Cholesterol-dependent Pore Formation of Clostridium difficile Toxin A

Giesemann, Torsten; Jank, Thomas; Gerhard, Ralf; Maier, Elke; Just, Ingo; Benz, Roland; Aktories, Klaus

doi:10.1074/jbc.m512720200

Cited by 80 publications

(78 citation statements)

References 57 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The observed current fluctuations were irregular and inhomogeneous, indicating that interaction of AIP56 with bilayer membranes does not lead to the formation of regular channels comparable to the ones formed by other AB toxins, such as anthrax toxins (65,66) or Clostridium botulinum C2 (65,66). The current fluctuations induced by AIP56 are similar to the ones reported to occur with the C. botulinum and Clostridium limosum C3 toxins (67) as well as with the Clostridium difficile TcdB and TcdA toxins (68,69) and may result from formation of transient channels.…”

Section: Figsupporting

confidence: 60%

Intracellular Trafficking of AIP56, an NF-κB-Cleaving Toxin from Photobacterium damselae subsp. piscicida

et al. 2014

Self Cite

View full text Add to dashboard Cite

f AIP56 (apoptosis-inducing protein of 56 kDa) is a metalloprotease AB toxin secreted by Photobacterium damselae subsp. piscicida that acts by cleaving NF-B. During infection, AIP56 spreads systemically and depletes phagocytes by postapoptotic secondary necrosis, impairing the host phagocytic defense and contributing to the genesis of infection-associated necrotic lesions. Here we show that mouse bone marrow-derived macrophages (mBMDM) intoxicated by AIP56 undergo NF-B p65 depletion and apoptosis. Similarly to what was reported for sea bass phagocytes, intoxication of mBMDM involves interaction of AIP56 C-terminal region with cell surface components, suggesting the existence of a conserved receptor. Biochemical approaches and confocal microscopy revealed that AIP56 undergoes clathrin-dependent endocytosis, reaches early endosomes, and follows the recycling pathway. Translocation of AIP56 into the cytosol requires endosome acidification, and an acidic pulse triggers translocation of cell surface-bound AIP56 into the cytosol. Accordingly, at acidic pH, AIP56 becomes more hydrophobic, interacting with artificial lipid bilayer membranes. Altogether, these data indicate that AIP56 is a short-trip toxin that reaches the cytosol using an acidic-pH-dependent mechanism, probably from early endosomes. Usually, for short-trip AB toxins, a minor pool reaches the cytosol by translocating from endosomes, whereas the rest is routed to lysosomes for degradation. Here we demonstrate that part of endocytosed AIP56 is recycled back and released extracellularly through a mechanism requiring phosphoinositide 3-kinase (PI3K) activity but independent of endosome acidification. So far, we have been unable to detect biological activity of recycled AIP56, thereby bringing into question its biological relevance as well as the importance of the recycling pathway.A IP56 (apoptosis-inducing protein of 56 kDa) is a plasmidencoded toxin of Photobacterium damselae subsp. piscicida (1), a Gram-negative bacterium that infects economically important fish species (2, 3) and is considered one of the most relevant pathogens in mariculture (2-5). In acute infections, a rapid septicemia develops, causing very high mortalities (2, 4, 6). Histopathological analysis of the diseased animals revealed cytotoxic alterations (4, 7-10) that were found to result from pathogeninduced apoptotic death of macrophages and neutrophils (11) and later associated with the activity of AIP56 (1). Indeed, it has been shown that in infected fish, the toxin is systemically distributed and depletes macrophages and neutrophils by postapoptotic secondary necrosis (12), leading to the impairment of the phagocytic defense of the host and consequently favoring P. damselae subsp. piscicida survival and dissemination. Furthermore, the occurrence of a secondary necrotic process in which the phagocytes undergoing apoptosis lyse and release their cytotoxic contents contributes to the genesis of the infection-associated necrotic lesions (12, 13). These observations, together with the fac...

show abstract

Section: Figsupporting

confidence: 60%

Intracellular Trafficking of AIP56, an NF-κB-Cleaving Toxin from Photobacterium damselae subsp. piscicida

et al. 2014

Self Cite

View full text Add to dashboard Cite

show abstract

“…hemolysin | intermedilysin | sterol | toxin | pore M embrane cholesterol is important to a variety of pathogenic processes that include virus fusion and budding (1) and the mechanisms of eukaryotic (2,3) and prokaryotic toxins (4)(5)(6)(7). Whether cholesterol is bound directly by these proteins as a receptor or it indirectly influences the binding or activity of the protein at the membrane surface remains unknown.…”

mentioning

confidence: 99%

Only two amino acids are essential for cytolytic toxin recognition of cholesterol at the membrane surface

