胞外絲胺酸蛋白酶對腸炎弧菌生物膜生成及遊走能力之影響

The Extracellular Serine Protease Affects the Biofilm Formation and Swarming Motility in Vibrio parahaemolyticus

張絜雯(Chieh-Wen Chang)

指導教授 ： 李佳音

國立臺灣大學/生物資源暨農學院/農業化學研究所/碩士(2009年)

https://doi.org/10.6342/NTU.2009.00924

摘要

前人研究(FEMS Microbiology Letter (2002) 209:31-37)已證實胞外絲胺酸蛋白酶PrtA可能為一毒力因子。已有報告指出，胞外蛋白酶會參與革蘭氏陽性菌的遊走能力。此外，遊走能力與生物膜的形成及致病能力有關，故本研究擬探討PrtA對於腸炎弧菌生物膜的形成及遊走能力扮演的角色。由於腸炎弧菌O3:K6菌株為我國與全球性食物中毒致病菌，本研究選擇三株腸炎弧菌O3:K6臨床菌株、一株type strain ATCC17802以及五株分離自台灣的環境菌株為研究材料，並建構O3:K6和type strain的prtA缺陷株和其補償株。首先利用接合生殖將帶有prtA缺失片段的自殺質體pDS132-mprtA送入腸炎弧菌中，篩選得到prtA缺陷株，再利用核酸序列定序、南方漬片和西方漬片進行確認。接著將帶有prtA的質體pSA19CP-MCS-prtA以電衝方式送入prtA缺陷株中製備補償株。在含3%NaCl之tryptic soy broth (TSB-3% NaCl)或是含5%NaCl之marine broth (MB-5% NaCl)，37 ℃的培養條件下觀察其生長情形，O3:K6和type strain的野生株、缺陷株和補償株的生長情形皆無顯著差異。將腸炎弧菌培養在37 ℃，TSB-3% NaCl和MB-5% NaCl兩種培養基中，測定胞外蛋白酶的活性，結果發現類似海洋環境的Marine broth才能誘導膠原蛋白酶產生，並有利於生物膜的形成。另在TSB-3% NaCl，37 ℃下培養，O3:K6菌株prtA缺陷株的生物膜形成能力明顯野生株增強，顯示prtA缺失能使腸炎弧菌在TSB-3% NaCl的環境中增加生物膜形成能力。但在MB-5% NaCl，30 ℃的條件下培養，腸炎弧菌ATCC17802的生物膜生成是需要prtA的參與，prtA缺失會造成生物膜生成量明顯降低。但相同的條件對O3:K6菌株而言，prtA缺失不會造成生物膜生成量的改變。藉由含0.25% agar的TSA-3%NaCl來測定菌株的遊走能力，結果發現prtA缺陷株的菌落大小比野生株明顯較小，顯示prtA缺失會造成遊走能力的降低。而補償株則生物膜生成能力與遊走能力則介於野生株和缺陷株之間。比較環境菌株以及臨床菌株，發現大部份環境菌株(4/5)生物膜的生成能力比臨床菌株強，但環境菌株整體的遊走能力則與臨床菌株無太大差異。當TSB培養基鹽濃度含1%和3%時，生物膜的生成量最高，而在MB培養基中，鹽濃度對於生物膜生成量則無明顯影響。在不同的條件培養下，由於不同的營養成分造成生物膜的生合成能力有所差異，而prtA不僅與致病能力有關，同時也和菌體在環境中的生存能力有關。

關鍵字

腸炎弧菌 ； 絲胺酸蛋白酶生物膜 ； 遊走能力

並列摘要

Protease A, serine protease of Vibrio parahaemolyticus, has been known to possess cytotoxicity and considered as a putative virulence factor. We constructed the prtA deletion mutants and their complement strains with three O3:K6 serotype strains and one V. parahaemolyticus type strain, ATCC17802. Campared the wild-type, prtA deletion mutants and their complement strains, their growth rates was not significant difference in TSB3 or MB5 medium at 37 ℃. But only strains cultured in MB5 medium, it could induce much protease activities from wild-type and a little protease activities from prtA deletion mutants. When comparing the biofilm formation between the wild type and the prtA deletion mutants, the prtA deletion mutants increased biofilm formation in TSB3 at 37 ℃ significantly. The mutants lacking prtA significantly reduced swarming motility on TSB3 semisolid agar at both 37 ℃ and 30 ℃. The type strain and most environmental strains had much more biofilm formation than those of clinical strains in MB5 medium at both 37 ℃ and 30 ℃. However, only prtA deletion mutant of type strain VP00 reduced half of biofilm formation on MB5 medium at 30 ℃ was observed. In different saline of TSB medium, the TSB1 and TSB3 medium induced most biofilm formation from V. parahaemolyticus strains. But no specific saline concentration of MB medium could induced most biofilm formation.These results indicated that prtA is growth independent and significantly affect the biofilm formation in TSB3 medium at 37 ℃ and the swarming motilities of V. parahaemolyticus. We conclude that when cultured at different temperatures and medium, PrtA leads to different microbial physiology and metabolism. The regulation of biofilm formation affected by culture temperature and medium slightly in O3:K6 strains but obviously in environmental strains from marine sources. Our results indicate that the regulation of biofilm formation in O3:K6 and in environmental strains had different.

並列關鍵字

Vibrio parahaemolyticus ； serine protease ； biofilm ； swarming motility

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