Study on the Responses of Vibrio parahaemolyticus to Sublethal Heat and Ethanol Shock Treatments
Date Issued
2007
Date
2007
Author(s)
Chiang, Ming-Lun
DOI
zh-TW
Abstract
In the present study, Vibrio parahaemolyticus 690, a clinical strain, was subjected to either heat shock at 42℃ or ethanol shock in the presence of 5% ethanol. The fatty acid profile, morphology and recovery of the heat-shocked and ethanol-shocked cells of V. parahaemolyticus on different media were investigated. The effects of heat shock and ethanol shock on the protein expression, thermostable direct hemolysin (TDH) production and survival of V. parahaemolyticus during subsequent exposure to various environmental stresses were examined. Furthermore, the superoxide dimutase (SOD), catalase (CAT) activities and growth behavior of V. parahaemolyticus in the presence of various nitrogen and carbon sources as affected by heat shock and ethanol shock were also determined.
It was found that the ratio of saturated fatty acid to unsaturated fatty acid (SFA/USFA) observed in the heat-shocked cells of V. parahaemolyticus was significantly higher than that in the control cells. Extensive cell-wall pitting and cell disruption, representing cell-surface damage, were also observed on the heat-shocked cells. The recovery of the heat-shocked cells of V. parahaemolyticus was significantly less on TCBS (thiosulfate-citrate-bile salts-sucrose agar) and APS agar (alkaline peptone salt broth supplemented with 1.5% agar) than on TSA-3% NaCl (tryptic soy agar supplemented with 3% NaCl). Heat shock decreased the tolerance of V. parahaemolyticus to organic acids (25 mM). While heat shock increased the survival of V. parahaemolyticus in the presence of 0.1% NaCl and made the test organism more susceptible to 20% NaCl.
The recovery of the ethanol-shocked cells of V. parahaemolyticus was significantly less on TCBS than on TSA-3% NaCl. A significantly marked increase of protein and nucleic acid materials in the supernatant of the cell suspension was found after cells of V. parahaemolyticus were subjected to ethanol shock. Extensive cell disruption, wrinkling and cell-wall pitting, indicative of cell-surface damage were also noted on the ethanol-shocked cells. The ethanol-shocked cells of V. parahaemolyticus exhibited a similar yet higher susceptibility at 4℃ and -18℃ compared with the control cells. Moreover, there was a marked increase in the thermal tolerance (47℃) and resistance to 8% ethanol with cells of V. parahaemolyticus after ethanol shock.
Ethanol shock decreased the SFA/USFA in cells of V. parahaemolyticus. The recovery of the ethanol-shocked cells on TSA plus 6% or 7.5% NaCl was significantly less than the control cells. Furthermore, ethanol shock increased the survival of V. parahaemolyticus in the presence of H2O2 (20 ppm), while made the test organism less resistant to crystal violet (3 ppm), high NaCl (20%) and organic acids (25 mM).
Analysis with one-dimensional SDS-PAGE indicated that three proteins with a molecular mass of 93, 77, and 58 kDa were found to be induced by heat shock and ethanol shock for V. parahaemolyticus compared with the control cells. Analysis with two-dimensional electrophoresis revealed that heat shock and ethanol shock affected the expression of a total of 28 proteins in V. parahaemolyticus. Among them four proteins with a molecular mass of 94, 32.1, 26.7 and 25.7 kDa were enhanced by heat shock and ethanol shock. Furthermore, immunoblotting analysis showed the presence of a GroEL-like protein with a molecular mass of 61 kDa in the heat-shocked, ethanol-shocked and control cells of the test organism. The heat-shocked and ethanol-shocked cells produced GroEL-like protein in a larger quantity than the control cells. However, DnaK-like protein was not detectable in cells of V. parahaemolyticus.
Heat shock and ethanol shock reduced the level of SOD and CAT activities in V. parahaemolyticus. Gel activity staining assay failed to detect the expression of CAT, while one SOD enzyme was detected in the control, heat-shocked and ethanol-shocked cells of V. parahaemolyticus. Heat shock for 15-60 min and ethanol shock for 45-60 min were found to enhance the synthesis of TDH by the test organism. The growth and TDH produced by the ethanol-shocked and control cells of V. parahaemolyticus in TSB-3% NaCl were identical, while a lower level of growth and TDH production was detected with the heat-shocked cells until after 200 min of cultivation. Furthermore, the growth of the ethanol-shocked cells was similar to that of the control cells, while the heat-shocked cells exhibited an extended lag period in the media with various nitrogen and carbon sources examined.
Subjects
腸炎弧菌
熱震處理
次致死乙醇處理
復甦
脂肪酸
蛋白質
耐熱性溶血素
Vibrio parahaemolyticus
Heat shock
Ethanol shock
Recovery
Fatty acid
Protein
Thermostable direct hemolysin
Type
thesis
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