On the cellular stress response to Staphylococcus aureus alpha-toxin

Staphylococcus aureus alpha-hemolysin was the first bacterial toxin recognized to form pores in the plasma membrane of eukaryotic cells. It is secreted as a water-soluble monomer that upon contact with target membranes forms an amphiphatic heptameric beta-barrel which perforates the bilayer. As a consequence, red cells undergo colloidosmotic lyses, while some nucleated cells may succumb to necrosis or programmed cell death. However, most cells are capable of repairing a limited number of membrane lesions, and then respond with productive transcriptional activation of NF-kB. In the present study, by using microarray and semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR), data from a previously performed serial analysis of gene expression (SAGE) were extended and verified, revealing that immediate early genes (IEGs) such as c-fos, c-jun and egr-1 are strongly induced at 2-8 h after transient toxin treatment. Activating protein 1 (AP-1: c-Fos, c-Jun) binding activity was increased accordingly. As IEGs are activated by growth factors, these findings led to the discovery that -toxin promotes cell cycle progression of perforated cells in an EGFR-dependent fashion. Although the amount of c-fos mRNA rose rapidly after toxin treatment, c-Fos protein expression was observed only after a lag of about 3 h. Since translation consumes much ATP, which transiently drops after transient membrane perforation, the suspicion arised that membrane-perforation caused global, but temporary downregulation of translation. In fact, eIF2α became heavily phosphorylated minutes after cells had been confronted with the toxin, resulting in shutdown of protein synthesis before cellular ATP levels reached the nadir. GCN2 emerged as a candidate eIF2α kinase, since its expression rapidly increased in toxin-treated cells. Two hours after toxin treatment, GADD34 transcripts, encoding a protein that targets the catalytic subunit of protein phosphatase 1 (PP1) to the endoplasmic reticulum, were overexpressed. This was followed by dephosphorylation of eIF2α and resumption of protein synthesis. Addition of tautomycetin, a specific inhibitor of PP1, led to marked hyperphosphorylation of eIF2α and significantly reduced the drop of ATP-levels in toxin-treated cells. A novel link between two major stress-induced signalling pathways emerged when it was found that both translational arrest and restart were under the control of stress-activated protein kinase (SAPK) p38. The data provide an explanation for the indispensible role of p38 for defence against the archetypal threat of membrane perforation by agents that produce small transmembrane-pores.

Induktion von Pro-Autophagie-Signalen durch einen extra- oder intrazellulären Alpha-Toxin-Angriff

