Characterization of pneumolysin from Streptococcus pneumoniae, interacting with carbohydrate moiety and cholesterol as a component of cell membrane

Lim, Jong Eun; Park, Seong Ah; Bong, Seoung Min; Min, Young; Lee, Ki Seog

doi:10.1016/j.bbrc.2012.11.095

Cited by 10 publications

(11 citation statements)

References 30 publications

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“…Although it has been shown that preincubation of Ply with cholesterol inhibits hemolytic activity against human RBCs (13,14), whether cholesterol is the actual cellular receptor for Ply has not been determined. Using glycan array analysis, we investigated the glycanbinding specificities of Ply.…”

Section: Resultsmentioning

confidence: 99%

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The cholesterol-dependent cytolysins pneumolysin and streptolysin O require binding to red blood cell glycans for hemolytic activity

Shewell

Harvey

Higgins

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

104

113

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The cholesterol-dependent cytolysin (CDC) pneumolysin (Ply) is a key virulence factor of Streptococcus pneumoniae. Membrane cholesterol is required for the cytolytic activity of this toxin, but it is not clear whether cholesterol is the only cellular receptor. Analysis of Ply binding to a glycan microarray revealed that Ply has lectin activity and binds glycans, including the Lewis histoblood group antigens. Surface plasmon resonance analysis showed that Ply has the highest affinity for the sialyl LewisX (sLeX) structure, with a K d of 1.88 × 10 −5 M. Ply hemolytic activity against human RBCs showed dose-dependent inhibition by sLeX. Flow cytometric analysis and Western blots showed that blocking binding of Ply to the sLeX glycolipid on RBCs prevents deposition of the toxin in the membrane. The lectin domain responsible for sLeX binding is in domain 4 of Ply, which contains candidate carbohydrate-binding sites. Mutagenesis of these predicted carbohydrate-binding residues of Ply resulted in a decrease in hemolytic activity and a reduced affinity for sLeX. This study reveals that this archetypal CDC requires interaction with the sLeX glycolipid cellular receptor as an essential step before membrane insertion. A similar analysis conducted on streptolysin O from Streptococcus pyogenes revealed that this CDC also has glycan-binding properties and that hemolytic activity against RBCs can be blocked with the glycan lacto-N-neotetraose by inhibiting binding to the cell surface. Together, these data support the emerging paradigm shift that pore-forming toxins, including CDCs, have cellular receptors other than cholesterol that define target cell tropism.S treptococcus pneumoniae is a leading cause of morbidity and mortality worldwide. This bacterial pathogen is responsible for a range of diseases, including pneumonia, meningitis, septicemia, and otitis media. One of the major virulence factors of S. pneumoniae is the multifunctional pore-forming toxin pneumolysin (Ply). Ply is produced by virtually all clinical isolates of S. pneumoniae and is a member of the cholesterol-dependent cytolysin (CDC) family of toxins (1). The key feature of the CDCs, which are expressed by a number of pathogenic Grampositive bacteria, is the ability to form pores in cholesterolcontaining cell membranes. The pore-forming mechanism of the CDCs is a multistep process that involves recognition and binding to the cholesterol-containing membrane by domain 4 of the toxin, oligomerization of ∼34-50 soluble monomers on the target cell membrane to form a large prepore complex (2), and penetration of the prepore structure into the membrane to become a transmembrane β-barrel pore (3-5).The cytolytic mechanism of the CDCs depends on the presence of cholesterol in the target cell membrane; hence, it was thought that cholesterol served as the cellular receptor for these toxins. The first suggestion of this cholesterol serving as the receptor occurred in the 1970s, when it was found that preincubation of the CDC of Streptococcus pyogenes, streptolysin O (SL...

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Section: Resultsmentioning

confidence: 99%

“…PlyL (domains 1-3) and PlyS (domain 4) cloning and recombinant protein production procedures have been described previously (13). Identical primers were used for PlyL306 and PlySL460D utilizing the appropriate template DNA described above.…”

Section: Methodsmentioning

confidence: 99%

The cholesterol-dependent cytolysins pneumolysin and streptolysin O require binding to red blood cell glycans for hemolytic activity

Shewell

Harvey

Higgins

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

104

113

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“…It was reported that domain four of PLY interacts directly with the 3β-hydroxyl group at the C-3 atom of cholesterol, which could explain the constitutive dependency of PLY on cholesterol in the membranes that are to be perforated [36]. Through binding experiments with truncated toxin subunits and different carbohydrates, it was also revealed that domain four has a high affinity for mannose, suggesting that mannose-containing glycoproteins or glycolipids might be receptors for the recognition of target cells.…”

Section: Pneumolysin: Structure and Activitiesmentioning

confidence: 99%

Mini-Review: Novel Therapeutic Strategies to Blunt Actions of Pneumolysin in the Lungs

