Aggregatibacter actinomycetemcomitans Leukotoxin Utilizes a Cholesterol Recognition/Amino Acid Consensus Site for Membrane Association

Brown, Angela C.; Balashova, Nataliya V.; Epand, Richard M.; Bragin, Alvina; Kachlany, Scott C.; Walters, Michael J.; Du, Yurong; Boesze‐Battaglia, Kathleen; Lally, Edward T.

doi:10.1074/jbc.m113.486654

Cited by 49 publications

(122 citation statements)

References 77 publications

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“…There is, however, evidence that the lipid composition is important for the embedding of LtxA in the membrane. The content of cholesterol in the membrane greatly improves the membrane binding, a feature that apparently requires the cholesterol recognition/ amino acid consensus site CRAC 336 , which is conserved in other RTX toxins (60). This clearly shows that it is not only the immediate affiliation to the membrane that determines the cellular effects of the toxin.…”

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confidence: 83%

Sialic Acid Residues Are Essential for Cell Lysis Mediated by Leukotoxin from Aggregatibacter actinomycetemcomitans

et al. 2014

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c Leukotoxin (LtxA) from Aggregatibacter actinomycetemcomitans is known to target and lyse ␤ 2 -integrin-expressing cells such as polymorphonuclear leukocytes and macrophages. LtxA is an important virulence factor that facilitates chronic inflammation and is strongly associated with a fast-progressing form of periodontitis caused by the JP2 clone of the bacterium. Here, we show that sialic acid residues are important for LtxA-induced cell lysis, regardless of whether the cell express ␤ 2 -integrin or not. Clearly, removal of sialic acid groups significantly reduces a ␤ 2 -integrin-specific LtxA-induced lysis. Moreover, sialic acid presented on alternative proteins, such as, for instance, on erythrocytes that do not express ␤ 2 -integrin, also makes the cells more sensitive to LtxA. The data also illustrate the importance of the negative charge in order for the sialic acid to associate LtxA with the membrane. Removal of sialic acid is in itself sufficient to significantly reduce the negative charge on the erythrocytes. Moreover, we found that on human erythrocytes there is a positive association between the sensitivity to LtxA and the amount of negative charge caused by sialic acid. Interestingly, these features are not shared by all RTX toxins, since ␣-hemolysin from Escherichia coli induced cell lysis of both ␤ 2 -integrin-expressing and nonexpressing cells and this lysis is independent of the presence of sialic acid residues. In conclusion, LtxA not only is cytotoxic to ␤ 2 -integrin-expressing cells but can potentially initiate cell lysis in all cells that present a sufficient density of sialic acid groups on their plasma membrane.

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confidence: 83%

Sialic Acid Residues Are Essential for Cell Lysis Mediated by Leukotoxin from Aggregatibacter actinomycetemcomitans

et al. 2014

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“…LFA-1 clustering to lipid rafts is initiated by a LtxA-induced increase in cytosolic calcium that triggers the activation of calpain, cleavage of talin, and mobilization of LFA-1 into cholesterol and sphingolipid-rich microdomains of the plasma membrane. More recently, Brown et al [380] demonstrated that LtxA contains two cholesterol recognition/amino acid consensus (CRAC) sites, one (CRAC336) highly conserved among RTX toxins, while the second (CRAC503) is unique to LtxA. They further showed that a peptide corresponding to CRAC336 inhibited the LtxA cytotoxic activity and demonstrated that a point mutation in the CRAC336 site abolished LtxA toxicity.…”

Section: Interaction With Cell Receptors and Trafficmentioning

confidence: 94%

Structure and function of RTX toxins

Chenal

Sotomayor-Pérez

Ladant

2015

The Comprehensive Sourcebook of Bacterial Protein Toxins

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“…Therefore, we expected not only that this residue was not critical to CRAC site function but also that the proline substitution would also not contribute to any change in cholesterol binding. Thus, CdtB mutation of the CRAC region within CdtC (33) and with those of other investigators who made similar CRAC site mutations of residues within the CdtC subunit of Cdts produced by other bacterial species as well as to other cholesterol-binding proteins (30,31,42,50). In each instance CRAC site mutations resulted in a loss of cholesterol binding and associated downstream biological effects; in contrast, CdtB R110P had no effect on Cdt binding and CdtB internalization, confirming our expectation for the relative role of each of these residues within the CRAC site and overall role in the CdtB-cholesterol interaction.…”

Section: Discussionmentioning

confidence: 99%

“…It is likely that cholera toxin simultaneously binds with high affinity to multiple receptors as a result of receptor concentration within the raft (56,57). Likewise, the pore-forming leukotoxin from A. actinomycetemcomitans recognizes cholesterol via a CRAC site, which facilitates clustering to its receptor in the context of lipid rafts (50). Another pore-forming toxin, the aerolysin from Aeromonas hydrophila, which binds to glycosylphosphatidylinositol (GPI)-anchored proteins, also utilizes the concentrating properties of rafts to facilitate oligomerization, a requisite for channel formation (56,58).…”

