Relationship between Kanagawa phenomenon and promoter sequence of the thermostable direct haemolysin (tdh) gene of Vibrio parahaemolyticus

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Relationship between Kanagawa phenomenon and promoter sequence of the thermostable direct haemolysin (tdh) gene of Vibrio parahaemolyticus


Author: Li, Kwok-cheung
Title: Relationship between Kanagawa phenomenon and promoter sequence of the thermostable direct haemolysin (tdh) gene of Vibrio parahaemolyticus
Degree: M.Sc.
Year: 2007
Subject: Hong Kong Polytechnic University -- Dissertations.
Vibrio parahaemolyticus.
Seafood -- Contamination -- Health aspects.
Department: Dept. of Health Technology and Informatics
Pages: xi, 57 leaves : ill. (some col.) ; 30 cm.
Language: English
InnoPac Record:
Abstract: Vibrio parahaemolyticus is a halophilic, estuarine bacterium capable of causing acute gastroenteritis in humans through consumption of contaminated raw or undercooked seafoods. The strains manifesting Kanagawa phenomenon (KP), a beta-type haemolysis on Wagatsuma agar, were considered clinically important in early studies, where KP was induced by a thermostable direct haemolysin (TDH). TDH is toxin composed of two identical 165-amino-acid subunits and encoded on the tdh gene. There are five variants of the tdh gene, tdhl to tdh5. The tdh genes share >97% sequence identity and encoded proteins with similar biological activities. Each KP-positive strain carries both the tdhl and tdh2 genes. Only tdh2 gene contributes to the majority of TDK expression. Although the level of TDK production varied with different KP-positive strains, the underlying reason has not been fully elucidated. In this project, the relationship between the KP and the promoter sequence of the tdh gene of V. parahaemolyticus was studied. A commercial kit KAP-RPLA was used to quantify the level of TDH secretion. The tdh2 promoter sequences were determined by PCR and DNA sequencing. Promoter sequences of all KP-positive V. parahaemolyticus studied were compared by multiple alignment. Among the 71 clinical V. parahaemolyticus isolates, 68 strains (95.8%) were KP and PCR positive while three strains (4.2%) were negative for both. The result of multiple alignments showed two different groups (group I and group II) of promoter sequences among the KP-positive strains. Group I composed of 75% KP-positive strains and group II, 25%. The promoter sequence of group I strains resembled that of the reference tdh2 sequence (Genbank accession number AB112355). However, the promoters of group II strains had a guanine (G) to adenine (A) substitution at nucleotide position -17 upstream of the transcription start site of tdh2. Besides, the group II promoter also had a deletion of thymine (T) at +40 region downstream of the transcription start site. The TDH titres detected in the KP-positive strains ranged from 16 to 1024. The average level of TDH detected in group II strains was 3.6-fold higher than that in group I. When unpaired t-test was used, an association between tdh2 promoter groups (group I or group II) and mean of TDH production levels of these two groups was demonstrated (p=0.0002). According to the findings, it is possible that a single base substitution or mutation in the promoter region might generate a significant difference in TDH production. Although the tdh gene of KP-positive strain has already been considered clinically important, it might mutate to a more virulent strain by a point mutation or substitution in the tdh2 promoters. As a result, this may cause an increase in the pathogenicity of V. parahaemolyticus and the severity of V. parahaemolyticus-associaied infection. The mutations identified in the tdh2 promoters are novel and have not been reported before. However, further confirmatory study, such as site-directed mutagenesis, is required to investigate the effect of point mutations in positions -17 and +40 of tdh2 promoter on TDH production. Besides, reverse-transcription PCR (RT-PCR) is recommended in order to accurately quantify the level of tdh2 expression in V. parahaemolyticus strains.

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