Sequence and phylogenetic analyses of Nsp2-HVII, ORF5, and ORF7 coding regions of highly pathogenic porcine reproductive and respiratory syndrome virus from Myanmar.

In this study, PRRSVs that spread during the outbreaks of 2011 in Myanmar were investigated. Sequences and phylogenetic analyses of the Nsp2 middle hypervariable region (Nsp2-HVII) encoding gene, ORF5, and ORF7 showed that they belonged to the North American (NA) genotype and were clustered with HP-PRRSV strains from other Southeast Asian countries. The discontinuous 30-amino acid deletions at positions 481 and 533-561 were found in the Nsp2-HVII of all Myanmarese PRRSVs, implying their derivation from HP-PRRSV. The phylogenetic trees also showed that Myanmarese strains were in the same group as other Southeast Asian strains from Cambodia, Thailand, and Laos suggesting their close relationships. Conversely, Vietnamese 07QN was in the same group as Chinese JXA1. The unique amino acid mutations found only in Myanmarese PRRSVs were L292F, P431S, and V621M in Nsp2-HVII and E170G in GP5, which may be used as a marker for monitoring genetic diversity of newly emerging HP-PRRSV strains.

Functional Contributions of Positive Charges in the Pore-Lining Helix 3 of the Bordetella pertussis CyaA-Hemolysin to Hemolytic Activity and Ion-Channel Opening.

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.

Functional importance of the Gly cluster in transmembrane helix 2 of the Bordetella pertussis CyaA-hemolysin: Implications for toxin oligomerization and pore formation.

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 (Gly(530), Gly(533), Gly(537) and Gly(544)) 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 Gly(530)_Gly(533)_Gly(537) 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 Gly(530)XXGly(533)XXXGly(537) could function as an important framework for toxin oligomerization. Altogether, our present data signify for the first time that the Gly(530)_Gly(533)_Gly(537) cluster in transmembrane helix 2 serves as a crucial constituent of the CyaA-Hly trimeric pore structure.

Membrane-Pore Forming Characteristics of the Bordetella pertussis CyaA-Hemolysin Domain.

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.

Functional significance of the highly conserved Glu(570) in the putative pore-forming helix 3 of the Bordetella pertussis haemolysin toxin.

Adenylate cyclase-haemolysin toxin (CyaA) is a virulence factor secreted from the etiologic agent of whooping cough, Bordetella pertussis. Previously, the haemolysin or pore-forming domain (CyaA-PF) has been shown to cause cell lysis of sheep erythrocytes independently, and the predicted helix 3((570-593)) within the PF-hydrophobic stretch could be a pore-lining constituent. Here, a plausible involvement in haemolytic activity of polar or charged residues (Glu(570), Gln(574), Glu(581), Ser(584) and Ser(585)) lining the hydrophilic side of CyaA-PF helix 3 was investigated via single-alanine substitutions. All the 126-kDa mutant proteins over-expressed in Escherichia coli were verified for toxin acylation as the results are corresponding to the wild-type toxin. When haemolytic activity of E. coli lysates containing soluble mutant proteins was tested against sheep erythrocytes, the importance of Glu(570), which is highly conserved among the pore-forming RTX cytotoxin family, was revealed for pore formation, conceivably for a general pore-lining residue involved in ion conduction.

Bordetella pertussis CyaA-RTX subdomain requires calcium ions for structural stability against proteolytic degradation.

Previously, the 126-kDa Bordetella pertussis CyaA pore-forming (CyaA-PF) domain expressed in Escherichia coli was shown to retain its hemolytic activity. Here, a 100-kDa RTX (Repeat-in-ToXin) subcloned fragment (CyaA-RTX) containing a number of putative calcium-binding repeats was further investigated. The recombinant CyaA-RTX protein, although expressed as a soluble form in a protease-deficient E. coli strain BL21(DE3)pLysS, was found to be highly sensitive to proteolytic degradation. Interestingly, the addition of calcium ions in a millimolar range into the CyaA-RTX preparation significantly prevented the degradation. Moreover, levels of proteolytic degradation were dependent on calcium concentrations, implying an important role for calcium-binding sites in the RTX subdomain for structural stability. Homology-based modeling of the repetitive blocks in the CyaA-RTX subdomain supports that this calcium-bound protein folds into a parallel ß-roll structure with calcium ions acting as a structural stabilizing bridge.