Autophagie ist ein konservierter, kataboler Mechanismus in allen eukaryoten Zellen. Unter anderem wird ihm eine wichtige Rolle als zellautonomer Abwehrmechanismus gegen Mikroorganismen zugeschrieben; von manchen Infektionserregern wird er jedoch unterlaufen oder sogar genutzt. Der stärkste Auslöser der Autophagie ist ein Mangel an Nährstoffen, insbesondere Aminosäuren. Über die Deaktivierung der Kinase mTORC1 und die Phosphorylierung des eukaryoten Translationsinitiationsfaktors eIF2α hemmt die Nährstoffknappheit die Proteinbiosynthese und aktiviert gleichzeitig Autophagie. Wie Mikroorganismen, insbesondere Bakterien, Autophagie auslösen oder manipulieren, ist derzeit Gegenstand intensiver Forschung. Modifikationen an Mikroben oder Phagosomen und Adapterproteine, die diese Veränderungen und Komponenten des Autophagieapparates erkennen, scheinen jedenfalls bei der selektiven Erkennung durch die Autophagie-Maschinerie wichtig zu sein. rnIn der vorliegenden Dissertationsarbeit wird die Rolle des membranporenbildenden α-Toxins von Staphylococcus aureus für die Induktion von Autophagie beleuchtet. Zum einen erwies sich die Akkumulation von (EGFP)-LC3(II), einem Marker der Autophagosomen, um intrazelluläre S. aureus als abhängig von α-Toxin. Zweitens, genügt extrazellulär appliziertes α-Toxin um (EGFP)-LC3(II)-positive Endosomen zu induzieren. Während der Angriff aus dem extrazellulären Raum jedoch binnen kurzer Zeit eine fokale Kumulation von phosphoryliertem eIF2α an der Plasmamembran induziert, die an der Internalisierung des Toxins beteiligt ist, findet sich am phagosomalen Kompartiment keine Toxin-abhängige Anhäufung von p-eIF2α oder proximalen Autophagieregulatoren. Dies impliziert, dass Toxin-Angriff auf die Plasmamembran, nicht aber auf das Phagosom, zu einer Reaktion führt, wie sie bei massivem Nährstoffmangel zu beobachten ist. Obwohl keine α-Toxin-abhängige Kumulation von p-eIF2α bei einem Angriff aus dem Phagosom erfolgt, findet sich um α-Toxin-produzierende Bakterien eine massive Kumulation von LC3 und Adapterprotein p62/Sequestosome1. Dies deutet daraufhin, dass der Ort des Angriffs - Plasmamembran oder Phagosom – für den Autophagie-induzierenden Mechanismus wichtig sein könnte. Der unterschiedliche Effekt auf die zellulären Ionenkonzentrationen, den ein Angriff auf die Plasmamembran oder auf ein Phagosom auslösen würde, bietet hierfür eine mögliche Erklärung. Die Aktivierung der Autophagie über Adapterproteine könnte dann als back-up Mechanismus fungieren, der auch dann greift, wenn eine Invasion ohne Schädigung der Plasmamembran erfolgt. Ein cross-talk der beiden Induktionswege ist angesichts der Bedeutung von p62 für die selektive und die Hunger-assoziierte Autophagie gut möglich; sezerniertes Toxin könnte durch die Aktivierung der basalen Autophagie Adapter-basierte Mechanismen verstärken.

IDENTIFICATION OF A PUTATIVE MEMBRANE-INSERTED SEGMENT IN THE ALPHA-TOXIN OF STAPHYLOCOCCUS-AUREUS

To gain a fuller understanding of the regions of the Staphylococcus aureus alpha-toxin important in pore formation, we have used Forster dipole-dipole energy transfer to demonstrate that a central glycine-rich region of alpha-toxin (the so-called ''hinge'' region) inserts deeply into the bilayer on association of toxin with liposomes. Mutant alpha-toxins with unique cysteine (C) residues at positions 69 and 130 [Palmer, M., et al. (1993) J. Biol. Chem. 268, 11959) were reacted with the C-specific fluorophore acrylodan, which acted as an energy donor. The chosen acceptor was N-(7-nitrobenz-2-oxa-13-diazol-4-yl)-1,2-bis(hexadecanoyl) -sn-glycero-3-phosphoethanolamine (NBD-PE). Measurement of the degree of donor quenching with increasing NBD-PE in the inner bilayer leaflet enables the distance of closest approach between donor and acceptor to be estimated. For toxin labeled with acrylodan at position 130 (in the hinge region), this distance is approximately 5 +/- 2 Angstrom, showing that the probe is close to the inner surface of the liposomes. A second probe labeled at position 69 (in the N-terminal domain) shows negligible energy transfer, indicating a distance of closest approach >40 Angstrom. This implies that this N-terminal region remains ''outside'' the liposome. We propose a model in which the central region of the alpha-toxin inserts into the membrane and possibly participates in forming the wall of the pore.

Levels of Alpha-Toxin Correlate with Distinct Phenotypic Response Profiles of Blood Mononuclear Cells and with agr Background of Community-Associated Staphylococcus aureus Isolates

