Biophysics Institute for Research and Development BIRD

Bangkok, Thailand

Biophysics Institute for Research and Development BIRD

Bangkok, Thailand
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Kurehong C.,Mahidol University | Kanchanawarin C.,Kasetsart University | Powthongchin B.,Silpakorn UniversityNakornpathom | Prangkio P.,Chiang Mai University | And 3 more authors.
Toxins | Year: 2017

The Bordetella pertussis CyaA‐hemolysin (CyaA‐Hly) domain was previously demonstrated to be an important determinant for hemolysis against target erythrocytes and ion‐channel formation in planar lipid bilayers (PLBs). Here, net‐charge variations in the pore‐lining helix of thirteen related RTX cytolysins including CyaA‐Hly were revealed by amino acid sequence alignments, reflecting their different degrees of hemolytic activity. To analyze possible functional effects of net‐charge alterations on hemolytic activity and channel formation of CyaA‐Hly, specific mutations were made at Gln574 or Glu581 in its pore‐lining α3 of which both residues are highly conserved Lys in the three highly active RTX cytolysins (i.e., Escherichia coli α‐hemolysin, Actinobacillus pleuropneumoniae toxin, and Aggregatibacter actinomycetemcomitans leukotoxin). All six constructed CyaA‐Hly mutants that were over‐expressed in E. coli as 126 kDa His‐tagged soluble proteins were successfully purified via immobilized Ni2+‐affinity chromatography. Both positive‐charge substitutions (Q574K, Q574R, E581K, E581R) and negative-charge elimination (E581Q) appeared to increase the kinetics of toxin‐induced hemolysis while the substitution with a negatively‐charged side‐chain (Q574E) completely abolished its hemolytic activity. When incorporated into PLBs under symmetrical conditions (1.0 M KCl, pH 7.4), all five mutant toxins with the increased hemolytic activity produced clearly‐resolved single channels with higher open probability and longer lifetime than the wild‐type toxin, albeit with a half decrease in their maximum conductance. Molecular dynamics simulations for 50 ns of a trimeric CyaA‐Hly pore model comprising three α2‐loop‐α3 transmembrane hairpins revealed a significant role of the positive charge at both target positions in the structural stability and enlarged diameter of the simulated pore. Altogether, our present data have disclosed functional contributions of positively‐charged side‐chains substituted at positions Gln574 and Glu581 in the pore‐lining α3 to the enhanced hemolytic activity and ion‐channel opening of CyaA‐Hly that actually mimics the highly‐active RTX (repeat‐in‐toxin) cytolysins. © 2017 by the authors. Licensee MDPI, Basel, Switzerland.

Raksanoh V.,Chiang Mai University | Shank L.,Chiang Mai University | Prangkio P.,Chiang Mai University | Yentongchai M.,Mahidol University | And 4 more authors.
Biochemical and Biophysical Research Communications | Year: 2017

Proteolytic degradation of the ∼100-kDa isolated RTX (Repeat-in-ToXin) subdomain (CyaA-RTX) of the Bordetella pertussis CyaA-hemolysin (CyaA-Hly) was evidently detected upon solely-prolonged incubation. Here, a truncated CyaA-Hly fragment (CyaA-HP/BI) containing hydrophobic and acylation regions connected with the first RTX block (BI1015-1088) was constructed as a putative precursor for investigating its potential autocatalysis. The 70-kDa His-tagged CyaA-HP/BI fragment which was over-expressed in Escherichia coli as insoluble aggregate was entirely solubilized with 4 M urea. After re-naturation in a Ni2+-NTA affinity column, the purified-refolded CyaA-HP/BI fragment in HEPES buffer (pH 7.4) supplemented with 2 mM CaCl2 was completely degraded upon incubation at 37 °C for 3 h. Addition of 1,10-phenanthroline-an inhibitor of Zn2+-dependent metalloproteases markedly reduced the extent of degradation for CyaA-HP/BI and CyaA-RTX, but the degradative effect was clearly enhanced by addition of 100 mM ZnCl2. Structural analysis of a plausible CyaA-HP/BI model revealed a potential Zn2+-binding His-Asp cluster located between the acylation region and RTX-BI1015-1088. Moreover, Arg997-one of the identified cleavage sites of the CyaA-RTX fragment was located in close proximity to the Zn2+-binding catalytic site. Overall results demonstrated for the first time that the observed proteolysis of CyaA-HP/BI and CyaA-RTX fragments is conceivably due to their Zn2+-dependent autocatalytic activity. © 2017 Elsevier Inc.

