Above and beyond C5a Receptor Targeting by Staphylococcal Leucotoxins: Retrograde Transport of Panton-Valentine Leucocidin and γ-Hemolysin.

Various membrane receptors associated with the innate immune response have recently been identified as mediators of the cellular action of Staphylococcus aureus leucotoxins. Two of these, the Panton-Valentine leucotoxin LukS-PV/LukF-PV and the γ-hemolysin HlgC/HlgB, bind the C5a complement-derived peptide receptor. These leucotoxins utilize the receptor to induce intracellular Ca2+ release from internal stores, other than those activated by C5a. The two leucotoxins are internalized with the phosphorylated receptor, but it is unknown whether they divert retrograde transport of the receptor or follow another pathway. Immunolabeling and confocal microscopic techniques were used to analyze the presence of leucotoxins in endosomes, lysosomes, endoplasmic reticulum, and Golgi. The two leucotoxins apparently followed retrograde transport similar to that of the C5a peptide-activated receptor. However, HlgC/HlgB reached the Golgi network very early, whereas LukS-PV/LukF-PV followed slower kinetics. The HlgC/HlgB leucotoxin remained in neutrophils 6 h after a 10-min incubation of the cells in the presence of the toxin with no signs of apoptosis, whereas apoptosis was observed 3 h after neutrophils were incubated with LukS-PV/LukF-PV. Such retrograde transport of leucotoxins provides a novel understanding of the cellular effects initiated by sublytic concentrations of these toxins.

Internalization of staphylococcal leukotoxins that bind and divert the C5a receptor is required for intracellular Ca(2+) mobilization by human neutrophils.

A growing number of receptors, often associated with the innate immune response, are being identified as targets for bacterial toxins of the beta-stranded pore-forming family. These findings raise the new question of whether the receptors are activated or merely used as docking points facilitating the formation of a pore. To elucidate whether the Staphylococcus aureus Panton-Valentine leukocidin and the leukotoxin HlgC/HlgB act through the C5a receptor (C5aR) as agonists, antagonists or differ from the C5a complement-derived peptide, their activity is explored on C5aR-expressing cells. Both leukotoxins equally bound C5aR in neutrophils and in stable transfected U937 cells and initiated mobilization of intracellular Ca(2+) . HlgC/HlgB requires the presence of robust intracellular acidic Ca(2+) stores in order to evoke a rise in free [Ca(2+) ]i , while the LukS-PV/LukF-PV directly altered reticular Ca(2+) stores. Intracellular target specificity is conferred by the F-subunit associated to the S-subunit binding the receptor. Furthermore, internalization of the two leukotoxin components (S- and F-subunits) associated to C5aR is required for the initiation of [Ca(2+) ]i mobilization. Electrophysiological recordings on living cells demonstrated that LukS-PV/LukF-PV does not alter the membrane resistance of C5aR-expressing cells. The present observations suggest that part of the pore-forming process occurs in distinct intracellular compartments rather than at the plasma membrane.

R-type calcium channels are crucial for semaphorin 3A-induced DRG axon growth cone collapse.

Semaphorin 3A (Sema3A) is a secreted protein involved in axon path-finding during nervous system development. Calcium signaling plays an important role during axonal growth in response to different guidance cues; however it remains unclear whether this is also the case for Sema3A. In this study we used intracellular calcium imaging to figure out whether Sema3A-induced growth cone collapse is a Ca2+ dependent process. Intracellular Ca2+ imaging results using Fura-2 AM showed Ca2+ increase in E15 mice dorsal root ganglia neurons upon Sema3A treatment. Consequently we analyzed Sema3A effect on growth cones after blocking or modifying intracellular and extracellular Ca2+ channels that are expressed in E15 mouse embryos. Our results demonstrate that Sema3A increased growth cone collapse rate is blocked by the non-selective R- and T- type Ca2+ channel blocker NiCl2 and by the selective R-type Ca2+ channel blocker SNX482. These Ca2+ channel blockers consistently decreased the Sema3A-induced intracellular Ca2+ concentration elevation. Overall, our results demonstrate that Sema3A-induced growth cone collapses are intimately related with increase in intracellular calcium concentration mediated by R-type calcium channels.

Residues essential for Panton-Valentine leukocidin S component binding to its cell receptor suggest both plasticity and adaptability in its interaction surface.

Panton-Valentine leukocidin (PVL), a bicomponent staphylococcal leukotoxin, is involved in the poor prognosis of necrotizing pneumonia. The present study aimed to elucidate the binding mechanism of PVL and in particular its cell-binding domain. The class S component of PVL, LukS-PV, is known to ensure cell targeting and exhibits the highest affinity for the neutrophil membrane (Kd∼10(-10) M) compared to the class F component of PVL, LukF-PV (Kd∼10(-9) M). Alanine scanning mutagenesis was used to identify the residues involved in LukS-PV binding to the neutrophil surface. Nineteen single alanine mutations were performed in the rim domain previously described as implicated in cell membrane interactions. Positions were chosen in order to replace polar or exposed charged residues and according to conservation between leukotoxin class S components. Characterization studies enabled to identify a cluster of residues essential for LukS-PV binding, localized on two loops of the rim domain. The mutations R73A, Y184A, T244A, H245A and Y250A led to dramatically reduced binding affinities for both human leukocytes and undifferentiated U937 cells expressing the C5a receptor. The three-dimensional structure of five of the mutants was determined using X-ray crystallography. Structure analysis identified residues Y184 and Y250 as crucial in providing structural flexibility in the receptor-binding domain of LukS-PV.

