Isolated CyaA-RTX subdomain from Bordetella pertussis: Structural and functional implications for its interaction with target erythrocyte membranes.
The 126-kDa Bordetella pertussis CyaA-hemolysin (CyaA-Hly) was previously expressed in Escherichia coli as a soluble precursor that can be acylated to retain hemolytic activity. Here, we investigated structural and functional characteristics of a ∼100-kDa isolated RTX (Repeat-in-ToXin) subdomain (CyaA-RTX) of CyaA-Hly. Initially, we succeeded in producing a large amount with high purity of the His-tagged CyaA-RTX fragment and in establishing the interaction of acylated CyaA-Hly with sheep red blood cell (sRBC) membranes by immuno-localization. Following pre-incubation of sRBCs with non-acylated CyaA-Hly or with the CyaA-RTX fragment that itself produces no hemolytic activity, there was a dramatic decrease in CyaA-Hly-induced hemolysis. When CyaA-RTX was pre-incubated with anti-CyaA-RTX antisera, the capability of CyaA-RTX to neutralize the hemolytic activity of CyaA-Hly was greatly decreased. A homology-based model of the 100-kDa CyaA-RTX subdomain revealed a loop structure in Linker II sharing sequence similarity to human WW domains. Sequence alignment of Linker II with the human WW-domain family revealed highly conserved aromatic residues important for protein-protein interactions. Altogether, our present study demonstrates that the recombinant CyaA-RTX subdomain retains its functionality with respect to binding to target erythrocyte membranes and the WW-homologous region in Linker II conceivably serves as a functional segment required for receptor-binding activity.
Graduate Program in Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand.
Riyaz Ahmad
RA
Pandit
Bacterial Protein Toxin Research Cluster, Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakornpathom 73170, Thailand.
Kanungsuk
K
Meetum
Graduate Program in Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand.
Kittipong
K
Suvarnapunya
Bacterial Protein Toxin Research Cluster, Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakornpathom 73170, Thailand.
Gerd
G
Katzenmeier
Department of Parasitology and Center of Excellence on Therapeutic Proteins and Antibody Engineering, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand.
Wanpen
W
Chaicumpa
Bacterial Protein Toxin Research Cluster, Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakornpathom 73170, Thailand; Laboratory of Molecular Biophysics and Structural Biochemistry, Biophysics Institute for Research and Development (BIRD), Bangkok 10160, Thailand. Electronic address: chanan.ang@mahidol.ac.th.
Chanan
C
Angsuthanasombat
D037361:Adenylate Cyclase Toxin / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001886:Bordetella pertussis / Q000502:physiology*; D004910:Erythrocyte Membrane / Q000382:microbiology*; D006461:Hemolysis; D054884:Host-Pathogen Interactions; D006801:Humans; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D012756:Sheep; D012757:Sheep Diseases / Q000378:metabolism; D014917:Whooping Cough / Q000378:metabolism* / Q000662:veterinary*
D037361:Adenylate Cyclase Toxin
Bordetella pertussis; CyaA-RTX; CyaA-hemolysin; Erythrocyte-membrane interaction; WW domain family