การศึกษาโปรตีนสารพิษ Alpha-Hemolysin (HlyA) ของเชื้อ Escherichia coli สายพันธุ์ที่ก่อให้เกิดโรคติดเชื้อในระบบทางเดินปัสสาวะ

Abstract

Uropathogenic Escherichia coli (UPEC) is the main cause of urinary tract infection (UTI) worldwide. The exotoxin hemolysin A (HlyA) is a virulence factor that plays important role in the mechanism of UTI and indicates the severity of UTI symptoms. HlyA induces the pore formation in membrane of the host cells. It functions as the active HlyA which is activated by HlyC in the acylation reaction. The main objective of this research is to express and purify both HlyA and HlyC proteins as well as to preliminary study the biochemical properties. Constructing the recombinant plasmids pET17b-hlyA encoding HlyA and pET17b-hlyC encoding HlyC by using UPEC genomics isolated from UTI patients admitted to the hospital in southern Thailand as a template revealed that nucleotide sequences of hlyA and hlyC genes were different from other UPEC strains i.e. CFT073, J96 and UTI89, with the most similarity to CFT073 corresponding to 99.2% and 98.8%, respectively. The UPEC strain of this research was therefore designated as strain MNT08. Both HlyA and HlyC proteins were overexpressed as insoluble proteins at 37 °C and 25 °C respectively in E. coli strain BL21(DE3). After a single-step purification using Ni-NTA affinity chromatography , the purified HlyA and HlyC proteins were analysed by SDS-PAGE revealing a molar mass of approximately 110 kDa and 19 kDa, respectively. Based on mass spectrometry, the purified HlyA protein was identified as hemolysin with 55% similarity to hemolysin encoded by hlyA gene from UPEC (Accession number: P09983) while HlyC had 56% similarity to hemolysin-activating lysine-acyltransferase HlyC from UPEC (Accession number: P09984). HlyA is expressed as an unfolded protein called pro-HlyA. Normally, pro-HlyA can folding in the condition with Ca2+, which is interaction on the RTX domain. HlyA was successfully folded in a buffer containing 50 mM Tris-HCl pH 7.4, 50 mM NaCl, and 20 mM CaCl2. A preliminary study on the structural and functional relationships of proteins. The three-dimensional structural of the HlyA protein was created using SWISS-MODEL. A homology model of HlyA protein was generated using the structure of CyaA from Bordetella pertussis, which has a T1SS secretion system like HlyA from E. coli, as the most suitable template. The model was predicted as a monomer covering 339 amino acids at the C-terminus (residues 612-950). Due to the limitation of short-length template, the model in this research was able to predict only one-third portion of HlyA structure. Meanwhile, ApxC of Actinobacillus pleuropneumoniae was used as a template for homology modeling of HlyC. The HlyC model was built as a homodimer covering 167 amino acids (residues 3-169), accounting for 98.2% of the total amino acid residues. To sum up, this research is only a preliminary study providing useful information for further functional and structural studies of the full length HlyA using X-ray crystallography in order to acquire a better understanding of the pore formation in UTI mechanism.