scholarly journals Engineered covalent leucotoxin heterodimers form functional pores: insights into S–F interactions

2006 ◽  
Vol 396 (2) ◽  
pp. 381-389 ◽  
Author(s):  
Olivier Joubert ◽  
Gabriella Viero ◽  
Daniel Keller ◽  
Eric Martinez ◽  
Didier A. Colin ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Pore Formation ◽  
Polymorphonuclear Neutrophils ◽  
Target Cells ◽  
Single Family ◽  
Wild Type ◽  
Subunit Interactions ◽  
Human Red Blood Cells ◽  
Large Unilamellar Vesicles ◽  
New Strategy

The staphylococcal α-toxin and bipartite leucotoxins belong to a single family of pore-forming toxins that are rich in β-strands, although the stoichiometry and electrophysiological characteristics of their pores are different. The different known structures show a common β-sandwich domain that plays a key role in subunit–subunit interactions, which could be targeted to inhibit oligomerization of these toxins. We used several cysteine mutants of both HlgA (γ-haemolysin A) and HlgB (γ-haemolysin B) to challenge 20 heterodimers linked by disulphide bridges. A new strategy was developed in order to obtain a good yield for S-S bond formation and dimer stabilization. Functions of the pores formed by 14 purified dimers were investigated on model membranes, i.e. planar lipid bilayers and large unilamellar vesicles, and on target cells, i.e. rabbit and human red blood cells and polymorphonuclear neutrophils. We observed that dimers HlgA T28C–HlgB N156C and HlgA T21C–HlgB T157C form pores with similar characteristics as the wild-type toxin, thus suggesting that the mutated residues are facing one another, allowing pore formation. Our results also confirm the octameric stoichiometry of the leucotoxin pores, as well as the parity of the two monomers in the pore. Correctly assembled heterodimers thus constitute the minimal functional unit of leucotoxins. We propose amino acids involved in interactions at one of the two interfaces for an assembled leucotoxin.

scholarly journals Translocation domain mutations affecting cellular toxicity identify the Clostridium difficile toxin B pore

2014 ◽  
Vol 111 (10) ◽  
pp. 3721-3726 ◽  
Author(s):  
Zhifen Zhang ◽  
Minyoung Park ◽  
John Tam ◽  
Anick Auger ◽  
Greg L. Beilhartz ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Lipid Bilayers ◽  
Pore Formation ◽  
Target Cells ◽  
Cellular Toxicity ◽  
Lack Of Information ◽  
Insertion Elements ◽  
Translocation Domain ◽  
Systematic Perturbation ◽  
Clostridium Difficile Toxin B

Disease associated with Clostridium difficile infection is caused by the actions of the homologous toxins TcdA and TcdB on colonic epithelial cells. Binding to target cells triggers toxin internalization into acidified vesicles, whereupon cryptic segments from within the 1,050-aa translocation domain unfurl and insert into the bounding membrane, creating a transmembrane passageway to the cytosol. Our current understanding of the mechanisms underlying pore formation and the subsequent translocation of the upstream cytotoxic domain to the cytosol is limited by the lack of information available regarding the identity and architecture of the transmembrane pore. Here, through systematic perturbation of conserved sites within predicted membrane-insertion elements of the translocation domain, we uncovered highly sensitive residues—clustered between amino acids 1,035 and 1,107—that when individually mutated, reduced cellular toxicity by as much as >1,000-fold. We demonstrate that defective variants are defined by impaired pore formation in planar lipid bilayers and biological membranes, resulting in an inability to intoxicate cells through either apoptotic or necrotic pathways. These findings along with the unexpected similarities uncovered between the pore-forming “hotspots” of TcdB and the well-characterized α-helical diphtheria toxin translocation domain provide insights into the structure and mechanism of formation of the translocation pore for this important class of pathogenic toxins.

scholarly journals Membrane Permeabilization by Pore-Forming RTX Toxins: What Kind of Lesions Do These Toxins Form?