Juntapremjit S.,Mahidol University | Thamwiriyasati N.,Burapha University | Kurehong C.,Mahidol University | Prangkio P.,Chiang Mai University | And 4 more authors.
Toxicon | Year: 2015

Adenylate cyclase-hemolysin (CyaA) is a major virulence factor of Bordetella pertussis causing whooping cough in humans. We previously showed that two transmembrane helices (α2 and α3) in the hemolysin domain (CyaA-Hly) are crucially involved in hemolytic activity. Here, PCR-based substitutions were employed to investigate a potential involvement in hemolysis of a series of four Gly residues (Gly530, Gly533, Gly537 and Gly544) which map onto one face of a helical wheel plot of pore-lining helix 2. All CyaA-Hly mutant toxins were over-expressed in Escherichia coli as 126-kDa soluble proteins at levels comparable to the wild-type toxin. A drastic reduction in hemolytic activity against sheep erythrocytes was observed for three CyaA-Hly mutants, i.e. G530A, G533A and G537A, but not G544A, suggesting a functional importance of the Gly530-Gly533-Gly537 cluster. A homology-based structure of the α2-loop-α3 hairpin revealed that this crucial Gly cluster arranged as a GXXGXXXG motif is conceivably involved in helix-helix association. Furthermore, a plausible pore model comprising three α2-loop-α3 hairpins implicated that Gly530XXGly533XXXGly537 could function as an important framework for toxin oligomerization. Altogether, our present data signify for the first time that the Gly530-Gly533-Gly537 cluster in transmembrane helix 2 serves as a crucial constituent of the CyaA-Hly trimeric pore structure. © 2015 Published by Elsevier Ltd.

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.

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.

Imtong C.,Mahidol University | Kanchanawarin C.,Kasetsart University | Katzenmeier G.,Mahidol University | Angsuthanasombat C.,Mahidol University | Angsuthanasombat C.,Biophysics Institute for Research and Development BIRD
Biochimica et Biophysica Acta - Proteins and Proteomics | Year: 2014

The long loop connecting transmembrane α4 and α5 of the Bacillus thuringiensis Cry4Aa toxin possesses a unique feature with Pro-rich sequence (Pro193Pro194-Pro196) which was shown to be crucial for toxicity. Here, the structural role in the intrinsic stability of the Pro-rich sequence toward toxin activity was investigated. Three Val-substituted mutants (P193V, P194V and P196V) and one Phe-substituted mutant (P193F) were generated and over-expressed in Escherichia coli as inclusions at levels equal to the wild-type. Bioassays demonstrated that all mutants, particularly P193V and P193F whose inclusions were hardly soluble in carbonate buffer (pH 9.0), exhibited reduced toxicity, suggesting an essential role in toxin function by the specific cyclic structure of individual Pro residues. Analysis of the 65-kDa Cry4Aa structure from 10-ns molecular dynamics (MD) simulations revealed that the α4-α5 loop is substantially stable as it showed low structural fluctuation with a 1.2-Å RMSF value. When the flexibility of the α4-α5 loop was increased through P193G, P194G and P196G substitutions, decreased toxicity was also observed for all mutants, mostly for the P193G mutant with low alkali-solubility, suggesting a functional importance of loop-rigidity attributed by individual Pro-cyclic side-chains, particularly Pro193. Further MD simulations revealed that the most critical residue-Pro193 for which mutations vastly affect toxin solubility and larval toxicity is in close contact with several surrounding residues, thus playing an additional role in the structural arrangement of the Cry4Aa toxin molecule. Altogether, our data signify that the intrinsic stability of the unique Cry4Aa α4-α5 loop structure comprising the Pro-rich sequence plays an important role in toxin activity. © 2014 Elsevier B.V.

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.

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.

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.

Visitsattapongse S.,Mahidol University | Sakdee S.,Mahidol University | Leetacheewa S.,Mahidol University | Angsuthanasombat C.,Mahidol University | Angsuthanasombat C.,Biophysics Institute for Research and Development BIRD
Biochemical and Biophysical Research Communications | Year: 2014

Bacillus thuringiensis Cry4Aa toxin was previously shown to be much more toxic to Culex mosquito-larvae than its closely related toxin - Cry4Ba, conceivably due to their sequence differences within the β10-β11 receptor-binding loop. Here, single-Ala substitutions of five residues (Pro 510, Thr512, Tyr513, Lys514 and Thr515) within the Cry4Aa β10-β11 loop revealed that only Lys514 corresponding to the relative position of Cry4Ba-Asp 454 is crucial for toxicity against Culex quinquefasciatus larvae. Interestingly, charge-reversal mutations at Cry4Ba-Asp454 (D454R and D454K) revealed a marked increase in toxicity against such less-susceptible larvae. In situ binding analyses revealed that both Cry4Ba-D454R and D454K mutants exhibited a significant increase in binding to apical microvilli of Culex larval midguts, albeit at lower-binding activity when compared with Cry4Aa. Altogether, our present data suggest that a positively charged side-chain near the tip of the β10-β11 loop plays a critical role in determining target specificity of Cry4Aa against Culex spp., and hence a great increase in the Culex larval toxicity of Cry4Ba was obtained toward an opposite-charge conversion of the corresponding Asp454. © 2014 Elsevier Inc. All rights reserved.

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