Diphtheria toxin and its mutant <i>crm</i> 197 differ in their interaction with lipids

Papini, Emanuele; Colonna, Raffaele; Schiavo, Giampietro; Cusinato, Federico; Tomasi, M.; Rappuoli, Rino; Montecucco, Cesare

doi:10.1016/0014-5793(87)80116-3

Cited by 32 publications

(18 citation statements)

References 33 publications

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“…These findings point to a tight interaction between the two fragments, and are in agreement with a number of previous observations: namely, (i) the CD spectra of DT and CRM197 are different [32,33]; (ii) CRM197 is more susceptible than DT to the action of proteases ([7] and unpublished observations); (iii) the interaction of fragment B of CRM197 with lipid membranes is stronger than that of the same fragment in DT [34]. This last property described in the recent work of Papini et al [34] could explain why the affinity of CRM197 for the cell receptors is apparently higher than that of DT [32].…”

Section: Discussionsupporting

confidence: 91%

Conformational changes in diphtheria toxoids

Bigio¹,

Rossi²,

Nucci³

et al. 1987

FEBS Letters

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Monoclonal antibodies (Mab) were raised against CRM197, a non-toxic mutant of diphtheria toxin (DT). The ability of four Mabs to bind DT and the six functional mutants CRM197, CRM176, CRM228, CRMlOOl, CRM45 and CRM30 was assessed by immunoblotting and by a radioimmunoassay in which the protein antigen in solution competes with labeled CRM197 for the Mab binding site. The results show that the peptides recognized by Mabl 1.3, Mab53 and Mab23 are accessible in the mutant molecules in solution but not when they are part of the native DT structure, which could therefore be described for this purpose as 'closed' in contrast with an 'open' conformation of CRM 197, CRM 176 and CRM228. In particular, the behaviour of Mab53 indicates that the single amino acid substitutions in the A fragments of CRM197 and CRM176 also affect the conformation of their B fragments.

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Section: Discussionsupporting

confidence: 91%

Conformational changes in diphtheria toxoids

Bigio¹,

Rossi²,

Nucci³

et al. 1987

FEBS Letters

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“…The finding that crm 45 and crm 197 penetrate into the lipid layer at neutral pH correlates with previous indirect evidence of a hydrophobic interaction of these two DT mutants with membranes [9,43]. Moreover it provides an explanation for the neurotoxicity of crm 45 that could result from a preferential location of crm 45 in the lipid-rich nervous tissue [16].…”

Section: Discussionsupporting

confidence: 73%

Lipid interaction of diphtheria toxin and mutants

Demel

Schiavo

Kruijff

et al. 1991

European Journal of Biochemistry

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To study the structural change of diphtheria toxin (DT) induced by low pH and its influence on the interaction with membrane lipids, protein and lipid monolayers were formed and characterized. DT at neutral and acidic pH forms stable monolayers, whose surface-pressure-increase curves allow an estimation of the apparent molecular area of 29.5 nm2/molecule at pH 7.4 (corresponding to a radius of 3.06 nm) and 34.5 nm2/molecule at pH 5.0 (corresponding to a radius of 3.32 nm).DT at pH 7.4 does not insert into phospholipid monolayers, while at pH 5.0 it penetrates into the lipid layer with a portion of apparent molecular area of 21.0 nm2/molecule (corresponding to a radius of 2.6 nm).The low-pH driven lipid interaction of the toxin is favoured by the presence of acidic phospholipids, without an apparent requirement for a particular class of negative lipids. The DT mutants crm 45 and crm 197 are capable of hydrophobic interaction already at neutral pH and cause an increase of surface pressure with a further increase upon acidification.Diphtheria toxin (DT) is responsible of most clinical symptoms of diphtheria [l]. It is a protein, produced by toxigenic strains of Corynebacterium diphtheriae as a single inactive polypeptide chain [2, 31. The toxin is activated by a specific proteolytic cleavage at an exposed loop that generates two chains held together by a single interchain disulfide bridge and by non-covalent forces [4]. Chain A (21 164 Da) ADPribosylates specifically elongation factor 2 of eukaryotes and archaebacteria, thus blocking protein synthesis [5]. Chain B (37 194 Da) binds via its carboxy-terminal region to a protein receptor [6, 71, but there is evidence that also acidic phospholipids may be involved in DT binding via headgroup interactions with both DT protomers [S, 91. After internalization, DT is included into intracellular acidic compartments, where by a mechanism yet unknown, chain A translocates across the membrane to reach the cytoplasm where it exerts its action [lo, 111. A key role in the intoxication process is played by toxin-lipid interactions because the toxin has to cross the lipid bilayer and also because of the suggested lipid involvement in DT cell binding.The interaction of DT with lipids has been studied with a variety of techniques (reviewed in [lo]). It is now clear that low pH induces a structural transition of DT, characterized by the exposure of hydrophobic surfaces and by an interaction with the fatty acid chains of phospholipids with a preference for acidic phospholipids. Both DT protomers are involved in this structural change, but at the present time the structural features of DT at acidic and neutral pH are poorly defined and data are lacking on the size of the part of DT that interacts with lipids and mediates the translocation of chain A in the In order to gain further insights on this poorly understood aspect of DT action we have used monolayers of both diphtheria toxin and lipids spread at the water-air interface and we have monitored the lipid interaction of DT by surfa...

