dependent cytolysin (CDC) family of toxins (Billington et al 1997), which includes listeriolysin O (LLO), perfringolysin O (PFO), pneumolysin (PLY) and streptolysin O (SLO).…”

Section: Factormentioning

confidence: 97%

“…A primary virulence factor of A. pyogenes is the potent extracellular toxin, pyolysin (PLO) (Billington et al 1997;Ding and La¨mmler 1996;Jost et al 1999). PLO is a haemolysin capable of lysing red blood cells of a variety of animals species, and is responsible for the characteristic betahaemolysis exhibited by A. pyogenes grown on blood-containing media (Pascual Ramos et al 1997).…”

Section: Factormentioning

confidence: 98%

Arcanobacterium pyogenes: molecular pathogenesis of an animal opportunist

Jost

Billington

2005

Antonie Van Leeuwenhoek

180

216

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Arcanobacterium pyogenes is a commensal and an opportunistic pathogen of economically important livestock, causing diseases as diverse as mastitis, liver abscessation and pneumonia. This organism possesses a number of virulence factors that contribute to its pathogenic potential. A. pyogenes expresses a cholesterol-dependent cytolysin, pyolysin, which is a haemolysin and is cytolytic for immune cells, including macrophages. Expression of pyolysin is required for virulence and this molecule is the most promising vaccine candidate identified to date. A. pyogenes also possesses a number of adherence mechanisms, including two neuraminidases, the action of which are required for full adhesion to epithelial cells, and several extracellular matrix-binding proteins, including a collagen-binding protein, which may be required for adhesion to collagen-rich tissue. A. pyogenes also expresses fimbriae, which are similar to the type 2 fimbriae of Actinomyces naeslundii, and forms biofilms. However, the role of these factors in the pathogenesis of A. pyogenes infections remains to be elucidated. A. pyogenes also invades and survives within epithelial cells and can survive within J774A.1 macrophages for up to 72 h, suggesting an important role for A. pyogenes interaction with host cells during pathogenesis. The two component regulatory system, PloSR, up-regulates pyolysin expression and biofilm formation but down-regulates expression of proteases, suggesting that it may act as a global regulator of A. pyogenes virulence. A. pyogenes is a versatile pathogen, with an arsenal of virulence determinants. However, most aspects of the pathogenesis of infection caused by this important opportunistic pathogen remain poorly characterized.

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“…pyogenes secretes a haemolytic exotoxin, pyolysin (PLO) (Billington et al, 1997 ;Ding & La$ mmler, 1996), which is an important virulence factor, as a knockout mutation in the plo gene resulted in reduced virulence of the mutant in a mouse model (Jost et al, 1999). Recombinant PLO is also an effective vaccine in the prevention of experimental infections in mice (Jost et al, 1999), although its ability to protect domestic animals from natural infection is still to be tested.…”

Section: Introductionmentioning

confidence: 99%

The variant undecapeptide sequence of the Arcanobacterium pyogenes haemolysin, pyolysin, is required for full cytolytic activity

2002

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The cholesterol-dependent cytolysins (CDCs) are characterized by an undecapeptide sequence (ECTGLAWEWWR) that is located near the C terminus and within domain 4 of these proteins. Pyolysin (PLO), the CDC of Arcanobacterium pyogenes, has a variant undecapeptide sequence (EATGLAWDPWW). Site-directed mutants were constructed in undecapeptide residues in a recombinant PLO molecule containing a hexahistidine tag (His-PLO). Mutations in each of the three undecapeptide tryptophan residues resulted in low haemolytic activity, confirming the importance of these residues in the protein. Deletion of a proline residue (P 499 ), inserted in PLO, or substitution of this residue with either phenylalanine or glycine resulted in mutant proteins with undetectable or low haemolytic activities, indicating that P 499 is essential for His-PLO haemolytic activity. Substitution of the PLO undecapeptide sequence with a consensus undecapeptide resulted in a His-PLO protein with only 01 % activity, confirming that the variant PLO undecapeptide is required for the full cytolytic activity of this toxin. The presence of the conserved undecapeptide cysteine residue either alone (His-PLO.C 492 ) or in a consensus sequence resulted in His-PLO molecules which were activated in the presence of reducing compounds, confirming the importance of this residue in the thiol-activated nature of many CDC toxins. The ability of His-PLO mutant proteins to bind cholesterol mimicked haemolytic activity, with the exception of His-PLO.C 492 , which, despite having reduced haemolytic activity, showed an increased ability to bind cholesterol compared to His-PLO. Despite reductions in haemolytic activity and cholesterol-binding, all mutant proteins were still able to bind to erythrocyte membranes, suggesting that other regions of PLO may recognize host-cell membranes, through receptors other than cholesterol.

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Purification and Further Characterization of a Haemolysin of Actinomyces pyogenes

Cited by 25 publications

References 31 publications

The Arcanobacterium (Actinomyces) pyogenes hemolysin, pyolysin, is a novel member of the thiol-activated cytolysin family

The Arcanobacterium (Actinomyces) pyogenes hemolysin, pyolysin, is a novel member of the thiol-activated cytolysin family

Arcanobacterium pyogenes: molecular pathogenesis of an animal opportunist

The variant undecapeptide sequence of the Arcanobacterium pyogenes haemolysin, pyolysin, is required for full cytolytic activity

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