Anthrax Toxin Targeting of Myeloid Cells through the CMG2 Receptor Is Essential for Establishment of Bacillus anthracis Infections in Mice

Liu, Shihui; Miller-Randolph, Sharmina; Crown, Devorah; Moayeri, Mahtab; Sastalla, Inka; Okugawa, Shu; Leppla, Stephen H.

doi:10.1016/j.chom.2010.10.004

Cited by 92 publications

(147 citation statements)

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“…EdTx, on the other hand, targets the intestinal epithelial cells and hepatocytes (33). Another surprising result was that mice generated with Cmg2 Ϫ/Ϫ myeloid cells were still sensitive to LeTx, whereas complete Cmg2 Ϫ/Ϫ mice were resistant to LeTx (28). However, mice with Cmg2 Ϫ/Ϫ myeloid cells were completely resistant to infection following anthrax spore challenge.…”

Section: Discussionmentioning

confidence: 85%

“…The roles of CMG2 and TEM8 as anthrax toxin receptors have been firmly established by others in vitro (7,8) and using in vivo mouse models (28,29). In the absence of these receptors, host cells are resistant to anthrax toxins.…”

Section: Discussionmentioning

confidence: 99%

“…However, mice with Cmg2 Ϫ/Ϫ myeloid cells were completely resistant to infection following anthrax spore challenge. A possible explanation is that anthrax toxins disable myeloid cells, including neutrophils and macrophages, thus evading the immune system and allowing for the establishment of infection (28). Anthrax toxins can suppress immune function in monocytes, macrophages, dendritic cells, B lymphocytes, and T lymphocytes (34) because their activities interfere with various signaling pathways, including those of the innate immunity (35).…”

Section: Discussionmentioning

confidence: 99%

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Targeted Silencing of Anthrax Toxin Receptors Protects against Anthrax Toxins

Arévalo

Navarro

Arico

et al. 2014

Journal of Biological Chemistry

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Background: Existing anthrax postexposure antibiotic treatments are inadequate because they do not clear the high levels of secreted anthrax toxins. Results: Susceptible cells treated with anthrax toxin receptor-targeted siRNAs became resistant to anthrax toxin-mediated cytotoxicity. Conclusion: RNAi-targeted silencing of anthrax toxin receptors prevents toxins from entering target cells and inducing pathogenesis. Significance: Toxin receptor-targeted RNAi can be developed as a postexposure treatment against anthrax.

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Section: Discussionmentioning

confidence: 85%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Targeted Silencing of Anthrax Toxin Receptors Protects against Anthrax Toxins

Arévalo

Navarro

Arico

et al. 2014

Journal of Biological Chemistry

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“…PA is the cellular binding moiety of anthrax toxin, which specifically binds to either capillary morphogenesis protein 2 (CMG2, major toxin receptor) or tumor endothelium marker 8 (TEM8, minor toxin receptor) on host cells (11)(12)(13)(14) causing the endocytosis and translocation of anthrax lethal factor (LF), edema factor, or FP59 into the cytosol of host cells. FP59 is a fusion protein of LF amino acids 1-254 and the catalytic domain of P. aeruginosa exotoxin A (15) that kills cells by ADP ribosylation of eEF2 after delivery to cytosol by PA.…”

mentioning

confidence: 99%

Diphthamide modification on eukaryotic elongation factor 2 is needed to assure fidelity of mRNA translation and mouse development

Liu

Bachran

Gupta

et al. 2012

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

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To study the role of the diphthamide modification on eukaryotic elongation factor 2 (eEF2), we generated an eEF2 Gly 717 Arg mutant mouse, in which the first step of diphthamide biosynthesis is prevented. Interestingly, the Gly 717 -to-Arg mutation partially compensates the eEF2 functional loss resulting from diphthamide deficiency, possibly because the added +1 charge compensates for the loss of the +1 charge on diphthamide. Therefore, in contrast to mouse embryonic fibroblasts (MEFs) from OVCA1 −/− mice, eEF2 G717R/G717R MEFs retain full activity in polypeptide elongation and have normal growth rates. Furthermore, eEF2 G717R/G717R mice showed milder phenotypes than OVCA1 −/− mice (which are 100% embryonic lethal) and a small fraction survived to adulthood without obvious abnormalities. Moreover, eEF2 G717R/G717R /OVCA1 −/− double mutant mice displayed the milder phenotypes of the eEF2 G717R/G717R mice, suggesting that the embryonic lethality of OVCA1 −/− mice is due to diphthamide deficiency. We confirmed that the diphthamide modification is essential for eEF2 to prevent −1 frameshifting during translation and show that the Gly 717 -to-Arg mutation cannot rescue this defect. E ukaryotic elongation factor 2 (eEF2) is a member of the GTPbinding translation elongation factor family, and an essential factor for protein synthesis and cell survival. eEF2 drives the GTP-dependent translocation of the nascent polypeptide chain from the A site to the P site of the ribosome and advances mRNA by three bases during the elongation cycle of protein synthesis (1). eEF2 is highly homologous in all eukaryotes. In fact, eEF2 of humans, rats, mice, hamsters, and other mammals have exactly the same amino acid sequence. Intriguingly, all eukaryotic eEF2 proteins contain a unique posttranslationally modified histidine residue termed diphthamide (2, 3). Diphthamide modification occurs after eEF2 is translated and is irreversible, marking the completion of the biosynthesis of eEF2.Although the physiological role of the diphthamide modification on eEF2 remains elusive, diphthamide is the well-known target for the adenosine diphosphate (ADP)-ribosylating toxins from bacterial pathogens, such as diphtheria toxin (DT) from Corynebacterium diphtheriae, Pseudomonas exotoxin A (ETA) from Pseudomonas aeruginosa, and the recently identified cholix toxin (CT) from Vibrio cholerae (4). As virulence factors, these ADP-ribosylating toxins catalyze transfer of the ADP ribose from nicotinamide adenine dinucleotide (NAD + ) to diphthamide on eEF2 (Fig. S1), thus inactivating eEF2, halting cellular protein synthesis, and causing cell death.Because the diphthamide modification is required for the action of the ADP-ribosylating toxins, the complex diphthamide biosynthesis pathway is amenable to genetic analysis, and mutants defective in diphthamide biosynthesis have been isolated in both Chinese hamster ovary (CHO) cells and yeast (Saccharomyces cerevisiae) by selection for resistance to DT or an engineered ADP-ribosylating toxin − anthrax protective a...

