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

Abstract

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.

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

“…An equal amount of erythrocytes lysed with 0.1% Triton X-100 was defined as 100% hemolysis. All samples were tested in triplicate for three independent experiments as previously described [21]. …”

“…However, we have recently demonstrated that such palmitoylation at Lys 983 is not required for the binding of CyaA-Hly to target erythrocyte membranes, but rather needed for stabilizing CyaA-Hly-induced pores/channels [18]. Several studies clearly shown that, without the AC part, the CyaA-Hly domain can independently exert hemolytic activity against sheep erythrocytes that lack the CD11b/CD18 receptor, suggesting an alternative mechanism of target cell recognition via the RTX subdomain [19,20,21,22]. Nevertheless, the exact molecular mechanism of translocation of the AC domain across target cell membranes still needs to be elucidated.…”

Esterase activity ofBordetella pertussis CyaC-acyltransferase against synthetic substrates: implications for catalytic mechanismin vivo

“…Escherichia coli BL21(DE3)pLysS harboring the recombinant plasmids pCyaAC‐PF encoding CyaA‐PF with CyaC, pCyaA‐PF (without CyaC) or pCyaC (Powthongchin & Angsuthanasombat, 2008), was grown at 30 °C in Luria–Bertani medium containing 100 μg mL −1 ampicillin. Protein expression was induced with 0.1 mM isopropyl‐β‐ d ‐thiogalactopyranoside (IPTG) for 5 h. Cultured cells were harvested by centrifugation at 5000 g (4 °C, 15 min), resuspended in 25 mM HEPES (pH 7.0) and disrupted via French Pressure Cell at 10 000 psi.…”

“…The pCyaC plasmid encoding the 21‐kDa CyaC‐acyltransferase (Powthongchin & Angsuthanasombat, 2008) was used as a template for single‐alanine substitutions at Ser 30 , His 33 and Tyr 66 , using a pair of mutagenic oligonucleotides as follows: S30A (f‐primer, 5′‐GATGAAC G CTCCCATGCA T CGCGACTGGCCGGT‐3′ and r‐primer, 5′‐GTCGCG A TGCATGGGAG C GTTCATCCACAGCCAG‐3′, with bold letters indicating changed nucleotides and underlined bases indicating a added NruI restriction site); H33A (f‐primer, 5′‐CCCATG GC CCGCGACTGG‐3′ and r‐primer, 5′‐CGCGG GC CATGGGAGAGT‐3′, with bold letters indicating changed nucleotides and underlined bases indicating an added NcoI restriction site); Y66A (f‐primer, 5′‐GTTGCA GCA TGCAGCTGGGC‐3′ and r‐primer, 5′‐GCTGCA TGC TGCAACCGGCA‐3′, with bold letters indicating changed nucleotides and underlined bases indicating a deleted PstI restriction site). All mutant plasmids were generated by PCR‐based directed mutagenesis using a high‐fidelity Pfu DNA polymerase, following the procedure of the QuickChange ™ Mutagenesis Kit (Stratagene).…”

“…In our previous studies, the recombinant CyaA‐PF protein (residues 482–1706) coexpressed with CyaC in Escherichia coli was found to be palmitoylated in vivo at Lys 983 to become hemolytically active (Powthongchin & Angsuthanasombat, 2008). However, the precise mechanism of CyaA acylation by CyaC‐acyltransferase has not yet been fully elucidated.…”