Section: Discussionmentioning

confidence: 99%

“…We have also demonstrated cholesterol-specific binding of Cdt to both model membranes and lymphocyte membranes; furthermore, this binding is dependent upon a cholesterol recognition sequence within the CdtC subunit. In this regard, numerous proteins have been shown to bind cholesterol; these include the benzodiazepine receptor, the HIV transmembrane protein gp41, caveolin, G protein-coupled receptors, components of Ca ϩϩ -and voltage-gated K ϩ channels, apolipoproteins, the translocator protein TSPO, and, more recently, the leukotoxin produced by A. actinomycetemcomitans (42)(43)(44)(45)(46)(47)(48)(49)(50). Each of these cholesterol-binding proteins contains a CRAC sequence, L/V-X 1-5 -Y-X 1-5 -R/K, where X 1-5 represents one to five residues of any amino acid.…”

Section: Discussionmentioning

confidence: 99%

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The Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin Active Subunit CdtB Contains a Cholesterol Recognition Sequence Required for Toxin Binding and Subunit Internalization

et al. 2015

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Induction of cell cycle arrest in lymphocytes following exposure to the Aggregatibacter actinomycetemcomitans cytolethal distending toxin (Cdt) is dependent upon the integrity of lipid membrane microdomains. Moreover, we have previously demonstrated that the association of Cdt with target cells involves the CdtC subunit which binds to cholesterol via a cholesterol recognition amino acid consensus sequence (CRAC site). In this study, we demonstrate that the active Cdt subunit, CdtB, also is capable of binding to large unilamellar vesicles ( A ggregatibacter actinomycetemcomitans is a Gram-negative organism that is associated with aggressive forms of periodontitis and other systemic infections (1-5). Periodontitis is a chronic infectious inflammatory disorder that ultimately leads to the destruction of tooth-supporting tissue. While the exact nature of the pathogenesis of periodontal disease and the contribution of bacteria to this process are not known, it is becoming increasingly clear that A. actinomycetemcomitans produces several potential virulence factors; these include adhesins and fimbria which have been shown to contribute to colonization of the human oral cavity as well as two exotoxins, cytolethal distending toxin (Cdt) and leukotoxin, both of which are capable of killing and/or altering the function of host immune cells (4,(6)(7)(8).The Cdts are a family of heat-labile protein cytotoxins produced by several additional bacterial species, including Campylobacter jejuni, Shigella species, Haemophilus ducreyi, and diarrheal disease-causing enteropathogens such as some Escherichia coli isolates (9-15). There is clear evidence that Cdts are encoded by three genes, designated cdtA, cdtB, and cdtC, which are arranged as an apparent operon (7,(15)(16)(17)(18)(19)(20). The Cdt holotoxin consists of three subunits, CdtA, CdtB, and CdtC, that form a heterotrimeric complex. Furthermore, there is considerable agreement among investigators that, regardless of the microbial source of Cdt, the heterotrimeric holotoxin functions as an AB 2 toxin where CdtB is the active (A) unit and the complex of CdtA and CdtC comprises the binding (B) unit (18,21,22). Indeed, several investigators have demonstrated that the internalization of CdtB requires the presence of both CdtA and CdtC (21,23,24).While several cell types and cell lines have been shown to be susceptible to the toxic actions of Cdt, tropism for specific cells and/or tissue remains to be identified. In this regard, we have demonstrated that lymphocytes in vitro are most susceptible, requiring very low concentrations of Cdt (picograms/milliliter) to induce cell cycle arrest and apoptosis versus other cell types that typically require as much as microgram quantities (25). Typically, susceptibility to bacterial toxins is dependent upon the expression of specific receptors or moieties that enable the toxin to preferentially associate with target host cells. Structural analysis of CdtA and CdtC identified ricin-like lectin domains, suggesting that these units interact wi...

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Aggregatibacter actinomycetemcomitans Leukotoxin Utilizes a Cholesterol Recognition/Amino Acid Consensus Site for Membrane Association

Cited by 49 publications

References 77 publications

Sialic Acid Residues Are Essential for Cell Lysis Mediated by Leukotoxin from Aggregatibacter actinomycetemcomitans

Sialic Acid Residues Are Essential for Cell Lysis Mediated by Leukotoxin from Aggregatibacter actinomycetemcomitans

Structure and function of RTX toxins

The Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin Active Subunit CdtB Contains a Cholesterol Recognition Sequence Required for Toxin Binding and Subunit Internalization

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