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Malik A.A.,Mahidol University | Malik A.A.,Therapeutic Proteins | Imtong C.,Mahidol University | Sookrung N.,Mahidol University | And 4 more authors.
Toxins | Year: 2016

Previously, the 126-kDa CyaA-hemolysin (CyaA-Hly) fragment cloned from Bordetella pertussis—the causative agent of whooping cough—and functionally expressed in Escherichia coli was revealed as a key determinant for CyaA-mediated hemolysis against target erythrocytes. Here, phagemid-transfected E. coli clones producing nanobodies capable of binding to CyaA-Hly were selected from a humanized-camel VH/VHH phage-display library. Subsequently verified for binding activities by indirect ELISA and Western blotting, four CyaA-Hly-specific nanobodies were obtained and designated according to the presence/absence of VHH-hallmark amino acids as VHH2, VH5, VH18 and VHH37. In vitro neutralization assay revealed that all four ~17-kDa His-tagged VH/VHH nanobodies, in particular VHH37, which were over-expressed as inclusions and successfully unfolded-refolded, were able to effectively inhibit CyaA-Hly-mediated hemolysis. Phage-mimotope searching revealed that only peptides with sequence homologous to Linker 1 connecting Blocks I and II within the CyaA-RTX subdomain were able to bind to these four CyaA-Hly-specific nanobodies. Structural analysis of VHH37 via homology modeling and intermolecular docking confirmed that this humanized nanobody directly interacts with CyaA-RTX/Linker 1 through multiple hydrogen and ionic bonds. Altogether, our present data demonstrate that CyaA-RTX/Linker 1 could serve as a potential epitope of CyaA-protective antigen that may be useful for development of peptide-based pertussis vaccines. Additionally, such toxin-specific nanobodies have a potential for test-driven development of a ready-to-use therapeutic in passive immunization for mitigation of disease severity. © 2016 by the authors; licensee MDPI, Basel, Switzerland.


Kurehong C.,Mahidol University | Kanchanawarin C.,Kasetsart University | Powthongchin B.,Silpakorn University | Katzenmeier G.,Mahidol University | And 2 more authors.
Toxins | Year: 2015

Previously, the 126-kDa Bordetella pertussis CyaA pore-forming/hemolysin (CyaA-Hly) domain was shown to retain its hemolytic activity causing lysis of susceptible erythrocytes. Here, we have succeeded in producing, at large quantity and high purity, the His-tagged CyaA-Hly domain over-expressed in Escherichia coli as a soluble hemolytically-active form. Quantitative assays of hemolysis against sheep erythrocytes revealed that the purified CyaA-Hly domain could function cooperatively by forming an oligomeric pore in the target cell membrane with a Hill coefficient of ~3. When the CyaA-Hly toxin was incorporated into planar lipid bilayers (PLBs) under symmetrical conditions at 1.0 M KCl, 10 mM HEPES buffer (pH 7.4), it produced a clearly resolved single channel with a maximum conductance of ~35 pS. PLB results also revealed that the CyaA-Hly induced channel was unidirectional and opened more frequently at higher negative membrane potentials. Altogether, our results first provide more insights into pore-forming characteristics of the CyaA-Hly domain as being the major pore-forming determinant of which the ability to induce such ion channels in receptor-free membranes could account for its cooperative hemolytic action on the target erythrocytes. © 2015 by the authors; licensee MDPI, Basel, Switzerland.


Kaikaew A.,Mahidol University | Promptmas C.,Mahidol University | Angsuthanasombat C.,Mahidol University | Angsuthanasombat C.,Biophysics Institute for Research and Development BIRD
Biochemical and Biophysical Research Communications | Year: 2016

Bacillus thuringiensis Cry4Ba mosquito-active toxin was previously shown to utilize two critical loop-residues, Tyr332 and Phe364 which are respectively located in β2-β3 and β4-β5 loops, for synergistic interactions with its alternative receptor-Cyt2Aa2. Here, structural analysis of the Cry4Ba-receptor-binding domain revealed tha 2015 Elsevier Inc. All rights reserved.t its N-terminal subdomain encompasses β2-β3 and β4-β5 hairpins which are stabilized by inter-hairpin hydrogen bonding between Thr328 in β2 and Thr369 in β5. Functional importance of these two side-chains was demonstrated by single-Ala substitutions (T328A and T369A), adversely affecting toxin activity against Aedes aegypti larvae. Unlike toxicity restoration of the inactive E417A/Y455A toxin mutated within another receptor-binding subdomain, defective bioactivity of T328A and T369A mutants cannot be restored by Cyt2Aa2 as also observed for β2-β3 (Y332A) and β4-β5 (F364A) loop-mutants. ELISA-based analysis further verified a loss in binding of all four bio-inactive mutants (T328A, Y332A, T369A and F364A) to the immobilized Cyt2Aa2. Protein-protein docking suggested that the two critical loop-residues (Tyr332 and Phe364) correspondingly located at β2-β3 and β4-β5 loops can clearly interact with four counterpart surface-exposed residues of Cyt2Aa2. Altogether, our present data demonstrate structural importance of Thr328 and Thr369 toward hydrogen-bonded stabilization of two receptor-binding hairpins (β2-β3 and β4-β5) for synergistic toxicity of Cry4Ba with Cyt2Aa2. © 2015 Elsevier Inc. All rights reserved.