Epidemiological studies of Staphylococcus aureus have shown a relation between certain clones and the presence of specific virulence genes, but how this translates into virulence-associated functional responses is not fully elucidated. Here we addressed this issue by analyses of community-acquired S. aureus strains characterized with respect to antibiotic resistance, ST types, agr types, and virulence gene profiles. Supernatants containing exotoxins were prepared from overnight bacterial cultures, and tested in proliferation assays using human peripheral blood mononuclear cells (PBMC). The strains displayed stable phenotypic response profiles, defined by either a proliferative or cytotoxic response. Although, virtually all strains elicited superantigen-mediated proliferative responses, the strains with a cytotoxic profile induced proliferation only in cultures with the most diluted supernatants. This indicated that the superantigen-response was masked by a cytotoxic effect which was also confirmed by flow cytometry analysis. The cytotoxic supernatants contained significantly higher levels of alpha-toxin than did the proliferative supernatants. Addition of alpha-toxin to supernatants characterized as proliferative switched the response into cytotoxic profiles. In contrast, no effect of Panton Valentine Leukocidin, delta-toxin or phenol soluble modulin alpha-3 was noted in the proliferative assay. Furthermore, a significant association between agr type and phenotypic profile was found, where agrII and agrIII strains had predominantly a proliferative profile whereas agrI and IV strains had a predominantly cytotoxic profile. The differential response profiles associated with specific S. aureus strains with varying toxin production could possibly have an impact on disease manifestations, and as such may reflect specific pathotypes.

A stability transition at mildly acidic pH in the alpha-hemolysin (alpha-toxin) from Staphylococcus aureus

The effects of mildly acidic conditions on the free energy of unfolding (Delta G(u)(buff)) of the pore-forming alpha-hemolysin (alpha HL) from Staphylococcus aureus were assessed between pH 5.0 and 7.5 by measuring intrinsic tryptophan fluorescence, circular dichroism and elution time in size exclusion chromatography during urea denaturation, Decreasing the pH from 7.0 to 5.0 reduced the calculated Delta G(u)(buff) from 8.9 to 4.2 kcal moI(-1), which correlates with an increased rate of pore formation previously observed over the same pH range, It is proposed that the lowered surface pH of biological membranes reduces the stability of alpha HL thereby modulating the rate of pore formation. (C) 1999 Federation of European Biochemical Societies.

AMINO-ACID-SEQUENCE OF TSTX-V, AN ALPHA-TOXIN FROM TITYUS-SERRULATUS SCORPION-VENOM, AND ITS EFFECT ON K+ PERMEABILITY OF BETA-CELLS FROM ISOLATED RAT ISLETS OF LANGERHANS

Highly purified Tityustoxin V (TsTX-V), an alpha-toxin isolated from the venom of the Brazilian scorpion Tityus serrulatus, was obtained by ion exchange chromatography on carboxymethylcellulose-52. It was shown to be homogeneous by reverse phase high performance liquid chromatography, N-terminal sequencing (first 39 residues) of the reduced and alkylated protein and by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate and tricine. Following enzymatic digestion, the complete amino acid sequence (64 residues) was determined. The sequence showed higher homology with the toxins from the venoms of the North African than with those of the North and South American scorpions. Using the rate of Rb-86(+) release from depolarized rat pancreatic beta-cells as a measure of K+ permeability changes, TsTX-V (5.6 mu g/ml) was found to increase by 2.0-2.4-fold the rate of marker outflow in the presence of 8.3 mM glucose. This effect was persistent and slowly reversible, showing similarity to that induced by 100 mu-M veratridine, an agent that increases the open period of Na+ channels, delaying their inactivation. It is suggested that, by extending the depolarized period, TsTX-V indirectly affects beta-cell voltage-dependent K+ channels, thus increasing K+ permeability.

Crystal structure of Staphylococcus aureus exfoliative toxin D-like protein: structural basis for the high specificity of exfoliative toxins

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Tertiary structural changes of the alpha-hemolysin from Staphylococcus aureus on association with liposome membranes

The interaction of alpha-hemolysin (also called alpha-toxin) from Staphylococcus aureus with mixed egg-yolk phosphatidylcholine/cholesterol liposomes has been investigated using the intrinsic tryptophan fluorescence emission (ITFE) signal. The ITFE intensity of alpha-hemolysin, which was obtained using a novel purification protocol, showed a triphasic increase on incubation with liposomes at low protein/lipid ratios. The first, rapid phase results in an increase in ITFE of 10%, which reflects rapid conformation changes in the alpha-hemolysin on association with the liposome membrane, the second phase of the ITFE increase is associated with a red shift from 334 to 339 nm in the maximum emission wavelength, suggesting the transition to a partially unfolded intermediate in the oligomerization process. The third phase of the ITFE intensity change demonstrates a temporal correlation with the appearance of SDS-stable oligomers. The results demonstrate the feasibility of identification of intermediate protein conformations in complex membrane-associated processes by manipulation of the liposomal membrane composition. (C) 1998 Academic Press.