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“…The intrinsic ability of DTx to directly interact with the lipid membrane (4, 20) could be responsible for this unexpected result. Furthermore, CRM 197 was shown to bind more strongly to the lipid bilayer than DTx (23). Alternatively, CRM 197 could be localized in inclusion bodies, which would precipitate together with cell wall components in the fractionation experiments.…”

Section: Discussionmentioning

confidence: 99%

Induction of Neutralizing Antibodies against Diphtheria Toxin by Priming with Recombinant Mycobacterium bovis BCG Expressing CRM ₁₉₇ , a Mutant Diphtheria Toxin

Miyaji¹,

Mazzantini²,

Dias³

et al. 2001

Infect Immun

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BCG, the attenuated strain of Mycobacterium bovis, has been widely used as a vaccine against tuberculosis and is thus an important candidate as a live carrier for multiple antigens. With the aim of developing a recombinant BCG (rBCG) vaccine against diphtheria, pertussis, and tetanus (DPT), we analyzed the potential of CRM 197 , a mutated nontoxic derivative of diphtheria toxin, as the recombinant antigen for a BCG-based vaccine against diphtheria. Expression of CRM 197 in rBCG was achieved using Escherichia coli-mycobacterium shuttle vectors under the control of pBlaF*, an upregulated ␤-lactamase promoter from Mycobacterium fortuitum. Immunization of mice with rBCG-CRM 197 elicited an anti-diphtheria toxoid antibody response, but the sera of immunized mice were not able to neutralize diphtheria toxin (DTx) activity. On the other hand, a subimmunizing dose of the conventional diphtheria-tetanus vaccine, administered in order to mimic an infection, showed that rBCG-CRM 197 was able to prime the induction of a humoral response within shorter periods. Interestingly, the antibodies produced showed neutralizing activity only when the vaccines had been given as a mixture in combination with rBCG expressing tetanus toxin fragment C (FC), suggesting an adjuvant effect of rBCG-FC on the immune response induced by rBCG-CRM 197 . Isotype analysis of the anti-diphtheria toxoid antibodies induced by the combined vaccines, but not rBCG-CRM 197 alone, showed an immunoglobulin G1-dominant profile, as did the conventional vaccine. Our results show that rBCG expressing CRM 197 can elicit a neutralizing humoral response and encourage further studies on the development of a DPT vaccine with rBCG.Many currently used vaccines require multiple doses to achieve maximum protection, which has led to reduced coverage of vaccination campaigns, especially in developing countries. The use of live viral or bacterial carriers for heterologous antigen presentation, such as vaccinia virus, Salmonella, and Mycobacterium bovis BCG (Bacille Calmette-Guérin), has been intensively investigated in an effort to reduce the number of doses required for immunization. M. bovis BCG is the most widely used live vaccine, having been administered as an antituberculosis vaccine to over 3 billion individuals. It has several features that have encouraged its use as a live carrier for recombinant antigens, such as low production cost, possibility of administration at birth with very strong adjuvant activity, induction of immunity after a single dose, and low frequency of side effects. The induction of humoral and cellular immune responses against antigens from several pathogens by recombinant BCG (rBCG) strains has been reported, such as rBCG expressing antigens from human immunodeficiency virus (25, 29), simian immunodeficiency virus (30), Leishmania major (1, 6), Plasmodium falciparum (13), Streptococcus pneumoniae (18), and Borrelia burgdorferi (26).The conventional diphtheria-pertussis-tetanus (DPT) vaccine was shown to be extremely efficient, and the recently...

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Diphtheria toxin and its mutant crm 197 differ in their interaction with lipids

Cited by 32 publications

References 33 publications

Conformational changes in diphtheria toxoids

Conformational changes in diphtheria toxoids

Lipid interaction of diphtheria toxin and mutants

Induction of Neutralizing Antibodies against Diphtheria Toxin by Priming with Recombinant Mycobacterium bovis BCG Expressing CRM ₁₉₇ , a Mutant Diphtheria Toxin

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Diphtheria toxin and its mutant crm 197 differ in their interaction with lipids

Cited by 32 publications

References 33 publications

Conformational changes in diphtheria toxoids

Conformational changes in diphtheria toxoids

Lipid interaction of diphtheria toxin and mutants

Induction of Neutralizing Antibodies against Diphtheria Toxin by Priming with Recombinant Mycobacterium bovis BCG Expressing CRM 197 , a Mutant Diphtheria Toxin

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Product

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Induction of Neutralizing Antibodies against Diphtheria Toxin by Priming with Recombinant Mycobacterium bovis BCG Expressing CRM ₁₉₇ , a Mutant Diphtheria Toxin