Juntadech T.,Mahidol University | Kanintronkul Y.,Mahidol University | Kanchanawarin C.,Kasetsart University | Katzenmeier G.,Mahidol University | And 2 more authors.
Biochimica et Biophysica Acta - Biomembranes | Year: 2014

Bacillus thuringiensis Cry4Ba toxin is lethal to mosquito-larvae by forming ion-permeable pores in the target midgut cell membrane. Previously, the polarity of Asn166 located within the α4-α5 loop composing the Cry4Ba pore-forming domain was shown to be crucial for larvicidal activity. Here, structurally stable-mutant toxins of both larvicidal-active (N166D) and inactive (N166A and N166I) mutants were FPLC-purified and characterized for their relative activities in liposomal-membrane permeation and single-channel formation. Similar to the 65-kDa trypsin-activated wild-type toxin, the N166D bio-active mutant toxin was still capable of releasing entrapped calcein from lipid vesicles. Conversely, the two other bio-inactive mutants showed a dramatic decrease in causing membrane permeation. When the N166D mutant was incorporated into planar lipid bilayers (under symmetrical conditions at 150 mM KCl, pH 8.5), it produced single-channel currents with a maximum conductance of about 425 pS comparable to the wild-type toxin. However, maximum conductances for single K+-channels formed by both bio-inactive mutants (N166I and N166A) were reduced to approximately 165-205 pS. Structural dynamics of 60-ns simulations of a trimeric α4-α5 pore model in a fully hydrated-DMPC system revealed that an open-pore structure could be observed only for the simulated pores of the wild type and N166D. Additionally, the number of lipid molecules interacting with both wild-type and N166D pores is relatively higher than those of N166A and N166I pores. Altogether, our results further signify that the polarity at the α4-α5 loop residue - Asn166 is directly involved in ion permeation through the Cry4Ba toxin-induced ionic pore and pore opening at the membrane-water interface. © 2013 Elsevier B.V.


Aroonkesorn A.,Mahidol University | Pootanakit K.,Mahidol University | Katzenmeier G.,Mahidol University | Angsuthanasombat C.,Mahidol University | Angsuthanasombat C.,Biophysics Institute for Research and Development BIRD
Biochemical and Biophysical Research Communications | Year: 2015

The interaction between Bacillus thuringiensis Cry toxins and their receptors on midgut cells of susceptible insect larvae is the critical determinant in toxin specificity. Besides GPI-linked alkaline phosphatase in Aedes aegypti mosquito-larval midguts, membrane-bound aminopeptidase N (AaeAPN) is widely thought to serve as a Cry4Ba receptor. Here, two full-length AaeAPN isoforms, AaeAPN2778 and AaeAPN2783, predicted to be GPI-linked were cloned and successfully expressed in Spodoptera frugiperda (Sf9) cells as 112- and 107-kDa membrane-bound proteins, respectively. In the cytotoxicity assay, Sf9 cells expressing each of the two AaeAPN isoforms showed increased sensitivity to the Cry4Ba mosquito-active toxin. Double immunolocalization revealed specific binding of Cry4Ba to each individual AaeAPN expressed on the cell membrane surface. Sequence analysis and homology-based modeling placed these two AaeAPNs to the M1 aminopeptidase family as they showed similar four-domain structures, with the most conserved domain II being the catalytic component. Additionally, the most variable domain IV containing negatively charged surface patches observed only in dipteran APNs could be involved in insect specificity. Overall results demonstrated that these two membrane-bound APN isoforms were responsible for mediating Cry4Ba toxicity against AaeAPN-expressed Sf9 cells, suggesting their important role as functional receptors for the toxin counterpart in A. aegypti mosquito larvae. © 2015 Elsevier Inc. All rights reserved.

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