Toxins ◽  
2019 ◽  
Vol 11 (6) ◽  
pp. 354 ◽  
Author(s):  
Helena Ostolaza ◽  
David González-Bullón ◽  
Kepa B. Uribe ◽  
Cesar Martín ◽  
Jone Amuategi ◽  
...  
Keyword(s):  
Cell Death ◽  
Lipid Bilayers ◽  
Pore Formation ◽  
Whooping Cough ◽  
Cell Types ◽  
Target Cells ◽  
Cation Selectivity ◽  
Protein Toxin ◽  
Current State ◽  
Rtx Toxins

Pore-forming toxins (PFTs) form nanoscale pores across target membranes causing cell death. The pore-forming cytolysins of the RTX (repeats in toxin) family belong to a steadily increasing family of proteins characterized by having in their primary sequences a number of glycine- and aspartate-rich nonapeptide repeats. They are secreted by a variety of Gram-negative bacteria and form ion-permeable pores in several cell types, such as immune cells, epithelial cells, or erythrocytes. Pore-formation by RTX-toxins leads to the dissipation of ionic gradients and membrane potential across the cytoplasmic membrane of target cells, which results in cell death. The pores formed in lipid bilayers by the RTX-toxins share some common properties such as cation selectivity and voltage-dependence. Hemolytic and cytolytic RTX-toxins are important virulence factors in the pathogenesis of the producing bacteria. And hence, understanding the function of these proteins at the molecular level is critical to elucidating their role in disease processes. In this review we summarize the current state of knowledge on pore-formation by RTX toxins, and include recent results from our own laboratory regarding the pore-forming activity of adenylate cyclase toxin (ACT or CyaA), a large protein toxin secreted by Bordetella pertussis, the bacterium causative of whooping cough.

scholarly journals A Histidine-to-Arginine Substitution in Panton-Valentine Leukocidin from USA300 Community-Acquired Methicillin-Resistant Staphylococcus aureus Does Not Impair Its Leukotoxicity

2009 ◽  
Vol 78 (1) ◽  
pp. 260-264 ◽  
Author(s):  
Timothée Besseyre des Horts ◽  
Oana Dumitrescu ◽  
Cédric Badiou ◽  
Damien Thomas ◽  
Yvonne Benito ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Pore Formation ◽  
Methicillin Resistant Staphylococcus Aureus ◽  
Polymorphonuclear Neutrophils ◽  
Wild Type ◽  
Different Types ◽  
Usa300 Strain ◽  
Culture Supernatants ◽  
Panton Valentine Leukocidin ◽  
Valentine Leukocidin

ABSTRACT Panton-Valentine leukocidin (PVL) is a synergohymenotropic toxin (SHT) produced by Staphylococcus aureus. At present, there are conflicting reports on the leukotoxic activity of PVL and its consequent role as a virulence factor in USA300. In this work, we compared the cytolytic effects induced by wild-type PVL and those of PVL harboring a histidine-to-arginine substitution at amino acid 176 in the S. aureus USA300 strain. We also investigated the capacity of wild-type and H176R LukS-PV to recruit and form pores with the F components of other SHTs. For this purpose, we assayed polymorphonuclear neutrophils for leukotoxicity after incubation with either culture supernatants from strains bearing different PVL haplotypes or recombinant toxins from different types of SHT. We show here that the H176R variation in the PVL sequence causes no change in leukotoxicity and that the R variant is as efficient as wild-type PVL at inducing pore formation in leukocytes.