Staphylococcal leukotoxins trigger free intracellular Ca(2+) rise in neurones, signalling through acidic stores and activation of store-operated channels.

Headache, muscle aches and chest pain of mild to medium intensity are among the most common clinical symptoms in moderate Staphylococcus aureus infections, with severe infections usually associated with worsening pain symptoms. These nociceptive responses of the body raise the question of how bacterial infection impinges on the nervous system. Does S. aureus, or its released virulence factors, act directly on neurones? To address this issue, we evaluated the potential effects on neurones of certain bi-component leukotoxins, which are virulent factors released by the bacterium. The activity of four different leukotoxins was verified by measuring the release of glutamate from rat cerebellar granular neurones. The bi-component γ-haemolysin HlgC/HlgB was the most potent leukotoxin, initiating transient rises in intracellular Ca(2+) concentration in cerebellar neurones and in primary sensory neurones from dorsal root ganglia, as probed with the Fura-2 Ca(2+) indicator dye. Using pharmacological antagonists of receptors and Ca(2+) channels, the variations in intracellular Ca(2+) concentration were found independent of the activation of voltage-operated Ca(2+) channels or glutamate receptors. Drugs targeting Sarco-Endoplasmic Reticulum Ca(2+)-ATPase (SERCA) or H(+)-ATPase and antagonists of the store-operated Ca(2+) entry complex blunted, or significantly reduced, the leukotoxin-induced elevation in intracellular Ca(2+). Moreover, activation of the ADP-ribosyl cyclase CD38 was also required to initiate the release of Ca(2+) from acidic stores. These findings suggest that, prior to forming a pore at the plasma membrane, leukotoxin HlgC/HlgB triggers a multistep process which initiates the release of Ca(2+) from lysosomes, modifies the steady-state level of reticular Ca(2+) stores and finally activates the Store-Operated Calcium Entry complex.

A label-free biosensor assay for botulinum neurotoxin B in food and human serum.

Botulinum neurotoxins (BoNTs) are among the most toxic substances known. Surveillance and diagnostics require methods for rapid detection of BoNTs in complex media such as foodstuffs and human serum. We have developed in vitro assays to specifically detect the protease activity of botulinum neurotoxin B (BoNT/B) on a time scale of minutes. Cleavage of the BoNT/B substrate VAMP2, a membrane SNARE protein associated with synaptic vesicles, was monitored using real-time surface plasmon resonance to measure vesicle capture by specific antibodies coupled to microchips. The assay is functional in low-ionic-strength buffers and stable over a wide range of pH values (5.5-9.0). Endoproteolytic cleavage of VAMP2 was detected in 10 min with 2 pM native BoNT/B holotoxin. Contamination of liquid food products such as carrot juice, apple juice, and milk with low picomolar amounts of BoNT/B was revealed within 3h. BoNT/B activity was detected in sera from patients with type B botulism but not in healthy controls or patients with other neurological diseases. This robust, sensitive, and rapid protein chip assay is appropriate for monitoring BoNT/B in food products and diagnostic tests for type B botulism and could replace the current in vivo mouse bioassay.

Clostridium perfringens epsilon toxin targets granule cells in the mouse cerebellum and stimulates glutamate release.

Epsilon toxin (ET) produced by C. perfringens types B and D is a highly potent pore-forming toxin. ET-intoxicated animals express severe neurological disorders that are thought to result from the formation of vasogenic brain edemas and indirect neuronal excitotoxicity. The cerebellum is a predilection site for ET damage. ET has been proposed to bind to glial cells such as astrocytes and oligodendrocytes. However, the possibility that ET binds and attacks the neurons remains an open question. Using specific anti-ET mouse polyclonal antibodies and mouse brain slices preincubated with ET, we found that several brain structures were labeled, the cerebellum being a prominent one. In cerebellar slices, we analyzed the co-staining of ET with specific cell markers, and found that ET binds to the cell body of granule cells, oligodendrocytes, but not astrocytes or nerve endings. Identification of granule cells as neuronal ET targets was confirmed by the observation that ET induced intracellular Ca(2+) rises and glutamate release in primary cultures of granule cells. In cultured cerebellar slices, whole cell patch-clamp recordings of synaptic currents in Purkinje cells revealed that ET greatly stimulates both spontaneous excitatory and inhibitory activities. However, pharmacological dissection of these effects indicated that they were only a result of an increased granule cell firing activity and did not involve a direct action of the toxin on glutamatergic nerve terminals or inhibitory interneurons. Patch-clamp recordings of granule cell somata showed that ET causes a decrease in neuronal membrane resistance associated with pore-opening and depolarization of the neuronal membrane, which subsequently lead to the firing of the neuronal network and stimulation of glutamate release. This work demonstrates that a subset of neurons can be directly targeted by ET, suggesting that part of ET-induced neuronal damage observed in neuronal tissue is due to a direct effect of ET on neurons.

