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Anthrax toxin triggers endocytosis of its receptor via a lipid raft-mediated clathrin-dependent process
Anthrax is a lethal disease mainly transmitted by animals, recently known for its implication in terrorist attacks in 2001.The bacterium Bacillus anthracis is responsible of anthrax by producing the “anthrax toxin” which is composed of 3 subunits : EF, LF and PA. EF and LF are active factors that need PA to exert their effects. PA acts as a carrier (EF and LF can not reach cytoplasmic targets alone).
The extracellular behaviour of PA has already been described : PA 83 (a 83 kD protein), binds ATR (Anthrax Toxin Receptor) on the target’s surface, then is cleaved by a furin (protease) and gives birth to PA 63 (a 63 kD protein). The consequences of the formation of PA 63 is its heptamerization. Heptamer can bind LF and/or EF. The internalization process includes a transport to endosomes where channel-heptamer (permitted by low pH) allows exit of active factors towards cytoplasm, where targets are located.
Nature of questions
They wonder about a specific mechanism (the entry of the toxin) that might help them to understand a little better how the Anthrax toxin triggers infection. However their primary motivation does not seem to be the finding of a therapeutic target (drugs) against anthrax.
Concretely, they want to stress endocytosis mechanisms and more precisely they wonder if cleavage of PA 83 into PA 63 causes a change in PA 63 distribution on cell membrane. Also they try to determine if this hypothetical change of distribution may deal with PA 63 entry into the targeted cells.
They rely on a comparison with aerolysin to find the items they need to test.
Indeed, the analogy with aerolysin (pore-making as well) led the researchers to seek if there was an association of PA 63 with lipid rafts (especially caveolae) that would trigger PA 63 internalization.
In fact, rafts seem to play a key role in aerolysin endocytosis caused by heptamerization and they want to know if it is possible to extend this mechanism to PA 63.
Western-blots (fig 1, 2, 4, 5)
Microscopy (fig 2, 3, 5). More precisely, electronic microscopy (fig 5E) and fluorescence microscopy (other figures). By the way, we have to admit that biochemical methods, immunofluorescence and electron microscopy are described too precisely for us to give a valuable opinion on.
Answer to questions with experiments
Figure 1 :
PA 63 is situated in lipid rafts, unlike PA 83 (1A).
More precisely, PA 63 is linked to cholesterol and sphingomyelin, i.e. linked to lipid rafts, which are not caveolae (1B).
They emphasize that ATR, before binding PA 63, is localized out of lipid rafts. It constitutes a major difference with aerolysin-receptor which is, by itself, constitutively located in rafts. So, PA 63 (linked to ATR) forces rafts association of ATR(1C).
More accurately rafts association is due to PA 63 heptamerization(1D).
Figure 2 : They verify that [ATR+PA 63 heptamerized] clustering explains lipid rafts association. They imitate clustering by heptamerization by using an artificial method : primary and secondary antibodies “sandwich”.
The team highlights that ATR-clustering by antibodies explains rafts association - like with PA 63 because they have the same behaviour (fig 2A to 2E). Moreover PA 83 clusters are not located in caveolae (fig 2F).
Figure 3 : Next they study internalization of PA 83 and whether it is caused by clustering.They prove that clustering with antibodies causes PA 83 internalization and thereby of PA 63 if prolonging the paralell explained in fig 2 (fig 3A and 3B : it requires antibodies sandwich and then adequate temperature).
Figure 4 : Then they focus on final sequence of anthrax infection that means LF exit of endosomes. They show that lipid rafts integrity is necessary for entry in endosomes and exit from it, hence the confirmation of the endocytosis of PA 63 heptamers thanks to lipid rafts (fig 4 : deletion of cholesterol).
Figure 5 : Finally the researchers at last confirm that PA (both PA 83 and PA 63 heptamer) internalization does not use caveolae (fig 5A, 5B, unpublished data about macrophages without caveolin, 5 E, 5 G and 5 H).
In the other hand they show that internalization occurs via a clathrin-dependent pathway (fig 5 C, 5D, 5E,). They confirm the role of clathrin, and show that no endocytosis happens via other pathway that clathrin-dependent or caveosomes (fig 5G and 5 H). They further indicate that PA 83 moves to endosomes after clathrin mediated endocytosis (fig 5F).
Researchers stress the fact that they do not know the physiological role of ATR but that their work helps to understand why ATR seems to be an ideal anthrax toxin receptor.
They detail all the points that make ATR a good receptor :
It stays at the surface for a long time,
the initial localization and the low endocytosis rate ensure the cleveage of PA83 by furin,
its capacity to redistribute to rafts to be internalized,
rapid clathrin mediated uptake once heptamerization is done.
They conclude by stating that the potential target for therapeutic they are thinking about are lipid rafts.
First of all we can discuss about the experiment page 6 in which the researchers were studying the presence of clustered PA SNKE in clathrin-coated vesicles. As a matter of fact, they mention that they found PA SNKE in nonclathrin coated invaginations without detailing much of this phenomena. We can wonder if these results could suggest an other pathway of endocytosis, if this exists in the physiological process with PA 63 and if this noncalthrin invaginations contribute to the transport toward endosomes.
An other element that was problematic to us concerns the Eps 15 dominant negative experiment. How can we explain the delay of the appearance of SDS-resistant PA 60 heptamer while the clathrin pathway is not totally inhibited ?
Finally we can mention some careless mistakes :
End of first column page 3 : « Fig. 2 D ; the majority of ATR-HA remains in detergent soluble fractions because of the cellular vast excess of receptors with respect to PA, and because intra-cellular receptors are unavailable for PA63 PA 83 binding). »
Beginning of legend of figure 3B : « intracellular accumulation of PA63 PA 83. »
Legend of figure 5 (third line starting from the end) : « plotted as in C D. »
However the references were really detailed in this article and the experiments were clearly exposed.experiments
Figure 5 : Finally the researchers