Sulfated glycosaminoglycans and low-density lipoprotein receptor contribute to Clostridium difficile toxin A entry into cells

Tao, Liang; Tian, Songhai; Zhang, Jie; Liu, Zhuoming; Robinson-McCarthy, Lindsey R.; Miyashita, Shin-Ichiro; Breault, David T.; Gerhard, Ralf; Oottamasathien, Siam; Whelan, Sean P. J.; Dong, Min

doi:10.1038/s41564-019-0464-z

Cited by 73 publications

(95 citation statements)

References 51 publications

(63 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…As a control, also binding of labeled Clostridium difficile toxin A (TcdA) was studied. It was recently shown that TcdA binds to sulfated GAG (14). In the presence of heparin, hyaluronic acid and dextran sulfate, respectively, there was reduced binding of TcdA to Hela cells ( Fig.…”

Section: Identification Of the Relevant Gagmentioning

confidence: 79%

“…This is in contrast to the recently proposed binding of the clostridial toxin TcdA to the cell surface. The authors could interfere with intoxication of cells by addition of surfen (14). HSPGs are extracellular matrix components (27), they are found in secreted vesicles of mast cells (serglycin, (28)), described as sequestering co-receptors for growth factors and cytokines (fibroblast growth factor (FGF), (29,30)) or binding partners for molecules undergoing transcellular transport (31).…”

Section: Discussionmentioning

confidence: 99%

“…The binding affinities between CNF and GAGs were measured using the BLI assay with the BLItz system (ForteBio) as previously described (14). Briefly, 100 μg/mL biotinylated heparin (Sigma-Aldrich, B9806), hyaluronic acid (Sigma, B1557), or cellulose (Creative PEGWorks, CE501) were immobilized onto capture biosensors (Dip and Read Streptavidin, ForteBio).…”

Section: Biolayer Interferometry (Bli) Spectroscopymentioning

confidence: 99%

See 2 more Smart Citations

Glycosaminoglycans are specific endosomal receptors forYersinia pseudotuberculosisCytotoxic Necrotizing Factor

Schmidt

Kowarschik

Schöllkopf

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

The Cytotoxic Necrotizing Factor Y (CNFY) is produced by the gram-negative, enteric pathogen Yersinia pseudotuberculosis. The bacterial toxin belongs to a family of deamidases, which constitutively activate Rho GTPases, thereby balancing inflammatory processes. We identified heparan sulfate proteoglycans as essential host cell factors for intoxication with CNFY. Using flow cytometry, microscopy, knockout cell lines, pulsed electron-electron double resonance and bio-layer interferometry, we studied the role of glucosaminoglycans in the intoxication process of CNFY. To analyze toxin-glucosaminoglycan interaction we utilized a truncated CNFY (CNFY 709-1014 ).Especially this C-terminal part of CNFY, which encompasses the catalytic activity, binds with high affinity to heparan sulfates. CNFY binding with the N-terminal domain to its protein receptor seems to induce a first conformational change supporting the interaction between the C-terminal domain and heparan sulfates, which seems sterically hindered in the full toxin. A second conformational change occurs by acidification of the endosome, probably allowing insertion of the hydrophobic regions of the toxin into the endosomal membrane. Our findings suggest that heparan sulfates play a major role for intoxication within the endosome, rather than being relevant for an interaction at the cell surface. Lastly, cleavage of heparin sulfate chains by heparanase is likely required for efficient uptake of the toxic enzyme into the cytosol of mammalian cells. Author SummaryThe RhoA deamidating Cytotoxic Necrotizing Factor Y (CNFY) from Yersinia pseudotuberculosis is a crucial virulence factor that is important for successful infection 3 of mammalian cells by the pathogen. The mode of action by which CNFY is able to intoxicate cells can be divided into the following steps: Binding to the cell surface, internalization, translocation from the endosome to the cytosol and deamidation of RhoA. We show, that CNFY uses heparan sulfates to maximize the amount of molecules entering the cytosol. While not being necessary for toxin binding and uptake, the sugars hold a key role in the intoxication process. We show that CNFY undergoes a conformational change at a low endosomal pH, allowing the C-terminal domain to be released from the endosomal membrane by the action of heparanase. This study reveals new insights into the CNFY-host interaction and promotes understanding of the complex intoxication process of bacterial toxins.

