Saniee Parastoo, Siavoshi Farideh, Nikbakht Broujeni Gholamreza, Khormali Mahmood, Sarrafnejad Abdolfatah, Malekzadeh Reza
Department of Microbiology, School of Biology, University College of Sciences, University of Tehran, Tehran,
Department of Microbiology, School of Biology, University College of Sciences, University of Tehran, Tehran, Iran.
Arch Iran Med. 2013 Dec;16(12):705-10.
Reports indicate that H.pylori is able to invade the eukaryotic cells and establish inside their vacuoles. In this study, FITC-conjugated IgY-Hp was used to localize H.pylori inside the vacuole of Candida yeast. Presence of intracellular H.pylori inside the new generations of yeast cells was also examined by light microscopy and Live/Dead BacLight staining method.
A single colony of fresh yeast culture was cultivated in a 100-µl medium containing yeast extract and N-acetylglucoseamine supplemented with fetal bovine serum. After 12-hr incubation at 37℃, FITC-conjugated IgY-Hp was added. After 3 hours, 10 µL of yeast suspension was smeared on a glass slide, air-dried and examined by fluorescent microscopy. Wet mounts of yeast culture and Live/Dead BacLight stained preparations were examined by light and fluorescent microscopy, respectively. Photographs were taken from the fast-moving H.pylori inside the yeast vacuoles.
Fluorescent microscopy showed that FITC-conjugated IgY-Hp could enter yeast cells and specifically react with H.pylori, localizing the bacterium inside the yeast vacuole. Photographs taken from wet mounts observed by light and fluorescent microscopy showed fast-moving H.pylori cells in the vacuole of mother as well as daughter yeast cells. The intravacuolar H.pylori cells stained green, showing their viability.
Intracellular life of prokaryotes inside eukaryotes has been described as an evolutionary phenomenon with a great impact on bacterial persistence despite environmental stresses. Results of this study demonstrated the specific interaction of FITC-conjugated IgY-Hp with H.pylori cells and the bacterial localization inside the Candida yeast vacuole. The intracellular bacteria were viable and existed in the vacuole of next generations of yeast cells. It appears that H.pylori is well-equipped to dwell within the vacuole of eukaryotic cells where it is protected from stressful conditions, including antibacterial therapy. Presence of H.pylori inside the vacuole of new generations of yeasts demonstrates the intimate relationship between the two microorganisms, resulting in bacterial inheritance as part of the vacuolar content of yeast cells.
报告表明，幽门螺杆菌能够侵入真核细胞并在其液泡内定植。在本研究中，使用异硫氰酸荧光素（FITC）偶联的抗幽门螺杆菌免疫球蛋白Y（IgY-Hp）来定位念珠菌酵母液泡内的幽门螺杆菌。还通过光学显微镜和活菌/死菌BacLight染色法检查新一代酵母细胞内细胞内幽门螺杆菌的存在情况。
将新鲜酵母培养物的单个菌落接种于含有酵母提取物和N-乙酰葡糖胺并补充胎牛血清的100μl培养基中。在37℃孵育12小时后，加入FITC偶联的IgY-Hp。3小时后，取10μL酵母悬液涂片于载玻片上，空气干燥后用荧光显微镜检查。酵母培养物的湿片和活菌/死菌BacLight染色制剂分别用光学显微镜和荧光显微镜检查。拍摄酵母液泡内快速移动的幽门螺杆菌照片。
荧光显微镜显示，FITC偶联的IgY-Hp可进入酵母细胞并与幽门螺杆菌特异性反应，将细菌定位在酵母液泡内。光学显微镜和荧光显微镜观察湿片拍摄的照片显示，母代和子代酵母细胞液泡中有快速移动的幽门螺杆菌细胞。液泡内的幽门螺杆菌细胞染成绿色，表明它们具有活力。
真核生物内原核生物的细胞内生活被描述为一种进化现象，尽管存在环境压力，但对细菌的持续存在有很大影响。本研究结果证明了FITC偶联的IgY-Hp与幽门螺杆菌细胞的特异性相互作用以及细菌在念珠菌酵母液泡内的定位。细胞内细菌具有活力，并存在于新一代酵母细胞的液泡中。看来幽门螺杆菌具备在真核细胞液泡内生存的良好条件，在其中它受到保护，免受包括抗菌治疗在内的应激条件影响。新一代酵母液泡内存在幽门螺杆菌证明了这两种微生物之间的密切关系，导致细菌作为酵母细胞液泡内容物的一部分遗传下来。