Esterase activity of Bordetella pertussis CyaC-acyltransferase against synthetic substrates: implications for catalytic mechanism in vivo.

Adenylate cyclase-hemolysin toxin (CyaA) produced from the human respiratory tract pathogen Bordetella pertussis requires fatty-acyl modification by CyaC-acyltransferase to become an active toxin. Previously, the recombinant CyaA pore-forming (CyaA-PF) fragment expressed in Escherichia coli was shown to be hemolytically active upon palmitoylation in vivo by cosynthesized CyaC. Here, the 21-kDa CyaC enzyme separately expressed in E. coli as an inclusion body was solubilized in 8 M urea and successfully refolded into an enzymatically active monomer. In addition to the capability of activating CyaA-PF in vitro, CyaC showed esterase activity against p-nitrophenyl acetate (pNPA) and p-nitrophenyl palmitate (pNPP), with preferential hydrolysis toward pNPP when compared with chymotrypsin. A homology-based CyaC structure suggested a conceivable role of a catalytic triad including Ser(30), His(33) and Tyr(66) in substrate catalysis. Alanine substitutions of these individual residues caused a drastic decrease in specific activities of all three mutant enzymes (S30A, H33A and Y66A) toward pNPP, signifying that CyaC-acyltransferase shares a similar mechanism of hydrolysis with a serine esterase in which Ser(30) is part of the catalytic triad.

Effects on haemolytic activity of single proline substitutions in the Bordetella pertussis CyaA pore-forming fragment.

The recombinant Bordetella pertussis CyaA pore-forming (CyaA-PF) fragment was previously shown to be expressed separately in Escherichia coli as a soluble precursor that can be in vivo palmitoylated to exert haemolytic activity. In this study, PCR-based mutagenesis was employed to investigate the contributions to haemolysis of five predicted helices within the N-terminal hydrophobic region of the CyaA-PF fragment. Single proline substitutions were made for alanine near the centre of each predicted helix as a means of disrupting local secondary structure. All mutant proteins were over-expressed in E. coli as a 126-kDa soluble protein at levels comparable to the wild-type. Marked reductions in haemolytic activity against sheep erythrocytes of mutants, A510P, A538P, A583P and A687P pertaining to the putative helices 1(500-522), 2(529-550), 3(571-593) and 5(678-698), respectively, were observed. However, a slight decrease in haemolytic activity was found for the proline replacement in the predicted helix 4(602-627) (A616P). MALDI-TOF-MS and LC-MS-MS analyses verified the palmitoylation at Lys983 of all five mutants as identical to that of the CyaA-PF wild-type protein, indicating that toxin modification via this acylation was not affected by the mutations. Altogether, these results suggest that structural integrity of the predicted helices 1, 2, 3 and 5, but not helix 4, is important for haemolytic activity, particularly for the putative transmembrane helices 2 and 3 that might conceivably be involved in pore formation of the CyaA-PF fragment.

High level of soluble expression in Escherichia coli and characterisation of the CyaA pore-forming fragment from a Bordetella pertussis Thai clinical isolate.

Bordetella pertussis adenylate cyclase toxin-haemolysin (CyaA) can permeabilise erythrocytes by forming lytic pores. Here, a gene segment encoding CyaA pore-forming (CyaA-PF) domain cloned from genomic DNA of B. pertussis Thai isolate was over-expressed in Escherichia coli as a 126-kDa soluble protein which cross-reacted with anti-RTX monoclonal antibody. By co-expressing with acyltransferase CyaC, the CyaA-PF protein was found palmitoylated at Lys(983). Unlike E. coli lysate with the non-acylated form, the lysate containing acylated CyaA-PF exhibited high haemolytic activity against sheep erythrocytes. This study presents that the recombinant CyaA-PF protein comprising pore-forming domain can be expressed separately as soluble native-folded precursor that conserves at least part of its functionality.