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

2008 ◽  
Vol 77 (1) ◽  
pp. 266-273 ◽  
Author(s):  
Florent Meyer ◽  
Raymonde Girardot ◽  
Yves Piémont ◽  
Gilles Prévost ◽  
Didier A. Colin
Keyword(s):  
Flow Cytometry ◽  
Dissociation Constant ◽  
Pore Formation ◽  
Specific Binding ◽  
Polymorphonuclear Neutrophils ◽  
Target Cells ◽  
Activity Time ◽  
High Affinity ◽  
Time Courses ◽  
Monocytes And Lymphocytes

ABSTRACT 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 Ca2+ 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 [K d ], 6.2 ± 1.9 nM; n = 8), monocytes (K d , 2.8 ± 0.8 nM; n = 7), and lymphocytes (K d , 1.2 ± 0.2 nM; n = 7). Specific binding of HlgB was observed only after addition of LukS-PV on PMNs (K d , 1.1 ± 0.2 nM; n = 4) and monocytes (K d , 0.84 ± 0.31 nM; n = 4) or after addition of HlgC on PMNs, monocytes, and lymphocytes. Addition of LukS-PV or HlgC induced a second specific binding of LukF-PV on PMNs. HlgB and LukD competed only with LukF-PV molecules bound after addition of LukS-PV. LukF-PV and LukD competed with HlgB in the presence of LukS-PV on PMNs and monocytes. Use of antibodies and comparisons between binding and activity time courses showed that the LukF-PV molecules that bound to target cells before addition of LukS-PV were the only LukF-PV molecules responsible for Ca2+ entry and pore formation. In contrast, the active HlgB molecules were the HlgB molecules bound after addition of LukS-PV. In conclusion, LukF-PV must be linked to LukS-PV and to a binding site of the membrane to have toxin activity.

scholarly journals Yersinia pseudotuberculosis-Induced Calcium Signaling in Neutrophils Is Blocked by the Virulence Effector YopH

1999 ◽  
Vol 67 (5) ◽  
pp. 2567-2574 ◽  
Author(s):  
Kerstin Andersson ◽  
Karl-Eric Magnusson ◽  
Meytham Majeed ◽  
Olle Stendahl ◽  
Maria Fällman
Keyword(s):  
Yersinia Pseudotuberculosis ◽  
Tyrosine Phosphatase ◽  
Polymorphonuclear Neutrophils ◽  
Human Neutrophils ◽  
Free Calcium ◽  
Target Cells ◽  
Wild Type ◽  
Mediated Effects ◽  
Intracellular Free Calcium Concentration ◽  
Free Calcium Concentration

ABSTRACT Pathogenic species of the genus Yersinia evade the bactericidal functions of phagocytes. This evasion is mediated through their virulence effectors, Yops, which act within target cells. In this study we investigated the effect of Yersinia pseudotuberculosis on Ca2+ signaling in polymorphonuclear neutrophils. The intracellular free calcium concentration in single adherent human neutrophils was monitored during bacterial infection and, in parallel, the encounter between the bacteria and cells was observed. When a plasmid-cured strain was used for infection, adherence of a single bacterium to the cellular surface induced a β1 integrin-dependent transient increase in the intracellular concentration of free calcium. This was, however, not seen with Yop-expressing wild-type bacteria, which adhered to the cell surface without generating any Ca2+ signal. Importantly, the overall Ca2+homeostasis was not affected by the wild-type strain; the Ca2+ signal mediated by the G-protein-coupled formyl-methionyl-leucyl-phenylalanine receptor was still functioning. Hence, the blocking effect was restricted to certain receptors and their signaling pathways. The use of different Yop mutant strains revealed that the protein tyrosine phosphatase YopH was responsible for the inhibition. This virulence determinant has previously been implicated in very rapid Yersinia-mediated effects on target cells as the key effector in the blockage of phagocytic uptake. The present finding, that Y. pseudotuberculosis, via YopH, specifically inhibits a self-induced immediate-early Ca2+ signal in neutrophils, offers more-detailed information concerning the effectiveness of this virulence effector and implies an effect on Ca2+-dependent, downstream signals.

