Cholesterol, It’s Not Just For Heart Disease Anymore Amy Kerzmann and Andrew L. Feig* Department of Chemistry, Indiana University, 800 East Kirkwood Avenue, Bloomington, Indiana 47405
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n insidious threat lurks in the dark corners of hospital wards. Because of the widespread use of broad-spectrum antibiotics, C. difficile, an opportunistic pathogen, has become one of the most common hospital-acquired infections in the United States and Canada (1–3 ). It is also a growing threat to patients in nursing homes and extended care facilities (2 ). C. difficile colonizes the underpopulated anaerobic niches in the GI tracts of patients after their normal microflora has been killed. This organism causes pseudomembraneous colitis and severe antibiotic-associated diarrhea, also called C. difficile-associated diarrhea (CDAD) (1 ). The organism secretes two toxins, Toxin A (TcdA) and Toxin B (TcdB), that are the virulence factors responsible for the cellular damage (4 ). It was estimated in 2002 that the U.S. medical community spends more than $1.1 billion annually combating these infections (5 ). Recent work by Giesemann and colleagues shows that membrane cholesterol levels play a significant role in the transport of the C. difficile toxins into eukaryotic cells (6 ). The data suggest the intriguing possibility that cholesterol may act as a small molecule chaperone to facilitate the insertion of the protein into the membrane, thus, generating the pore necessary to translocate the catalytic component of the toxin into the cytoplasm. TcdA and TcdB are very large proteins (on the order of 300 kDa). They share 48% identity and in vitro behave in a very similar fashion. Each toxin is comprised of three functional domains (Figure 1, www.acschemicalbiolog y.o rg
panel a) (7 ). A C-terminal domain binds cell surface receptors for target recognition, and an N-terminal domain carries a glucosyltransferase functionality that targets a family of Ras-like G-proteins, disrupting their function upon modification. The large central domain participates in the translocation process. It creates the pore necessary to transport the catalytic domain across the endosomal membrane after endocytosis. Once internalized, it is believed that the catalytic domain is proteolytically processed by a cellular protease around residue 543, freeing it from the remainder of the toxin which, having completed its function, remains in the endosomal membrane (8 ). Interrupting any of these three major steps (cellular recognition, internalization, or catalysis) could in theory disrupt intoxication. The Internalization Process. One of the critical events during intoxication by the bacterial toxin is a protein transduction step, the movement of the catalytic domain from outside the cell into the cytoplasm (Figure 1, panel b, steps 3–6) (9, 10 ). In this process, the toxin must trigger endo cytosis and then escape from the endosome into the cytoplasm. The toxins that use endosome-mediated uptake can be divided into two classes. Cholera and Shiga toxins exemplify one class. They travel from the endosome into the Golgi body and eventually to the endoplasmic reticulum before leaving the vacuole and entering the cytoplasm. This mode of uptake is called the long-trip model. TcdA and TcdB are members of the other class of toxins that
A b s t r a c t Recent studies have shown that cholesterol plays a significant role in the ability of Toxin A from Clostridium difficile to enter eukaryotic cells. The translocation process is one of three major steps during intoxication that could be targeted for intervention against the severe antibiotic-associated diarrhea caused by C. difficile.
*To whom correspondence should be addressed. E-mail:
[email protected].
Published online April 21, 2006 10.1021/cb600133b CCC: $33.50 © 2006 by American Chemical Society
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Figure 1. a) The three functional regions of TcdA: the N-terminal enzymatic domain (red), the central translocation region (orange), and the C-terminal repetitive oligopeptide (CROP) domain (green). There is also a small C-terminal hydrophobic region present after the CROP region in TcdA (white), not observed in TcdB. The highlighted residues are essential for UDP-Glc binding (W102) and/or catalysis (DxD motif at 286–288). b) Cellular intoxication by C. difficile TcdA and TcdB. (Step 1) TcdA and TcdB are exported from the bacterium; (step 2) the C-terminal CROP motif binds to cell-surface carbohydrates; (steps 3 and 4) the toxin–receptor complex is internalized through receptor-mediated endocytosis; (step 5) acidification of the maturing endosome by V-type ATPases drives a pH-dependent conformational change of the central translocation domain, resulting in insertion into cholesterolcontaining endosomal membranes; (step 6) the N‑terminal catalytic fragment is released into the cytosol; (step 7) once in the cytosol, the toxin fragment catalyzes the transfer of glucose from UDP-glucose to a conserved threonine residue of specific Ras-like GTPases; and (step 8) monoglucosylation of the G-proteins blocks the conformational changes that normally occur as the protein switches between GDP- and GTP-bound states. By preventing these conformational changes, these proteins are effectively “turned off” and are unable to interact with their effector proteins leading to depolymerization of the actin cytoskeleton, cellular rounding, and ultimately cell death.