[Measurement of botulinum toxin activity: towards a new cellular culture assay?]. / Dosage de l'activité toxine botulique : vers un nouveau dosage sur culture cellulaire ?

UNLABELLED: The actual gold standard of Botulin A toxin (BoTx A) batches qualification is the mouse lethality assay. With this assay it is nevertheless impossible to set a therapeutic value unit. AIMS: The goal of this research was to study the effects of BoTx A increasing concentrations on glutamatergic rat neurons. EXPERIMENTAL PROCEDURE: We studied the glutamate release with increasing concentrations of BoTx A. We also studied the BoTx A target cleavage with a western blot technique. RESULTS: Our results proved that it is possible to establish a dose-response - like curve of BoTx A effects on glutamate release. Moreover the cleavage of the target protein was visible for the same toxin concentrations that inhibited the glutamate release. CONCLUSION: This technique could be the first step toward a new way of setting a better pharmaceutical profile for toxin batches.

Isoflavonoid-based bone-sparing treatments exert a low activity on reproductive organs and on hepatic metabolism of estradiol in ovariectomized rats.

The use of soy isoflavones is a potential alternative to hormone replacement therapy in post-menopausal bone-loss prevention. Nevertheless, phytoestrogens can target other organs and may disrupt cell proliferation, or could modify endogenous steroid hormone metabolism. These mechanisms could be linked to an increased risk of developing cancer. We therefore studied the possible side effects of such treatments in an experimental model of menopause. Forty adult female Wistar rats were ovariectomized and fed with a genistein-, daidzein- or equol-supplemented diet at bone-sparing levels (10 mg/kg BW/day) for 3 months. The estrogenic effects were assessed by histological and molecular analyses on reproductive organs. The impact on the oxidative metabolism of estradiol and on associated cytochrome P450 (CYP) activities was evaluated in liver microsomes. The relative wet weights of both the uterus and the vagina were increased in the equol group, but no significant changes in proliferating cell nuclear antigen or hormone receptor mRNA expression were noticed. In contrast, genistein and daidzein did not induce uterotrophy but caused an overexpression of estrogen receptor alpha mRNA which could correspond to a long-lasting effect of physiological concentrations of estrogens. The hepatic metabolism of estradiol was influenced by daidzein which increased the synthesis of putative mutagenic derivatives. At the same time, genistein favored estrogen 2-hydroxylation, and equol decreased 4-hydroxyestrogen production. Surprisingly, no significant alteration in hepatic CYP activities was detected. Taken together, these results demonstrate that isoflavonoid-based bone-sparing treatments are able to cause side effects on other estrogen-sensitive target organs when given in the long-term.

Effect of a cyclohexenonic long-chain fatty alcohol on calcium mobilization.

Cyclohexenonic long-chain fatty alcohols constitute a family of synthetic compounds with trophic, secretagogue and antioxidant properties. Despite their multiple biological actions in neuronal and non-neuronal tissues, the intracellular mechanisms underlying CFA activity remain unknown. In the present study, we show that 3-(15-hydroxypentadecyl)-2,4,4-trimethyl-2-cyclohexen-1-one (tCFA15) directly mobilizes Ca(2+) in the pituitary neural lobe synaptosomes and in primary sensory neurons from dorsal root ganglia. This effect is dependent on the presence of extracellular Ca(2+), but does not involve transmembrane voltage-operated calcium channels. Using a combination of pharmacological agents that block or deplete intracellular Ca(2+) stores, our results suggest the implication of a calcium induced-calcium release mechanism evoked by tCFA15-induced Ca(2+) influx. To our knowledge, these findings constitute the first attempt towards the comprehension of the biological actions of cyclohexenonic long-chain fatty alcohols at a molecular level.

Expression of tetrodotoxin-sensitive and resistant sodium channels by rat melanotrophs.

Rat melanotrophs fire Na+ and Ca2(+)-dependent action potentials. Whereas the molecular identity of Ca2+ channels expressed by these cells is well documented, less is known about Na channels. We characterize the expression of seven sodium channel alpha-subunit and the beta1- and beta2-subunit mRNAs. The tetrodotoxin-resistant Nav1.8 and Nav1.9 alpha subunit mRNAs are detected in the newborn intermediate lobe and in cultured melanotrophs. Electrophysiological recordings further demonstrate the expression of both tetrodotoxin-sensitive and tetrodotoxin-resistant currents by dissociated melanotrophs. Moreover, activated sodium channels are able to elicit intracellular calcium waves, both in the absence or in the presence of tetrodotoxin. This work shows that rat melanotrophs express functional tetrodotoxin-resistant sodium channels, whose activation can lead to the generation of intracellular calcium waves.