Granzyme B and perforin: constitutive expression in human polymorphonuclear neutrophils

Blood ◽  
2004 ◽  
Vol 103 (3) ◽  
pp. 1099-1104 ◽  
Author(s):  
Christof Wagner ◽  
Christof Iking-Konert ◽  
Birgit Denefleh ◽  
Sabine Stegmaier ◽  
Friederike Hug ◽  
...  
Keyword(s):  
Granzyme B ◽  
Constitutive Expression ◽  
Jurkat Cells ◽  
Polymorphonuclear Neutrophils ◽  
Target Cells ◽  
Dependent Manner ◽  
Cellular Cytotoxicity ◽  
Antibody Dependent Cellular Cytotoxicity ◽  
Cytotoxic Molecules ◽  
Chicken Erythrocytes

AbstractPolymorphonuclear neutrophils (PMNs) produce an abundance of bactericidal and cytotoxic molecules consistent with their role as first-line defense against bacterial infection. PMNs, however, also cause efficient cellular cytotoxicity when targeted through Fc receptors to appropriate antibody-coated target cells. Although this so-called antibody-dependent cellular cytotoxicity (ADCC) was described many years ago, the mechanism of killing is still elusive. We now have found that PMNs contain perforin and granzyme B, the 2 molecules known as the cytotoxic entity of natural killer cells and of cytotoxic T lymphocytes as well. Lysates of PMNs were lytic for chicken erythrocytes in a time-, temperature-, and Ca2+-dependent manner. Moreover, apoptosis of Jurkat cells was induced, consistent with the observation that the PMN lysates contain enzymatically active granzyme B. Taken together, our data provide evidence for the presence of perforin and granzyme B within the cytotoxic arsenal of PMNs. (Blood. 2004;103:1099-1104)

scholarly journals Glycoprotein D-Independent Infectivity of Pseudorabies Virus Results in an Alteration of In Vivo Host Range and Correlates with Mutations in Glycoproteins B and H

2001 ◽  
Vol 75 (21) ◽  
pp. 10054-10064 ◽  
Author(s):  
Jerg Schmidt ◽  
Volker Gerdts ◽  
Jörg Beyer ◽  
Barbara G. Klupp ◽  
Thomas C. Mettenleiter
Keyword(s):  
Host Range ◽  
Pseudorabies Virus ◽  
Natural Host ◽  
Target Cells ◽  
Glycoprotein D ◽  
Wild Type ◽  
Cellular Receptors ◽  
Receptor Usage

ABSTRACT Infection of cells by herpesviruses is initiated by the interaction of viral envelope glycoproteins with cellular receptors. In the alphaherpesvirus pseudorabies virus (PrV), the causative agent of Aujeszky's disease in pigs, the essential glycoprotein D (gD) mediates secondary attachment of virions to target cells by binding to newly identified cellular receptors (R. J. Geraghty, C. Krummenacher, G. H. Cohen, R. J. Eisenberg, and P. G. Spear, Science 280:1618–1620, 1998). However, in the presence of compensatory mutations, infection can also occur in the absence of gD, as evidenced by the isolation in cell culture of an infectious gD-negative PrV mutant (PrV-gD− Pass) (J. Schmidt, B. G. Klupp, A. Karger, and T. C. Mettenleiter, J. Virol. 71:17–24, 1997). PrV-gD− Pass is replication competent with an only moderate reduction in specific infectivity but appears to bind to receptors different from those recognized by wild-type PrV (A. Karger, J. Schmidt, and T. C. Mettenleiter, J. Virol. 72:7341–7348, 1998). To analyze whether this alteration in receptor usage in vitro influences infection in vivo, the model host mouse and the natural host pig were intranasally infected with PrV-gD− Pass and were compared to animals infected by wild-type PrV. For mice, a comparable progress of disease was observed, and all animals infected with mutant virus died, although they exhibited a slight delay in the onset of symptoms and, correspondingly, a longer time to death. In contrast, whereas wild-type PrV-infected pigs showed clinical signs and histological and histopathological findings typical of PrV infection, no signs of disease were observed after infection with PrV-gD− Pass. Moreover, in these animals, virus-infected cells were not detectable by immunohistochemical staining of different organ samples and no virus could be isolated from nasal swabs. Mutations in glycoproteins B and H were found to correlate with, and probably contribute to, gD-independent infectivity. In conclusion, although PrV-gD− Pass is virulent in mice, it is apparently unable to infect the natural host, the pig. This altered host range in vivo correlates with a difference of receptor usage in vitro and demonstrates for the first time the importance of gD receptors in alphaherpesvirus infection of an animal host.