Lucas

Czikora

Sridhar

et al. 2013

Toxins

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Severe pneumonia is the main single cause of death worldwide in children under five years of age. The main etiological agent of pneumonia is the G+ bacterium Streptococcus pneumoniae, which accounts for up to 45% of all cases. Intriguingly, patients can still die days after commencing antibiotic treatment due to the development of permeability edema, although the pathogen was successfully cleared from their lungs. This condition is characterized by a dramatically impaired alveolar epithelial-capillary barrier function and a dysfunction of the sodium transporters required for edema reabsorption, including the apically expressed epithelial sodium channel (ENaC) and the basolaterally expressed sodium potassium pump (Na+-K+-ATPase). The main agent inducing this edema formation is the virulence factor pneumolysin, a cholesterol-binding pore-forming toxin, released in the alveolar compartment of the lungs when pneumococci are being lysed by antibiotic treatment or upon autolysis. Sub-lytic concentrations of pneumolysin can cause endothelial barrier dysfunction and can impair ENaC-mediated sodium uptake in type II alveolar epithelial cells. These events significantly contribute to the formation of permeability edema, for which currently no standard therapy is available. This review focuses on discussing some recent developments in the search for the novel therapeutic agents able to improve lung function despite the presence of pore-forming toxins. Such treatments could reduce the potentially lethal complications occurring after antibiotic treatment of patients with severe pneumonia.

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“…It has been established that the presence of membrane cholesterol is an essential condition for successful insertion of PLY into membranes (1) and depletion of membrane cholesterol impairs the hemolytic activity of PLY (52). Natural variation in cholesterol content between different cells could then be responsible for the different binding of PLY to RBCs (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…RBCs were washed three times in DPBS/BSA, resus- pended in DPBS/BSA at a concentration of 3 ϫ 10 7 cells/ml, and cells (90 l) were incubated with PLY at a final concentration of 118 ng/ml (2.2 nM) for 30 min at room temperature, similar to the dose (1 nM) frequently used in the literature under comparable experimental conditions (40,52). The time course of hemolysis was monitored by phase contrast microscopy (Zeiss Axiophot upright microscope equipped with a Plan Neofluar ϫ20/0.5 air objective).…”

Section: Expression and Purification Of Egfp-⌬6plymentioning

confidence: 99%

Red Blood Cell Susceptibility to Pneumolysin

Bokori-Brown

Petrov

Khafaji

et al. 2016

Journal of Biological Chemistry

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This study investigated the effect of the biochemical and biophysical properties of the plasma membrane as well as membrane morphology on the susceptibility of human red blood cells to the cholesterol-dependent cytolysin pneumolysin, a key virulence factor of Streptococcus pneumoniae, using single cell studies. We show a correlation between the physical properties of the membrane (bending rigidity and surface and dipole electrostatic potentials) and the susceptibility of red blood cells to pneumolysin-induced hemolysis. We demonstrate that biochemical modifications of the membrane induced by oxidative stress, lipid scrambling, and artificial cell aging modulate the cell response to the toxin. We provide evidence that the diversity of response to pneumolysin in diabetic red blood cells correlates with levels of glycated hemoglobin and that the mechanical properties of the red blood cell plasma membrane are altered in diabetes. Finally, we show that diabetic red blood cells are more resistant to pneumolysin and the related toxin perfringolysin O relative to healthy red blood cells. Taken together, these studies indicate that the diversity of cell response to pneumolysin within a population of human red blood cells is influenced by the biophysical and biochemical status of the plasma membrane and the chemical and/or oxidative stress pre-history of the cell.The interaction of toxins with their target cells is generally characterized by a dose-response curve that is sigmoidal in shape (1). This shape is taken to reflect the differing susceptibilities of individual cells within the population. The fact that red blood cells (RBCs), with their less developed glycocalyx compared with other cell types (2), exhibit such a response is evidence that the initial interaction between the toxin and the plasma membrane is subject to, and probably the main determinant of, such variability. Therefore, it is important to understand the biochemical and biophysical factors affecting toxin-membrane interactions and the resulting variability of susceptibility within the same cell population.Individual RBCs within a population differ significantly (3) with respect to their shape, volume, and surface area. Some of these changes are related to cell age, due to the variety of mechanical and chemical stresses that an RBC undergoes in its life span of ϳ120 days (4, 5), and others can be associated with diseases, such as diabetes and with acute conditions, such as sepsis, among many others (6), and thus are likely to be present in the membranes of many other cell types.Previous work on bulk cell preparations revealed that in addition to its normal discocytic shape, the RBC at rest can assume a variety of other distinct shapes, such as echinocytes and acanthocytes, characterized by exterior projections, and stomatocytes with cup-shaped invaginations (7). Many of these unusual shapes appear in normal blood at a frequency of about 1%. However, during blood bank storage and in many inflammatory, degenerative, and microvascular disorders, the frequ...

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Characterization of pneumolysin from Streptococcus pneumoniae, interacting with carbohydrate moiety and cholesterol as a component of cell membrane

Cited by 10 publications

References 30 publications

The cholesterol-dependent cytolysins pneumolysin and streptolysin O require binding to red blood cell glycans for hemolytic activity

The cholesterol-dependent cytolysins pneumolysin and streptolysin O require binding to red blood cell glycans for hemolytic activity

Mini-Review: Novel Therapeutic Strategies to Blunt Actions of Pneumolysin in the Lungs

Red Blood Cell Susceptibility to Pneumolysin

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