Evidence That Clustered Phosphocholine Head Groups Serve as Sites for Binding and Assembly of an Oligomeric Protein Pore

Valeva, Angela; Hellmann, Nadja; Walev, Iwan; Strand, Dennis; Plate, Markus; Boukhallouk, Fatima; Brack, Antje; Hanada, Kentaro; Decker, Heinz; Bhakdi, Sucharit

doi:10.1074/jbc.m601960200

Cited by 107 publications

(121 citation statements)

References 43 publications

(34 reference statements)

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“…Recently, a new concept has been proposed to explain the binding of alpha-toxin, another member of the family of staphylococcal pore-forming toxins which forms homotypic heptamers. Besides low-affinity binding sites, the role of high-affinity receptors for this toxin would be played by clusters of phosphocholine head groups in sphingomyelin-cholesterol-rich microdomains (20) and consequently would not be protein mediated. The present study did not result in any final conclusions concerning the receptors of bicomponent leukotoxins.…”

Section: Discussionmentioning

confidence: 99%

Analysis of the Specificity of Panton-Valentine Leucocidin and Gamma-Hemolysin F Component Binding

et al. 2009

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In this study, the binding of F components of the staphylococcal bicomponent leukotoxins Panton-Valentine leucocidin (LukF-PV) and gamma-hemolysin (HlgB) on polymorphonuclear neutrophils (PMNs), monocytes, and lymphocytes was determined using labeled mutants and flow cytometry. Leukotoxin activity was evaluated by measuring Ca 2؉ entry or pore formation using spectrofluorometry or flow cytometry. Although HlgB had no affinity for cells in the absence of an S component, LukF-PV had high affinity for PMNs (dissociation constant Staphylococcus aureus secretes water-soluble protein monomers which assemble to form well-characterized two-component pore-forming leukotoxins that define a toxin subfamily (17). Each leukotoxin is formed by a class S protein (LukS-PV, HlgA, HlgC, or LukE) that is associated with a class F protein (LukF-PV, HlgB, or LukD). The Panton-Valentine leucocidin (PVL) (16), composed of LukS-PV and LukF-PV, is secreted by strains isolated from humans suffering from abscesses, furuncles (6, 8), and necrotizing pneumonia (11,12). PVL has been shown to contribute to the severity of acute hematogenous osteomyelitis caused by community-acquired methicillinresistant S. aureus in children (2). Moreover, reports of hospital-acquired and community-acquired methicillin-resistant S. aureus possessing PVL genes are increasing (3). The LukELukD-secreting S. aureus strains have been shown to be associated with postantibiotic diarrhea (10). However, gamma-hemolysin (HlgA-HlgB and HlgC-HlgB) is produced by all human clinical S. aureus strains.[The main target cells of staphylococcal leukotoxins are human polymorphonuclear cells (PMNs), monocytes, and lymphocytes (13). However, PVL is not toxic for lymphocytes, and gamma-hemolysin is hemolytic. After binding to the membrane, leukotoxins assemble as a ring-shaped prepore (15) consisting of heterologous octamers with a molar ratio of 1:1 (14). They induce an increase in the intracellular Ca 2ϩ level by opening Ca 2ϩ channels (19, 1), and the deployment of stems forms beta-barrels that create transmembrane pores in target cells (7, 1). Previous perfusion studies of PMNs showed that the initial binding of S components was a prerequisite for the binding of F components to obtain toxic activity on PMNs (4). Conversely, Yokota and Kamio (22) showed that the binding of the F component LukF (HlgB) on human erythrocytes was a prerequisite for the subsequent binding of Hlg2 (HlgA). Later, Gauduchon et al. (9) determined a dissociation constant (K d ) of 0.07 nM for LukS-PV binding on PMNs and showed that HlgC competed with LukS-PV for binding but HlgA and LukE did not compete. However, no data are available yet for the binding of F components.The present study was performed to analyze the binding of the F components of leukotoxins, particularly the binding of LukF-PV compared with that of HlgB, two F components which exhibit 71% identity. LukF-PV* and HlgB* leukotoxins with cysteine mutations were labeled with fluorescein to follow their binding to human leukocytes by flow cy...

