的高分辨电子显微<em&gt;幽门螺杆菌</em&gt;恰IV型分泌系统的霹雳产于铁可用性的条件变

摘要

Here we describe a method to visualize the oncogenic bacterial organelle known as the Cag Type IV Secretion System (Cag-T4SS). We find that the Cag-T4SS is differentially produced on the surface of H. pylori in response to varying conditions of iron availability.

摘要

Helicobacter pylori is a helical-shaped, gram negative bacterium that colonizes the human gastric niche of half of the human population^1,2. H. pylori is the primary cause of gastric cancer, the second leading cause of cancer-related deaths worldwide³. One virulence factor that has been associated with increased risk of gastric disease is the Cag-pathogenicity island, a 40-kb region within the chromosome of H. pylori that encodes a type IV secretion system and the cognate effector molecule, CagA^4,5. The Cag-T4SS is responsible for translocating CagA and peptidoglycan into host epithelial cells^5,6. The activity of the Cag-T4SS results in numerous changes in host cell biology including upregulation of cytokine expression, activation of proinflammatory pathways, cytoskeletal remodeling, and induction of oncogenic cell-signaling networks^5-8. The Cag-T4SS is a macromolecular machine comprised of sub-assembly components spanning the inner and outer membrane and extending outward from the cell into the extracellular space. The extracellular portion of the Cag-T4SS is referred to as the “pilus”⁵. Numerous studies have demonstrated that the Cag-T4SS pili are formed at the host-pathogen interface^9,10. However, the environmental features that regulate the biogenesis of this important organelle remain largely obscure. Recently, we reported that conditions of low iron availability increased the Cag-T4SS activity and pilus biogenesis. Here we present an optimized protocol to grow H. pylori in varying conditions of iron availability prior to co-culture with human gastric epithelial cells. Further, we present the comprehensive protocol for visualization of the hyper-piliated phenotype exhibited in iron restricted conditions by high resolution scanning electron microscopy analyses.

引言

H. pylori infection is a significant risk factor for gastric cancer¹. However, disease outcomes vary and depend on numerous factors such as host genetics, genetic diversity of H. pylori strains, and environmental elements such as host diet¹¹. Previous reports have established that a correlation exists between H. pylori infection, iron deficiency (as measured by decreased blood ferritin and hemoglobin concentrations), and increased proinflammatory cytokine production, including IL-8 secretion, which ultimately leads to increased gastric disease progression¹². Acute H. pylori infection is also associated with hypochlorhydria which impairs the host’s ability to absorb nutrient iron, and ultimately leads to changes in iron homeostasis¹³. These clinical findings suggest that iron availability within the gastic niche could be an important factor in disease outcome. In fact, animal models of H. pylori infection have demonstrated that low dietary iron consumption exacerbates gastric disease¹⁴. The reduced iron levels in these animals necessitate that H. pylori induce an iron-acquisition response in order to obtain the iron needed for bacterial replication. H. pylori has the capacity to perturb iron trafficking within host cells to facilitate bacterial replication in a CagA-dependent fashion¹⁵. Interestingly, the cag-pathogenicity island has been shown to be regulated by the iron-responsive transcription factor Fur^16,17. Furthermore, Cag+ strains are associated with increased inflammation and gastric diseases such as cancer¹. These findings support a model whereby H. pylori alters Cag-T4SS expression in an effort to obtain iron from host cells that reside in an iron deplete environment resulting in exacerbated disease outcomes.

Two factors that increase inflammation and morbidity are Cag expression and low dietary iron intake. These facts support the hypothesis that reduced iron availability increases the production of Cag-T4SS pili at the host pathogen interface resulting in worse gastric disease^11-14. The goal of the method provided in this manuscript is to establish the role of the micronutrient iron in the regulation of the Cag-T4SS pilus biogenesis. In previous work, we utilized two approaches to observe an iron-dependent increase in Cag-T4SS expression. First, output strains from animals maintained on high and low iron diets were analyzed and revealed that low-iron diet output strains produced more Cag-T4SS pili than high-iron diet strains¹⁴. Second, growing the H. pylori 7.13 strain in vitro in iron replete conditions resulted in reduced pili formation while cells grown in the presence of an iron chelator produced significantly more pili.