show abstract

Section: Identification Of the Relevant Gagmentioning

confidence: 79%

Section: Discussionmentioning

confidence: 99%

Section: Biolayer Interferometry (Bli) Spectroscopymentioning

confidence: 99%

See 1 more Smart Citation

Glycosaminoglycans are specific endosomal receptors forYersinia pseudotuberculosisCytotoxic Necrotizing Factor

Schmidt

Kowarschik

Schöllkopf

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…Notably, despite high sequence similarity between TcsL and TcdB, our screen did not identify Frizzled receptors, or CSPG4 or PVRL3, two other TcdB-associated receptors (7,10,11). Neither did we identify known receptors for C. perfringens TpeL or C. difficile TcdA, other related LCTs (12,13), although all these receptors are expressed in Hap1 cells ( fig. S1B).…”

mentioning

confidence: 69%

“…We cannot exclude the possibility that, like other clostridial toxins (7,(10)(11)(12), TcsL also binds additional receptors on host cells. However, we did not identify additional factors in CRISPR/Cas9 screens in cells that do not express SEMA6A or SEMA6B.…”

Section: Figure 2 Sema6a Ectodomain Protects Lung Endothelial Cells mentioning

confidence: 99%

Identification of the C. sordellii lethal toxin receptor elucidates principles of receptor specificity in clostridial toxins

Lee

Beilhartz

Kucharska

et al. 2019

Preprint

View full text Add to dashboard Cite

Clostridium sordellii lethal toxin (TcsL) is responsible for an almost invariably lethal toxic shock syndrome associated with gynecological C. sordellii infections. Here, using CRISPR/Cas9 screening, we identify semaphorins SEMA6A and SEMA6B as the cellular receptors for TcsL and demonstrate that soluble extracellular SEMA6A can protect mice from TcsL-induced edema. A 3.3 Å cryo-EM structure shows that TcsL binds SEMA6A with the same region that the highly related C. difficile TcdB toxin uses to bind structurally unrelated Frizzled receptors. Remarkably, reciprocal mutations in this evolutionarily divergent surface are sufficient to switch receptor specificity between the toxins. Our findings establish semaphorins as physiologically relevant receptors for TcsL, and reveal the molecular basis for the difference in tissue targeting and disease pathogenesis between highly related toxins.Clostridium sordellii is an anaerobic gram-positive bacterium found in soil and in the gastrointestinal and vaginal tracts of animals and humans. C. sordellii is present in the rectal or vaginal tract of 3-4% of women, but vaginal colonization rate after childbirth or abortion is as high as 29% (1, 2). While the majority of carriers are asymptomatic, pathogenic C. sordellii infections arise rapidly and are highly lethal. Most C. sordellii infections occur in women following childbirth, medically induced abortion, or miscarriage, leading to a toxic shock syndrome with almost 100% mortality within days (1-4). The primary cause of the high mortality associated with C. sordellii infections is the lethal toxin TcsL (5), which belongs to the large clostridial toxin (LCT) family together with toxins from related species such as Clostridium difficile (6). LCTs enter the host cell by receptor-mediated endocytosis followed by translocation into the cytoplasm, where they potently modulate host cell function by glucosylating small Ras family GTPases (6).Although all LCTs are highly similar at the sequence level, they differ in their tissue specificity and in their effects on cell morphology, physiology, and viability. TcsL is most closely related to the C. difficile cytotoxin TcdB, sharing almost 90% sequence similarity. TcdB binds Frizzled family receptors FZD1/FZD2/FZD7 expressed in the colonic epithelium (7,8), the primary site of C. difficile infection. C. sordellii, however, does not infect or damage colonic epithelium, suggesting that TcsL binds a different cell-surface receptor.

show abstract

CRISPRScreens

D’Gama

Park

Waldor

2022

Crispr

View full text Add to dashboard Cite

Sulfated glycosaminoglycans and low-density lipoprotein receptor contribute to Clostridium difficile toxin A entry into cells

Cited by 73 publications

References 51 publications

Glycosaminoglycans are specific endosomal receptors forYersinia pseudotuberculosisCytotoxic Necrotizing Factor

Glycosaminoglycans are specific endosomal receptors forYersinia pseudotuberculosisCytotoxic Necrotizing Factor

Identification of the C. sordellii lethal toxin receptor elucidates principles of receptor specificity in clostridial toxins

CRISPRScreens

Contact Info

Product

Resources

About