Structural characteristics and autocatalysis of bordetella pertussis CyaA-hemolysin

สมบัติเชิงโครงสร้างและการย่อยตัวเองของไซยาเอ-ฮีโมไลซินจาก Bordetella pertussis

Name: Veerada Raksanoh

ThaSH: Bacteria

Classification :.DDC: 589.9

Abstract: Adenylate cyclase (CyaA) toxin, a virulence factor of Bordetella pertussis, consists of adenylate cyclase and pore forming domains. Here, a truncated segment of pore forming domain (CyaA-HP/BI) containing hydrophobic and acylation site with first RTX block (Bl1015-1088) was studied for its characteristics and toxicity. The overexpressed 70- kDa CyaA-HP/BI with His-tag aggregated in inclusion bodies with CyaC acyltransferasefatty acid donor for palmitoylation. The proteins were solubilized with 4 M urea prior to refolding and purification via Ni2+-NTA network in affinity column in HEPES buffer, pH 7.4 and 2 mM CaCl2. Soluble CyaA-HP/BI did not lyse sheep erythrocytes (5 ± 2% of complete hemolysis), albeit with sufficient components for pore forming ability. Here, association between CyaA-HP/BI and CyaC was hypothesized to be a cause of hemolytic interruption. Size exclusion chromatography and semi-native gel electrophoresis revealed that no association between CyaA-HP/BI and CyaC although they were coeluted. In addition, the uncovered first block of RTX was found to likely be a cause of autocatalysis of protein in RTX family. Purified CyaA-HP/BI without protease preservation entirely degraded after incubation at 37 o C for 3 h. 1,10-Phenanthroline viii (PNT), a zinc metalloprotease inhibitor, could reduce the protein degradation, while phenylmethylsulphonylfluoride (PMSF), a serine protease inhibitor, could not prevent protein degradation. Overall data suggested that pore forming activity of CyaA-HP/BI was absent due to lack of association of CyaA-HP/BI with CyaC and proteolysis of CyaAHP/BI fragment is a Zn2+-dependent autocatalytic activity.

Chiang Mai University Library

Address: Chiang Mai

Email: cmulibref@cmu.ac.th

Name: Nalin Wongkattiya

Role: Cairman

Name: Chatchai Tayapiwatana

Role: Member

Name: Chanan Angsuthanasombat

Role: Member

Name: Piyarat Nimmanping

Role: Member

Name: Panchika Prangkio

Role: Member

Created: 2017

Modified: 2020-01-07

Issued: 2020-01-07

วิทยานิพนธ์/Thesis

application/pdf

CallNumber: Th 589.9 V419S

eng

DegreeName: Doctor of Philosophy

Level: Doctoral Degree

Descipline: Biotechnology

Grantor: Chiang Mai University

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