We have continued to investigate the iron-dependent regulation of Cag-T4SS pili phenotype and offer the following optimized protocol and representative results performed with an additional Helicobacter pylori strain, PMSS1. The rationale behind the development of this technique was to correlate increased Cag-T4SS activity in conditions of iron-limitation with increased Cag-T4SS pilus formation. The broader implication and use of this technique will provide optimized culture conditions that result in elevated production of the Cag-T4SS pili. This assay will be useful to researchers seeking to determine the composition and architecture of the Cag-T4SS by enriching for this important bacterial surface feature. The sample preparation and visualization by field-emission gun electron microscopy has numerous advantages over alternative techniques such as light-microscopy methods to visualize the Cag-T4SS and will be appropriate to investigators interested in studying the regulation of this organelle¹⁰.

研究方案

1. H.幽门螺旋杆菌生长的铁可用性和共培养的人胃癌上皮细胞的各种条件

1. 选择H.幽门螺杆菌菌株PMSS1对于这些研究，因为它有一个完整的cag致病岛，并表示一个功能IV型分泌系统。还利用同基因的突变体保持架 （PMSSIΔ 笼 ）作为阴性对照。上生长，在5％CO ₂的存在下补充有5％绵羊血（血琼脂平板上）放置24小时，在37℃TSA平板的细菌。
   注:准备试剂和作为材料和设备表所列组装材料。最终浓度为各试剂列在括号中。
2. 刮用无菌棉签从板菌，接种到由以下成分制备改性的布鲁氏肉汤（见材料和​​设备表），并补充有胆固醇。隔夜孵育培养37°下在室温空气中补充了5％CO ₂的振荡下每分钟（rpm）200旋转。
   注:胆固醇代替胎牛血清由于这一事实，即血清是复杂的;含有大量养分铁源如血红素引起变性的结果。
3. 对细菌培养的天，种子AGS人胃上皮细胞（ATCC CRL-1739）上在12孔板（每孔约1×10 ⁵个细胞）聚-D-赖氨酸处理过的盖玻片。
4. 第二天，稀释细菌培养物，以0.3的OD 600在单独或补充有100μM的_氯化铁，200μM联吡啶（一种合成的铁螯合剂）或200μM的联吡啶加250微米的FeCl ₃改性布鲁氏肉汤中。室内空气中补充有5％的CO ₂与200rpm振荡孵育这些培养4小时，在37℃下。
5. 细菌离心机在1000×g离心收集细胞，重悬我之前取出上清液为n的新鲜改性布鲁氏肉汤等体积的补充有胆固醇。该培养物（OD 600 = 1.0 = 5.5×10 ⁸个细胞/ ml）的测量OD 600和添加细菌的上皮细胞在感染20的复数（MOI）的多重性:1。进行连续稀释和平板的菌体到血琼脂平板上，以评估细菌细胞生存力。
6. 孵育H.幽门螺杆菌和AGS人胃上皮细胞共培养4小时，在37℃下在5％CO ₂的静态条件下进行扫描电子显微镜（SEM）样品制备之前的存在。

2.扫描电镜样品制备可视化H.幽门螺杆菌 CAG-T4SS霹雳

1. 删除H.幽门螺杆菌和AGS共培养物从培养箱和倾析培养物上清液（保存为IL-8的ELISA法）。用0.05M二甲胂酸钠缓冲液（pH7.4）轻轻洗涤三次。固定的样品在室温下2-4小时，在2.0％多聚甲醛的溶液，2.5％戊二醛，和0.05M的二甲胂酸钠缓冲液中。主固定后，冲洗样本三次用0.05M二甲胂酸钠缓冲液中。
2. 在两个连续的10分钟的固定步骤，可以执行用0.1％四氧化锇的辅助固定在0.05M二甲胂酸钠缓冲液中。次级固定后，冲洗样本三次用0.05M二甲胂酸钠缓冲液中。
3. 初级和次级固定后，脱水通过顺序洗涤样品以增加乙醇的浓度按表1。
4. 乙醇脱水后，进行液体二氧化碳的三个相继洗涤。在一个关键点上使用了一个临界点干燥机在31.1℃的高温1073 PSI和压力，然后干燥样品。
5. 山盖玻片上铝的SEM样品存根并用细线胶体银在样品边缘漆，以达到适当的接地和避免在扫描电镜成像充电。
6. 涂层样品为5nm金 - 钯，用溅射涂层机，以增加样品的二次电子信号和边缘效应。

高分辨率扫描电镜分析3.成像参数

1. 鉴于样品与场致发射枪扫描电子显微镜（FEG-SEM）。
2. 图像中在5-10毫米的工作距离的高真空状态。
3. 加速电压设定为5kV。
4. 设定光斑大小为2-2.5。
5. 倾斜15〜25度之间的样品来实现宿主 - 病原体界面的更好的视野。
6. 优先考虑成像的细菌粘附于上皮细胞的高倍率视野的边缘，作为宿主 - 病原体相互作用的这些区域富含菌毛形成的细菌。