Prevalence of methicillin resistance and virulence determinants of Staphylococcus aureus in diabetic foot ulcers

Background Diabetic foot ulceration (DFU) is a multifactorial process and is responsible for considerable morbidity and contributes to the increasing cost of health care worldwide. The diagnosis and identification of these ulcers remains a complex problem. Bacterial infection is promoted in the diabetic foot wound by decreased vascular supply and impaired host immune response. As conventional clinical microbiological methods are time-consuming and only identifies about 1% of the wound microbiota, detection of bacteria present in DFUs using molecular methods is highly advantageous and efficient. The aim of this study was to assess the virulence and methicillin resistance profiles of Staphylococcus aureus detected in DFUs using DNA-based methods. Methods A total of 223 swab samples were collected from 30 patients from March to October 2012. Bacterial DNA was extracted from the swab samples using standard procedures and was used to perform polymerase chain reaction (PCR) using specific oligonucleotide primers. The products were visualized using agarose gel electrophoresis. Results S. aureus was detected in 44.8% of samples. 25% of the S. aureus was methicillin-resistant S. aureus harboring the mecA gene. The alpha-toxin gene was present in 85% of the S. aureus positive samples. 61% of the S. aureus present in DFU samples harbored the exfoliatin factor A gene. Both the fibronectin factor A and fibronectin factor B gene were detected in 71% and 74% of the S. aureus positive samples. Conclusions DNA-based detection and characterization of bacteria in DFUs are rapid and efficient and can assist in accurate, targeted antibiotic therapy of DFU infections. The majority of S. aureus detected in this study were highly virulent and also resistant to methicillin. Further studies are required to understand the role of S. aureus in DFU trajectory.

Modelling staphylococcal pneumonia in a human 3D lung tissue model system delineates toxin-mediated pathology

Staphylococcus aureus necrotizing pneumonia is recognized as a toxin-mediated disease, yet the tissue-destructive events remain elusive, partly as a result of lack of mechanistic studies in human lung tissue. In this study, a three-dimensional (3D) tissue model composed of human lung epithelial cells and fibroblasts was used to delineate the role of specific staphylococcal exotoxins in tissue pathology associated with severe pneumonia. To this end, the models were exposed to the mixture of exotoxins produced by S. aureus strains isolated from patients with varying severity of lung infection, namely necrotizing pneumonia or lung empyema, or to purified toxins. The necrotizing pneumonia strains secreted high levels of alpha-toxin and Panton-Valentine leukocidin (PVL), and triggered high cytotoxicity, inflammation, necrosis and loss of E-cadherin from the lung epithelium. In contrast, the lung empyema strain produced moderate levels of PVL, but negligible amounts of alpha-toxin, and triggered limited tissue damage. alpha-toxin had a direct damaging effect on the epithelium, as verified using toxin-deficient mutants and pure alpha-toxin. Moreover, PVL contributed to pathology through the lysis of neutrophils. A combination of alpha-toxin and PVL resulted in the most severe epithelial injury. In addition, toxin-induced release of pro-inflammatory mediators from lung tissue models resulted in enhanced neutrophil migration. Using a collection of 31 strains from patients with staphylococcal pneumonia revealed that strains producing high levels of alpha-toxin and PVL were cytotoxic and associated with fatal outcome. Also, the strains that produced the highest toxin levels induced significantly greater epithelial disruption. Of importance, toxin-mediated lung epithelium destruction could be inhibited by polyspecific intravenous immunoglobulin containing antibodies against alpha-toxin and PVL. This study introduces a novel model system for study of staphylococcal pneumonia in a human setting. The results reveal that the combination and levels of alpha-toxin and PVL correlate with tissue pathology and clinical outcome associated with pneumonia.

