腸炎弧菌缺乏磷脂醯絲胺酸脫羧酶改變細胞型態與細胞膜性質

Abstract

大多數革蘭氏陰性菌的細胞膜組成主要成分有磷脂醯甘油脂(PG)、心磷脂(CL)與磷脂醯乙醇胺(PE)，其中以磷酸乙醇胺(PE)的比例最高，負責維持細胞膜的功能與完整性。原核細菌中，PE的生合成途徑包括兩個重要酵素: 磷脂醯絲胺酸合成酶(Pss)與磷脂醯絲胺酸脫羧酶 (Psd)，前者將CDP-二醯甘油與絲胺酸進行縮和反應，合成磷脂醯絲胺酸(PS)；後者催化PS的脫羧反應，合成PE。先前研究指出Pss與Psd對於大腸桿菌(Escherichia coli)的化學趨性與鞭毛基因表現是被需要的。腸炎弧菌(Vibrio parahaemolyticus)為一海洋弧菌，經汙染海鮮食物感染人類的消化道，在世界各地也廣泛流行。本研究中，我們將探討Psd對於腸炎弧菌的細胞型態與細胞膜性質的影響。生物資訊學分析的結果顯示腸炎弧菌No. 93的Psd與大腸桿菌的Psd的活性區及疏水區都具有高度的保守性。另外，我們在腸炎弧菌No. 93上，以同源基因重組的方式建構一株Δpsd的突變株，並與野生菌株進行比較。薄層層析分析細胞膜上的磷脂質組成比例，PE較野生株僅有微量的下降。而Δpsd的生長速率與野生株相當，但最終菌量只有野生株的1/3。在含有5% 蔗糖的TSA3培養基上，發現菌落型態發生改變，呈現半透明水狀。Δpsd對氯黴素及介面活性劑Triton X-100、CHAPS耐受性提升。在TSA3培養基上游走與泳動實驗的結果顯示，野生株與Δpsd只有在游走能力有差異。總結以上結果，psd基因的缺失會影響腸炎弧菌的生長，細胞膜對疏水性抗生素的通透性、雙電性及非離子性介面活性劑的耐受性提升，菌落型態改變及游走能力變差。

Most Gram negative bacteria contain phosphatidylglycerol, cardiolipin, phosphatidylcholine, and phosphatidylethanoamine (PE) as major membrane lipids. Among them, PE is the major composition of cell membrane and contributes to function and integrity of membranes. In bacteria, phosphatidylserine synthase (Pss) and phosphatidylserine decarboxylase (Psd) are involved in the biosynthesis pathway of PE. Pss condense CDP-diacylglycerol and serine into phosphatidylserine (PS) and Psd catalyze the decarboxylation of PS into PE. It was reported that Pss and Psd are required for expression of chemotaxis and flagellum genes in E. coli. Marine bacterium Vibrio paraheamolyticus is a major food-borne human pathogen in many countries. In this report, we show that Psd is required for normal cell morphology and membrane property in V. paraheamolyticus. The results of bioinformatics analysis showed that the catalytic site and hydrophobic site of V. parahaemolyticus No. 93 were was highly conserved to E. coli. To investigate the physiological role of Psd, we used homologous gene-replacement to construct a psd deletion mutant (Δpsd) in V. parahaemolyticus No. 93. Thin layer chromatography analysis showed the amount of PE in Δpsd is not significantly different from wild type. The growth rate of both wild type and Δpsd was the same, but the final biomass of Δpsd was one third of wild type. Surprisingly, we found the colony morphology of Δpsd on TSA3 medium supplemented with 5% sucrose become transparent and liqidity comparing to wild type strain. Interestingly, the Δpsd has higher resistant ability to chloramphenicol, Triton X-100 and CHAPS than wild type. Swimming ability was decreased in Δpsd in motility assay. Concluded the results, lacking of psd gene affect cell growth, resistance of hydrophobic antibiotics, non-ionic and zwitterionic surfactant compound, swimming ability decreased and alter the cell morphology in V. parahaemolyticus.