次致死熱處理影響阪崎腸桿菌蛋白質之表現與脂肪酸組成

Abstract

阪崎腸桿菌 (Cronobacter sakazakii) 為革蘭氏陰性菌，是一種新興食源性病原菌，常能存活於嬰兒配方奶粉等食品中，感染引發新生兒之腦膜炎、壞死性小腸結腸炎和菌血症等疾病，並具有40%~80% 之高致死率。本研究將C. sakazakii BCRC 13988進行47℃-15 min之熱震處理，探討熱震處理菌體於蛋白質表現、脂肪酸組成與其於含銅離子牛奶中之存活性。 以一維電泳分析發現，熱震處理後有11種蛋白質表現上升而1種蛋白質表現下降。二維電泳結果顯示，熱震處理影響C. sakazakii BCRC 13988 菌體25種蛋白質之表現，其中有16種菌體蛋白質表現量增加，9種蛋白質表現量減少。將4種因熱震而表現量上升之蛋白質以胰蛋白酶進行酵素水解，並利用液態串聯式質譜儀技術結合資料庫搜尋對蛋白質進行鑑定，這些蛋白質分別為IbpB、OmpA、OmpX和FoF1 ATP synthase α；蛋白質IbpB 可提供菌體於後續熱壓力下之保護效果，OmpA 與OmpX可能與細胞滲透壓之調節、菌體附著力與生物膜形成相關，FoF1 ATP synthase α之表現則與菌體能量生成有關。這些表現量上升之蛋白質不只能夠提高菌體在不同壓力環境下之存活，亦可能參與菌體內部生理反應之調整作用。此外發現熱震後會顯著地 (p<0.05) 提高菌體飽和-不飽和脂肪酸之比例。並可提高C. sakazakii於100 μg ml-1含銅離子牛乳中之存活。

Cronobacter sakazakii is a Gram-negative, emerging opportunistic pathogen which has been associated with life-threatening cases in infants, with infant formulas been implicated as the mode of transmission, and with high mortality rate of 40-80%. In the present study, C. sakazakii BCRC 13988, the type strain, was subjected to heat shock treatments (47℃, 15 min). The effect of heat shock on the protein expression, fatty acid profile as well as growth and survival behavior of heat-shocked C. sakazakii in the milk containing copper ion were examined. Analysis with one-dimensional SDS-PAGE showed that expression of 11 proteins bands was enhanced while 1 protein was reduced in the heat-shocked cells of C. sakazakii compared with control cells. The protein patterns revealed by two-dimensional electrophoresis showed that heat shock treatment affected the expression of 25 proteins, the levels of 16 proteins increased and 9 proteins decreased. Four proteins with enhanced expression level were subjected to tryptic digestion, LC/MS/MS analysis and protein identification using database search. They were identified as chaperone IbpB, OmpA, OmpX and FoF1 ATP synthase subunit α. IbpB exhibited protection effect in thermal environment. OmpA and OmpX involved in cellular osmotic pressure regulation, bacterial adhesion, or biofilm formation. FoF1 ATP synthase subunit α was related to energetic production. These proteins may not only correlate closely with the higher tolerance of C. sakazakii under various stress conditions but also involve in some other physiological regulation. It was also noted that heat shock significantly enhanced (p<0.05) the ratio of saturated fatty acids to unsaturated fatty acids in the cells of C. sakazakii. Besides, heat shock also enhanced the survival of C. sakazakii in sterile milk containing 100 μg ml-1 copper ion.