The long-term interest of the group has been to understand the molecular mechanisms that govern the interaction between bacterial toxins and target mammalian cells, with a focus on pore-forming toxins and the anthrax toxin. Pore-forming toxins are the most widespread toxins; they are produced by diverse pathogenic bacteria and other pathogenic organisms. They act on the plasma membrane by rendering it permeable to small ions and, for certain toxins such as those dependent on cholesterol, to proteins. Their contribution to virulence varies from one bacterium to another. Anthrax toxin is a tripartite toxin composed of a receptor-binding subunit—the protective antigen—and an enzymatic subunit—the lethal factor, which is a metalloprotease that cleaves MAP kinase kinases. Anthrax toxin, which is produced by Bacillus anthracis, exerts its effect by impairing the function of the immune system.
An essential step in the mode of action of all toxins with enzymatic activity, with the exception of lipases, is to access the cytoplasm where their targets reside. Using a variety of biochemical and cell-biological techniques, we have outlined the unusual and novel entry route taken by anthrax toxin. Unlike most toxins that hitch themselves to their receptor to enter cells by constitutive endocytosis, we found that anthrax toxin triggers signaling events that lead to its subsequent endocytosis, much like EGF-triggered endocytosis of its receptors. Multiple toxin-triggered post-translational modifications of the cytoplasmic tail of the receptors, such as monoubiquitination, are involved in the uptake process and are under investigation.
Most toxins are released into the cytoplasm upon reaching early endosomes, the first station of the endocytic pathway. By contrast, anthrax toxin penetrates deeply into the cell, reaching late endosomes, which are complex multivesicular organelles; from there the toxin is released into the cytoplasm. We found that the enzymatic subunits of the toxin do not reside in the lumen of late endosomes, which constitute an inhospitable environment because of the low pH (less than 5.5) and the presence of hydrolases. Instead, the toxin localizes to the lumen of the intraluminal vesicles found within the late endosomes, where it is protected from degradation. Thus, the toxin is safely transported to the perinuclear region of the cell. Through a poorly characterized intra-endosomal transport step known as back-fusion, the enzymatic subunits are then transported from the lumen of these vesicles to the cytoplasm. Our working model is that this localized delivery system brings the enzymatic subunits of the toxin into proximity of specific substrates and that regulation of substrate modification in time and space is important for the physiological outcome.
Recently, we investigated if and how cells sense the action of toxins and whether they mount a response. Interestingly, we found that although cellular responses vary depending on the type of target cell and the toxin, cells use similar devices to sense the effects of the pore-forming toxins and the anthrax toxin. Specifically, we found that, through a change in cytoplasmic ion composition, cells sense pore formation by aerolysin from Aeromonas hydrophila or by alpha-toxin from Staphylococcus aureus. In particular, the decline in intracellular potassium levels leads to activation of a cellular danger-sensing device called the inflammasome. The inflammasome is a multiprotein complex, whose central element is a member of the Nod-like receptor family, or NLR. In analogy to the Toll-like receptors involved in detecting the presence of extracellular pathogens, NLRs are involved in detecting intracellular pathogens or threats in general. Pore formation leads to the creation of inflammasomes containing the NAPL3 or IPAF NLR. The role of these inflammasomes is to mediate activation of caspase-1, a pro-inflammatory caspase. This cysteine protease is known to activate pro-interleukin 1β to form interleukin 1β. We found, however, that one of the downstream effects of this pathway was to mediate cell survival in response to pore formation by activating lipid-metabolic pathways; the downstream effectors remain to be identified. Thus, pore formation leads to potassium efflux, activation of a NALP3-containing inflammasome, activation of caspase-1, and promotion of cell survival.
We found that anthrax toxin also leads to the activation of caspase-1 in mouse dendritic cells, in a manner that depends on the genetic background. In marked contrast to the cellular response to aerolysin, caspase-1 activation by anthrax lethal toxin is mediated by NALP1, but not by NALP3, and, instead of promoting survival, leads to cell death. These observations also show that cell death is due to the cellular response and not the action of the toxin. Preliminary experiments suggest that in cells treated with lethal toxin, additional pathways are triggered, some of which promote cell survival.
Taken together, our parallel studies on the cellular responses of two markedly different toxins, aerolysin and anthrax toxin, are shaping our view of the highly complex and modular sensing systems with which cells sense threats. Our working model is that multiple NOD-like receptor family members sense the effects of a given toxin, leading to the onset of different signaling cascades, whose efficiencies are modulated by the genetic background, the cell type, and the nature and amplitude of the toxic insult.
Last updated August 2008