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“…Mice having each receptor deleted and cells isolated from them provide a well defined genetic system for examining the receptor specificity of the PA variants (7,8,21). Cytotoxicity tests of the PA variants having substitutions at Ile-656 (from Table 3) on MEFs isolated from these mice clearly demonstrated the high specificity of the PA Ile-656 variants for CMG2 (experiment 1 in Table 6).…”

Section: Pad4 Variants Having Substitutions In Multiplementioning

confidence: 99%

Anthrax Toxin Protective Antigen Variants That Selectively Utilize either the CMG2 or TEM8 Receptors for Cellular Uptake and Tumor Targeting

Chen

Liu

Leysath

et al. 2016

Journal of Biological Chemistry

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The protective antigen (PA) moiety of anthrax toxin binds to cellular receptors and mediates the translocation of the two enzymatic moieties of the toxin to the cytosol. Two PA receptors are known, with capillary morphogenesis protein 2 (CMG2) being the more important for pathogenesis and tumor endothelial marker 8 (TEM8) playing a minor role. The C-terminal PA domain 4 (PAD4) has extensive interactions with the receptors and is required for binding. Our previous study identified PAD4 variants having enhanced TEM8 binding specificity. To obtain PA variants that selectively bind to CMG2, here we performed phage display selections using magnetic beads having bound CMG2. We found that PA residue isoleucine 656 plays a critical role in PA binding to TEM8 but has a much lesser effect on PA binding to CMG2. We further characterized the role of residue 656 in distinguishing PA binding to CMG2 versus TEM8 by substituting it with the other 19 amino acids. Of the resulting variants, PA I656Q and PA I656V had significantly reduced activity on TEM8-expressing CHO cells but maintained their activity on CMG2-expressing CHO cells. The preference of these PA mutants for CMG2 over TEM8 was further demonstrated using mouse embryonic fibroblast cells and mice deficient in the CMG2 and/or the TEM8 receptors. The structural basis of the alterations in the receptor binding activities of these mutants is also discussed.Anthrax toxin is composed of three nontoxic proteins: protective antigen (PA), 3 lethal factor (LF), and edema factor (EF), which are released from Bacillus anthracis as monomers and assemble into toxic complexes on the surface of animal cells (1-3). PA is the moiety which binds to cell surface receptors and facilitates entry of the enzymatic EF and LF moieties into the host cell cytosol. Two anthrax toxin receptors have been identified; capillary morphogenesis protein 2 (CMG2, or anthrax toxin receptor 2) is the physiologically relevant toxin receptor mediating most of the in vivo toxicity of the toxin, whereas tumor endothelial marker 8 (TEM8, or anthrax toxin receptor 1) functions only as a minor anthrax toxin receptor (4 -8). Upon binding to the toxin receptors, PA is cleaved at the sequence 164 RKKR 167 by cell surface furin or furin-like proteases (9, 10), yielding the C-terminal 63-kDa fragment (PA63), which assembles to form a ring-shaped oligomer. The PA63 oligomer then binds LF and/or EF, and the toxin complex is internalized by receptor-mediated endocytosis. LF and EF are translocated into the cytosol from early or late endosomes to exert their cytotoxic effects. Therefore, PA plays the crucial role in anthrax toxin pathogenesis, serving as the delivery vehicle for translocation of LF and EF into the cytosol of the cell.The absolute requirement for cell surface proteolytic activation of PA for the toxin's action prompted our group and others to reengineer PA to make it depend on tumor-associated proteases for activation, thereby achieving high specificity for tumors (11)(12)(13)(14)(15). In a recent example, ...

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Anthrax Toxin Targeting of Myeloid Cells through the CMG2 Receptor Is Essential for Establishment of Bacillus anthracis Infections in Mice

Cited by 92 publications

References 36 publications

Targeted Silencing of Anthrax Toxin Receptors Protects against Anthrax Toxins

Targeted Silencing of Anthrax Toxin Receptors Protects against Anthrax Toxins

Diphthamide modification on eukaryotic elongation factor 2 is needed to assure fidelity of mRNA translation and mouse development

Anthrax Toxin Protective Antigen Variants That Selectively Utilize either the CMG2 or TEM8 Receptors for Cellular Uptake and Tumor Targeting

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