also includes the diphtheria and anthrax toxins. These toxins escape directly from the early endosomes (Figure 1b, step 6). The endosomal compartment is acidified by vacuolar ATPases as it begins to travel toward the Golgi body. The toxin uses the acidification process as its cue to insert into the membrane, forming a pore and extruding its catalytic domain into the cytoplasm. Giesemann and colleagues have explored the translocation phase of TcdA intoxication and found it to be highly dependent upon the presence of membrane cholesterol (6 ). The authors preloaded cells with 86Rb+. They then used 86Rb+ efflux measurements and single channel conductance to probe whether the toxin success fully inserted into the membrane and formed a channel. By analyzing intoxication of cell lines that differed in membrane cholesterol content, they could compare the relative susceptibility to the toxin. They also treated cells with methyl-b-cyclodextrin 142
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(MbCD), a reagent that binds to and depletes cholesterol from the plasma membrane. Cells treated with MbCD showed marked reductions in their mortality after exposure to the toxin. The toxin still bound to the surface of the cholesterol-depleted membranes, but failed to form pores, and the catalytic domain was incapable of being transported into the cytosol. One possible reason for cholesteroldependent toxin uptake would be involvement of lipid raft structures or the receptors associated with them. Lipid rafts are specialized microdomains in the membranes that have a high concentration of both cholesterol and membrane proteins (11, 12 ). By depleting cells of cholesterol, one might disrupt these rafts and thereby disperse the receptors contained therein. To address this possibility, Giesemann and colleagues treated cells with a phosphatidylinositol-specific phospholipase C (PI-PLC). This enzyme hydrolyzes the proteins and Kerzmann and Feig
polysaccharide receptors from glycosyl phosphatidylinositol (GPI)-anchored structures, leaving the remainder of the lipid raft intact. HT-29 cells treated with PI‑PLC were just as susceptible to intoxication as the untreated cells. This result demonstrated that GPI-anchored receptors are not required for uptake, but the remainder of the lipid raft might still play a role. So what is the function of cholesterol in toxin translocation? Are there direct interactions between cholesterol and the toxin or does cholesterol simply affect the physical properties of the membrane making it more susceptible to protein insertion? A clue may come from examining the role of cholesterol in cytolysins, another class of bacterial toxins exemplified by perfringolysin O (PFO) (13 ). Cytolysins kill eukaryotic cells by forming oligomeric structures that breach the plasma membrane with large pores up to 300 Å across (13 ). It has been hypothesized that stable folding of w w w. a c s c h e m i ca l biology.org
Figure 2. Structural biology of C. difficile toxin fragments. a) Model resulting from the X-ray crystal structure of a catalytically active N-terminal fragment of TcdB consisting of residues 1–543 (reprinted with permission from ref 16, Copyright 2005 Elsevier B.V.). The conserved GT-A fold is shown in blue. The catalytic DxD motif is shown in ball-and-stick mode, as well as UDP, glucose, and the catalytic Mn2+ ion. b) A structural model of the CROP domain from TcdA. The model is based on a crystal structure of a 127 amino acid fragment (residues 2573–2709) revealing stacked pairs of b-hairpins in a b-solenoid fold (reprinted with permission from ref 17, Copyright 2005 National Academy of Sciences, U.S.A.).
the monomeric PFO prevents premature aggregation of the cytolysins in solution. In cases where cholesterol has been depleted from target membranes, PFO oligomerizes on the membrane surface, but fails to insert into the membrane (14 ). Thus, cholesterol then may help to unfold the preinsertion structure of the toxin during the initial stages of membrane interaction (15 ). C. difficile toxins do not need to aggregate the way PFO and related cytolysins do. However, the translocation domains of TcdA and TcdB do need to refold from their initial solution conformation to their membraneinserted pore conformation at the proper time during entry into the cytosol (Figure 1, panel b, step 5). If the toxin refolds too early, it may be subject to aggregation and precipitation. If it occurs too late, it will have lost its chance to escape from the endosome. It is possible that acidification alone is insuffi cient to destabilize the preinsertion structures of TcdA and TcdB. Giesemann’s results www.acschemicalbiolog y.o rg
are consistent with a mechanism whereby cholesterol may chaperone the membrane insertion process. The search for the mechanism by which cholesterol facilitates intoxication may provide a detailed window into the nature of protein insertion into membranes in addition to providing a potential mode for therapeutic intervention for CDAD. Receptor Binding and Catalysis. Recent work has also expanded our understanding of the two other steps in the intoxication process. A high-resolution crystal structure of the catalytically active N-terminal domain consisting of residues 1–543 of TcdB was recently reported (Figure 2, panel a) (16 ). The model shows an extensive network of b structure at its core surrounded by a cluster of a helices. The core of the fold is homologous to the GT-A family of glycosyltransferases that also includes glycogenin, a3-GalT, and LgtC. Whereas the basic glucosyltransferase behavior is probably quite similar to that of other members
of this family of enzymes, the toxins are unique in their exquisite selectivity for their protein acceptors. Besides TcdA and TcdB, there are several other large clostridial toxins, and each has a unique set of cellu lar targets (4 ). The potential to use this selectivity in specifically targeting the toxins for inhibition is so far underutilized. A 127 amino acid fragment from the C‑terminus of TcdA has also been structurally characterized (Figure 2, panel b) (17 ). This fragment derives from the CROP region of the protein responsible for binding receptors on the surface of colonic epithelial cells (Figure 1, panel b, step 2). This repetitive sequence folds into a series of b hairpins that stack on top of one another to form an extended filamentous assembly with a significant helical twist. This domain most likely protrudes from the body of the toxin in search of an appropriate cell-surface receptor, believed to be a short oligosaccharide motif. Several candi date trisaccharides have been reported as potential targets for TcdA binding, including Gal-a1,3-Galb-1,4-GlcNAc (18 ) and GalNAc-b1,3-Gal-b1,4-GlcNAc (19 ), but it remains unclear whether these are in fact the biologically relevant motifs. Much work remains to be done on this aspect of the cellular recognition problem. Together, these most recent studies on the role of cholesterol and the mechanism by which the C. difficile toxins recognize, penetrate, and kill host cells will facilitate a host of additional experiments on these systems. Scientists have been working tirelessly to develop novel antibiotics and immunization against C. difficile, and this route still holds significant promise for long-term efficacy. The deeper understanding of the molecular details of intoxication, however, may allow direct targeting of the toxins in the ongoing battle against CDAD. Acknowledgment: The authors acknowledge support from NIH Training Grant GM-007757 (to A.K.) and a Women in Science Fellowship (to A.K.). A.L.F. is a Cottrell Scholar of Research Corporation. VOL.1 NO. 3 • 141—144 • 2 0 0 6
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