4.统计分析，以评估霹雳量化值

1. 分析H.使用ImageJ软件幽门恰-T4SS菌毛量化菌毛/细胞的细胞数目以及百分比表现出菌毛苯丙氨酸按下面的说明NoType在。
2. 在ImageJ的软件打开的显微照片文件。识别菌毛作为细菌细胞和宿主细胞，以均匀的宽度（10-13纳米）和长度（60-150纳米）之间形成的结构。
   注:菌毛结构上同基因的菌株，如Δ 笼突变株，其中海港基因的失活编码主要ATP酶的权力IV型分泌菌毛装配存在于野生菌株，但缺席。
3. 通过绘制与直线工具行，然后单击"分析"选项卡上的校准测量工具。从下拉菜单中选择"设置缩放"，然后输入放大倍率栏数值成箱标有"已知的距离"和长度的单位设置为相应的设置（纳米）。点击"确定"。
4. 使用直线工具绘制过菌毛的长度或宽度测量CAG-T4SS菌毛，然后按"Ctrl-M"来衡量每个菌毛。观察一个对话框，与测得的值。记录这些值或复制并粘贴到电子表格程序。
5. 使用的长度和宽度参数预先确定，不适合恰-T4SS的轮廓^24，无视特征。然后量化的基础上是10-13纳米的宽度和60-150纳米的长度截断部位内的值菌毛的数目。
6. 通过使用GraphPad Prism软件的双尾学生t检验进行统计分析菌毛的量化。

5.可选:评估IL-8分泌的关联与菌毛生产

1. 利用上清液从步骤2.1，按照制造商的试剂盒说明书（见材料和​​设备表）执行IL-8 ELISA。
2. 分析IL-8的由宿主细胞分泌的，并计算百分比的IL-8的诱导相对于WT PMSS1单独在培养基中生长。通过执行使用GraphPad Prism让双尾t检验进行统计分析ftware。

结果

在这份报告中，我们已经证明，不同的铁可用性的条件下具有调节H.能力幽门螺杆菌 CAG-T4SS菌毛的生物合成在主机病原体接口。当在中单独培养，H。幽门螺旋杆菌形成平均3菌毛/细胞。当H.幽门螺旋杆菌是生长在铁耗尽条件（用合成的螯合剂联吡啶基），这些亚抑制细菌生长（ 图1）中，细菌产生无数恰-T4SS菌毛（〜7菌毛/细胞）共培养人胃癌细胞（ 图2...

讨论

铁是大多数生命形式，包括细菌病原体必需的微量营养素。在努力限制入侵微生物的生存能力，脊椎动物宿主螯合营养铁在被称为"营养免疫^"18的处理。响应于此，细菌病原体已经进化为使用铁作为全局信号传导分子以感测周围的环境并调节阐述毒力的功能，如铁采集系统，毒素和毒素分泌机械^19,20。

幽门螺杆菌需要微量营养素如铁，锌和镁来生长^2...

材料

Name	Company	Catalog Number	Comments
Modified brucella broth
Peptone from casein (10 g/L)	Sigma	70172
Peptic digest of animal tissue (10 g/L)	Sigma	70174
Yeast extract (2 g/L)	Sigma	92144
Dextrose (1 g/L)	Sigma	D9434
Sodium chloride (5 g/L)	Thermo Fisher	S271-10
Cholesterol (250X) (4 ml/L)	Life Technologies	12531018
Ferric chloride (100 or 250 uM)	Sigma	157740-100G
Dipyridyl (200 uM)	Sigma	D216305-100G
Modified RPMI
RPMI+HEPES (1X)	Life Technologies	22400-121
Fetal bovine serum (100 ml/L)	Life Technologies	10438-026
Electron Microscopy Preparation
Paraformaldehyde (2.0% aqueous)	Electron Microscopy Sciences	15713
Gluteraldehyde (2.5% aqueous)	Electron Microscopy Sciences	16220
Sodium cacodylate (0.05 M)	Electron Microscopy Sciences	12300
Osmium tetroxide (0.1% aqueous)	Electron Microscopy Sciences	19150
Ethanol (absolute)	Sigma	E7023
Colloidal silver paint	Electron Microscopy Sciences	12630
SEM sample stubs	Electron Microscopy Sciences	75220
Coverslips	Thermo Fisher	08-774-383
IL-8 Secretion Evaluation
Quantikine IL-8 ELISA kit	R&D Systems	D8000C