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Section: Discussionmentioning

confidence: 99%

Analysis of the Specificity of Panton-Valentine Leucocidin and Gamma-Hemolysin F Component Binding

et al. 2009

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“…Such small clusters of cholesterol could serve as docking sites for LLO and facilitate oligomerization, as shown by our results. Clustered lipids as binding sites for proteins emerge as a general concept in PFT; i.e., the binding and permeabilizing activity of ostreolysin, a PFT from oyster mushroom, is optimal above 40% cholesterol in sphingomyelin-containing membranes (61), and Valeva et al have recently shown that clustered phosphocholine headgroups lead to rapid oligomerization of staphylococcal R-toxin (62).…”

Section: Discussionmentioning

confidence: 99%

Sterol and pH Interdependence in the Binding, Oligomerization, and Pore Formation of Listeriolysin O

et al. 2007

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Listeriolysin O (LLO) is the most important virulence factor of the intracellular pathogen Listeria monocytogenes. Its main task is to enable escape of bacteria from the phagosomal vacuole into the cytoplasm. LLO belongs to the cholesterol-dependent cytolysin (CDC) family but differs from other members, as it exhibits optimal activity at low pH. Its pore forming ability at higher pH values has been largely disregarded in Listeria pathogenesis. Here we show that high cholesterol concentrations in the membrane restore the low activity of LLO at high pH values. LLO binds to lipid membranes, at physiological or even slightly basic pH values, in a cholesterol-dependent fashion. Binding, insertion into lipid monolayers, and permeabilization of calcein-loaded liposomes are maximal above approximately 35 mol % cholesterol, a concentration range typically found in lipid rafts. The narrow transition region of cholesterol concentration separating low and high activity indicates that cholesterol not only allows the binding of LLO to membranes but also affects other steps in pore formation. We were able to detect some of these by surface plasmon resonance-based assays. In particular, we show that LLO recognition of cholesterol is determined by the most exposed 3 -hydroxy group of cholesterol. In addition, LLO binds and permeabilizes J774 cells and human erythrocytes in a cholesterol-dependent fashion at physiological or slightly basic pH values. The results clearly show that LLO activity at physiological pH cannot be neglected and that its action at sites distal to cell entry may have important physiological consequences for Listeria pathogenesis.

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“…This feature is not unique to HlyA, as the sensitivity to other poreformers such as ␣-toxin from Staphylococcus aureus also shows great interspecies variability (7). Regarding S. aureus, the interspecies variation was explained through differences in expression levels of a specific receptor for ␣-toxin (8). This option has also been suggested for HlyA-induced hemolysis (9) but is not yet generally accepted (10).…”

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confidence: 99%

α-Hemolysin from Escherichia coli uses endogenous amplification through P2X receptor activation to induce hemolysis

Skals

Jørgensen

Leipziger

et al. 2009

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

117

165

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Escherichia coli is the dominant facultative bacterium in the normal intestinal flora. E. coli is, however, also responsible for the majority of serious extraintestinal infections. There are distinct serotypical differences between facultative and invasive E. coli strains. Invasive strains frequently produce virulence factors such as ␣-hemolysin (HlyA), which causes hemolysis by forming pores in the erythrocyte membrane. The present study reveals that this pore formation triggers purinergic receptor activation to mediate the full hemolytic action. Non-selective ATP-receptor (P2) antagonists (PPADS, suramin) and ATP scavengers (apyrase, hexokinase) concentration dependently inhibited HlyA-induced lysis of equine, murine, and human erythrocytes. The pattern of responsiveness to more selective P2-antagonists implies that both P2X 1 and P2X7 receptors are involved in HlyA-induced hemolysis in all three species. In addition, our results also propose a role for the pore protein pannexin1 in HlyA-induced hemolysis, as non-selective inhibitors of this channel significantly reduced hemolysis in the three species. In conclusion, activation of P2X receptors and possibly also pannexins augment hemolysis induced by the bacterial toxin, HlyA. These findings potentially have clinical perspectives as P2 antagonists may ameliorate symptoms during sepsis with hemolytic bacteria.alpha-hemolysin ͉ E. coli ͉ erythrocytes ͉ hemolysis ͉ P2X

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Evidence That Clustered Phosphocholine Head Groups Serve as Sites for Binding and Assembly of an Oligomeric Protein Pore

Cited by 107 publications

References 43 publications

Analysis of the Specificity of Panton-Valentine Leucocidin and Gamma-Hemolysin F Component Binding

Analysis of the Specificity of Panton-Valentine Leucocidin and Gamma-Hemolysin F Component Binding

Sterol and pH Interdependence in the Binding, Oligomerization, and Pore Formation of Listeriolysin O

α-Hemolysin from Escherichia coli uses endogenous amplification through P2X receptor activation to induce hemolysis

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