scholarly journals Protease-Dependent Uncoating of a Complex Retrovirus

2005 ◽  
Vol 79 (14) ◽  
pp. 9244-9253 ◽  
Author(s):  
Jacqueline Lehmann-Che ◽  
Marie-Lou Giron ◽  
Olivier Delelis ◽  
Martin Löchelt ◽  
Patricia Bittoun ◽  
...  
Keyword(s):  
Nuclear Import ◽  
Viral Genome ◽  
Productive Infection ◽  
Wild Type Virus ◽  
Target Cells ◽  
Wild Type ◽  
Microtubule Organizing Center ◽  
Viral Protease ◽  
Virus Life Cycle ◽  
Organizing Center

ABSTRACT Although retrovirus egress and budding have been partly unraveled, little is known about early stages of the replication cycle. In particular, retroviral uncoating, a process during which incoming retroviral cores are altered to allow the integration of the viral genome into host chromosomes, is poorly understood. To get insights into these early events of the retroviral cycle, we have used foamy complex retroviruses as a model. In this report, we show that a protease-defective foamy retrovirus is noninfectious, although it is still able to bud and enter target cells efficiently. Similarly, a retrovirus mutated in an essential viral protease-dependent cleavage site in the central part of Gag is noninfectious. Following entry, wild-type and mutant retroviruses are able to traffic along microtubules towards the microtubule-organizing center (MTOC). However, whereas nuclear import of Gag and of the viral genome was observed for the wild-type virus as early as 8 hours postinfection, incoming capsids and genome from mutant viruses remained at the MTOC. Interestingly, a specific viral protease-dependent Gag cleavage product was detected only for the wild-type retrovirus early after infection, demonstrating that cleavage of Gag by the viral protease at this stage of the virus life cycle is absolutely required for productive infection, an unprecedented observation among retroviruses.

scholarly journals Subunit Interactions in the mariner Transposase

Genetics ◽  
1996 ◽  
Vol 144 (3) ◽  
pp. 1087-1095 ◽  
Author(s):  
Allan R Lohe ◽  
David T Sullivan ◽  
Daniel L Hartl
Keyword(s):  
Hybrid System ◽  
Catalytic Domain ◽  
Wild Type ◽  
Target Element ◽  
Yeast Two Hybrid ◽  
Subunit Interactions ◽  
Hsp70 Promoter ◽  
Yeast Two Hybrid System ◽  
Transposition Reaction ◽  
Two Hybrid

Abstract We have studied the Mos1 transposase encoded by the transposable element mariner. This transposase is a member of the “D,D(35)E” superfamily of proteins exhibiting the motif D,D(34)D. It is not known whether this transposase, or other eukaryote transposases manifesting the D,D(35)E domain, functions in a multimeric form. Evidence for oligomerization was found in the negative complementation of Mos1 by an EMS-induced transposase mutation in the catalytic domain. The transposase produced by this mutation has a glycine-to-arginine replacement at position 292. The G292R mutation strongly interferes with the ability of wild-type transposase to catalyze excision of a target element. Negative complementation was also observed for two other EMS mutations, although the effect was weaker than observed with G292R. Results from the yeast two-hybrid system also imply that Mos1 subunits interact, suggesting the possibility of subunit oligomerization in the transposition reaction. Overproduction of Mos1 subunits through an hsp70 promoter also inhibits excision of the target element, possibly through autoregulatory feedback on transcription or through formation of inactive or less active oligomers. The effects of both negative complementation and overproduction may contribute to the regulation of mariner transposition.

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