Farrand

LaChapelle

Hotze

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

195

279

View full text Add to dashboard Cite

The recognition and binding of cholesterol is an important feature of many eukaryotic, viral, and prokaryotic proteins, but the molecular details of such interactions are understood only for a few proteins. The pore-forming cholesterol-dependent cytolysins (CDCs) contribute to the pathogenic mechanisms of a large number of Grampositive bacteria. Cholesterol dependence of the CDC mechanism is a hallmark of these toxins, yet the identity of the CDC cholesterol recognition motif has remained elusive. A detailed analysis of membrane interactive structures at the tip of perfringolysin O (PFO) domain 4 reveals that a threonine-leucine pair mediates CDC recognition of and binding to membrane cholesterol. This motif is conserved in all known CDCs and conservative changes in its sequence or order are not well tolerated. Thus, the Thr-Leu pair constitutes a common structural basis for mediating CDC-cholesterol recognition and binding, and defines a unique paradigm for membrane cholesterol recognition by surface-binding proteins.M embrane cholesterol is important to a variety of pathogenic processes that include virus fusion and budding (1) and the mechanisms of eukaryotic (2, 3) and prokaryotic toxins (4-7). Whether cholesterol is bound directly by these proteins as a receptor or it indirectly influences the binding or activity of the protein at the membrane surface remains unknown. The cholesterol-dependent cytolysins (CDCs) use cholesterol as their receptor at the membrane surface (7) and contribute to the pathogenesis of a large number of Gram-positive bacterial pathogens (8). The CDC-sterol interaction initiates a cascade of secondary and tertiary structural changes that lead to the formation of a large oligomeric complex, and ultimately a pore in the membrane of eukaryotic cells (9-13). Although significant progress has been made in understanding the assembly of the CDC pore complex, the structural basis for recognition and binding to cholesterol-rich membranes remains elusive.Early studies with the Clostridium perfringens perfringolysin O (PFO) suggested that the highly conserved tryptophan-rich undecapeptide sequence at the base of domain 4 (14, 15) (Fig. S1) mediated the PFO-cholesterol interaction. However, recent studies by Soltani et al. (16) uncoupled cholesterol binding from the undecapeptide and showed that the membrane insertion of loops L1-L3 at the base of domain 4 was cholesterol dependent (Fig. S1). These observations are also consistent with a lack of conservation of the 3D structures of the undecapeptide in the closely related CDCs PFO (17) and Bacillus anthracis anthrolysin O (ALO) (18) (Fig. S1). These studies suggest the residues that comprise the cholesterol recognition motif are located within L1-L3 because these loops and the undecapeptide are the only structures at the tip of domain 4 exposed to the nonpolar bilayer core; the rest of the domain 4 surface is surrounded by water (19).Cholesterol was thought to function as the sole CDC receptor until the discovery of intermedilysin (ILY), a CDC fr...

show abstract

“…Interaction between the C terminus and the host cell receptors is believed to initiate receptormediated endocytosis (11,25,63). Although the intracellular mode of action remains unclear, it has been proposed that the toxins undergo a conformational change at low pH in the endosomal compartment, leading to membrane insertion and channel formation (12,15,17,47). A host cofactor is then required to trigger a second structural change, which is accompanied by immediate autocatalytic cleavage and release of the glucosyltransferase domain into the cytosol (44,49,52).…”

mentioning

confidence: 99%

Antibody-Enhanced, Fc Gamma Receptor-Mediated Endocytosis of Clostridium difficile Toxin A

Sun

Wang

et al. 2009

Infect Immun

View full text Add to dashboard Cite

Clostridium difficile has emerged as a leading cause of hospital-acquired enteric infections whose annual health care costs are rapidly escalating in the United States (33). The severity of C. difficile-associated infections ranges from mild diarrhea to life-threatening pseudomembranous colitis (2, 3). Several hospital outbreaks of C. difficile-associated diarrhea (CDAD) with high rates of morbidity and mortality in the past few years in North America have been attributed to the widespread use of broad-spectrum antibiotics. The emergence of more virulent C. difficile strains is also contributing to the increased incidence and severity of disease (38, 39).Toxin A (TcdA) and toxin B (TcdB) are the two major virulence factors in pathogenic C. difficile strains. These toxins are enterotoxic, induce intestinal epithelial cell damage, and disrupt epithelium tight junctions, leading to increased mucosal permeability (46,51,55). Moreover, these toxins induce production of immune mediators, leading to subsequent neutrophil infiltration and severe colitis (28,29). TcdA and TcdB are structurally homologous and putatively contain an N-terminal glucosyltransferase domain, a cysteine proteinase domain, a transmembrane domain, and a C-terminal receptor binding domain (21,65,66). Interaction between the C terminus and the host cell receptors is believed to initiate receptormediated endocytosis (11,25,63). Although the intracellular mode of action remains unclear, it has been proposed that the toxins undergo a conformational change at low pH in the endosomal compartment, leading to membrane insertion and channel formation (12,15,17,47). A host cofactor is then required to trigger a second structural change, which is accompanied by immediate autocatalytic cleavage and release of the glucosyltransferase domain into the cytosol (44,49,52). Once the glucosyltransferase domain reaches the cytosol, it inactivates proteins belonging to the Rho/Rac family, leading to alterations in the cytoskeleton and ultimately cell death (23,57).The clinical manifestations of CDAD are highly variable and range from asymptomatic carriage to mild self-limiting diarrhea to the more severe disease pseudomembranous colitis. Systemic complications and death are increasingly common in CDAD patients (58). In life-threatening cases of CDAD, systemic complications that include cardiopulmonary arrest (22), acute respiratory distress syndrome (20), multiple organ failure (9), renal failure (6), and liver damage (53) are observed. The exact reason for these complications is unclear, but the toxin's entry into the circulation and systemic dissemination have been suggested as possible causes (16).Protection against C. difficile appears to be conferred by antitoxin antibodies, which are present in the general population in individuals over 2 years of age; the levels of these antibodies are higher and relapse is less frequent in less severe cases (27,30,35,62,64). Disease progression and recurrence

show abstract

Cholesterol-dependent Pore Formation of Clostridium difficile Toxin A

Cited by 80 publications

References 57 publications

Intracellular Trafficking of AIP56, an NF-κB-Cleaving Toxin from Photobacterium damselae subsp. piscicida

Intracellular Trafficking of AIP56, an NF-κB-Cleaving Toxin from Photobacterium damselae subsp. piscicida

Only two amino acids are essential for cytolytic toxin recognition of cholesterol at the membrane surface

Antibody-Enhanced, Fc Gamma Receptor-Mediated Endocytosis of Clostridium difficile Toxin A

Contact Info

Product

Resources

About