Convergencia de vías de señalización en un modelo de apoptosis

La sepsis es un evento inflamatorio generalizado del organismo inducido por un daño causado generalmente por un agente infeccioso. El patógeno más frecuentemente asociado con esta entidad es el Staphylococcus aureus, responsable de la inducción de apoptosis en células endoteliales debida a la producción de ceramida. Se ha descrito el efecto protector de la proteína C activada (PCA) en sepsis y su relación con la disminución de la apoptosis de las células endoteliales. En este trabajo se analizó la activación de las quinasas AKT, ASK1, SAPK/JNK y p38 en un modelo de apoptosis endotelial usando las técnicas de Western Blotting y ELISA. Las células endoteliales (EA.hy926), se trataron con C2-ceramida (130μM) en presencia de inhibidores químicos de cada una de estas quinasas y PCA. La supervivencia de las células en presencia de inhibidores químicos y PCA fue evaluada por medio de ensayos de activación de las caspasas 3, 7 y 9, que verificaban la muerte celular por apoptosis. Los resultados evidencian que la ceramida reduce la activación de AKT y aumenta la activación de las quinasas ASK, SAPK/JNK y p38, en tanto que PCA ejerce el efecto contrario. Adicionalmente se encontró que la tiorredoxina incrementa la activación/fosforilación de AKT, mientras que la quinasa p38 induce la defosforilación de AKT.

Etiolofia, epidemiologia e prognóstico das pneumonias associadas à ventilação mecânica em um hospital de ensino, com ênfase no estudo molecular e virulência de Staphylococcus aureus

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Etiolofia, epidemiologia e prognóstico das pneumonias associadas à ventilação mecânica em um hospital de ensino, com ênfase no estudo molecular e virulência de Staphylococcus aureus

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Clostridium difficile: Entdeckung und Charakterisierung der autokatalytischen Prozessierung von Toxin B

Clostridium difficile, der Auslöser der nosokomialen Antibiotika-assoziierten Durchfälle und der Pseudomembranösen Kolitis, besitzt zwei Hauptvirulenzfaktoren: die Toxine A und B. In vorangegangenen Veröffentlichungen wurde gezeigt, dass Toxin B durch einen zytosolischen Faktor der eukaryotischen Zielzelle während des Aufnahmeweges in die Zelle gespalten wird. Nur die N-terminale katalytische Domäne erreicht das Zytosol. Hierbei wurde davon ausgegangen, dass eine Protease der Zielzelle die Spaltung katalysiert. In dieser Arbeit konnte gezeigt werden, dass die Spaltung von Toxin B ein intramolekularer Prozess ist, der zytosolisches Inositolphosphat der Zielzelle als Kofaktor zur Aktivierung der intrinsischen Protease benötigt. Die Freisetzung der katalytischen Domäne durch Inositolphosphat-induzierte Spaltung ist nicht nur das Prinzip des Clostridium difficile Toxin B sondern auch des Toxin A, als auch des alpha Toxin von Clostridium novyi und das Letale Toxin von Clostridium sordellii. Der kovalente Inhibitor von Aspartatproteasen 1,2-epoxy-3-(p-nitrophenoxy)propan (EPNP), wurde dazu verwendet die intrinsische Protease von Toxin B zu blockieren und ermöglichte die Identifikation des katalytischen Zentrums. EPNP modifiziertes Toxin B verliert die intrinsische Proteaseaktivität und Zytotoxizität, aber wenn es direkt in das Zytosol der Wirtszelle injiziert ist, bleibt die Toxizität erhalten. Diese ist damit der erste Bericht eines bakteriellen Toxins, das eukaryotische Signale zur induzierten Autoproteolyse nutzt, um seine katalytisch-toxische Domäne in das Zytosol der Zielzelle freizusetzen. Durch diese Ergebnisse kann das Modell der Toxin-Prozessierung nun um einen weiteren entscheidenden Schritt vervollständigt werden.