生物膜启动对病毒感染的细胞测定细菌和真菌
Summary
A method is described herein for the determination of inter-Kingdom association and competition (bacterial and fungal) for adherence to virus-infected HeLa cell monolayers. This protocol can be extended to multiple combinations of prokaryotes, eukaryotes, and viruses.
Abstract
整个分类王国，包括真菌，细菌，病毒等多种微生物相互作用的研究没有先前关于审查的微生物病毒的成员是如何影响这些病毒感染的宿主细胞随后微生物相互作用。与细菌和真菌病毒的同居主要是存在于口腔和生殖道的粘膜表面上。粘膜细胞，尤其是那些具有持久的慢性或持续性潜病毒感染，可以有一个显著对微生物的成员通过病毒改变数量和表达的受体的影响的类型。变形例在宿主细胞膜结构会导致正常菌群和机会致病菌的后续成员的以引发在生物膜的形成， 即 ，粘附在第一步骤改变的能力。这项研究描述了BIOFIL的启动为HSV的效果定量和视觉检查方法S. m的形成（粘附） 金黄色葡萄球菌和C.白色念珠菌 。
Introduction
人类微生物包括来自身体共享地理区域的多个分类王国不同的生物体。附着到细胞表面是在生物膜的形成，这是微生物定植过程的一部分重要的第一步。包括在微生物可以是引起慢性和持续感染病毒。慢性细胞感染这些病毒可以在推定受体可用性引起的改变。1,2-此外，通过细胞内病原体进入细胞也可能影响宿主细胞膜的流动性/疏水性这反过来又可以改变其他微生物部件的附着，包括细菌和真菌。为了理解可在这些多种病原体在人类宿主的同一地理区域该共定位之间发生的相互作用，我们必须能够学习的表示存在于粘膜表面生物分类王国的频谱病原体的相互作用。
t“的>的疱疹病毒是存在于人类的微生物的永久成员100％的微生物的一个家族3,4-。此外，它们也可以持续在症状的存在和缺乏棚两者。具体地说，单纯疱疹病毒-1和单纯疱疹病毒-2（HSV-1和HSV-2，分别）是在oronasopharynx和生殖道的微生物的永久成员。在免疫能力的个体，无论是HSV-1和HSV-2引起龈，以及生殖器疱疹5-8。在这些站点，单纯疱疹病毒引起潜伏感染为特征的慢性持续性无症状病毒脱落的HSV 9。加入到在改变细胞的结果在nectins，硫酸乙酰肝素，脂筏和疱疹病毒条目介体的表面表达/肿瘤坏死因子受体（HVEM / TNFR）10-25。这些潜在代表共享受体某些细菌和真菌， 如金黄色葡萄球菌和白色念珠菌，这同时致病菌，也可以驻留作为oronasopharynx 26,27的粘膜微生物的成员。内oronasopharynx S.金黄色葡萄球菌和C.白色念珠菌定植占据两个不同的地点。与天然齿的主机，口腔粘膜用HSV-1和C共享白色念珠菌，而前鼻鼻孔都是由S.占用金黄色葡萄球菌 28。然而，尽管体外研究结果，即S。金黄色葡萄球菌坚持口上皮细胞，29,30 S.当正常组织存在金黄色葡萄球菌 29,30不经常口腔标本中分离。鲜为人知的是关于超越的临床研究结果，即生殖道共定植龛称为S.金黄色葡萄球菌与有氧阴道炎，其特点是生殖器炎症，分泌物和性交痛，而C.白色念珠菌产生粘膜病变类似于在口腔中31-35观察。因此，虽然口腔和生殖器microbi的这些部件青梅交叉分类王国小关于它们的相互作用是已知的，因为它影响其通过加入引发生物膜形成与宿主细胞表面5的能力。该协议得到了有效的应用，以确定共同定植/感染的功能性后果。Protocol
1，HSV毒株和处理注：重组非扩频HSV-1（KOS）gL86和HSV-2（KOS）333gJ -与所使用的五Twiari 36,37分别提供β-半乳糖苷报告基因活性。 从单一的很多并储存在-80℃，使用的病毒以1：Dulbecco改良的Eagle培养基（DMEM），用20％胎牛血清（FBS）的比例为1和脱脂牛奶直到使用。病毒大量的存储之前，由邻 -硝基苯基β-D-吡喃半乳糖苷（ONPG）和5-溴-4-氯-3-吲哚基β-D-吡喃半乳糖苷（X…
Representative Results
从这份报告中所描述的系统获得数据的稳健性的级别如图2所示AF 38。通过使用这种系统的与病毒感染的细胞和它们彼此的粘附效果葡萄球菌和真菌相互作用的调制可以划定。这些类型的研究需要的相互作用的显微镜检查中，以确定多种微生物相互作用是否在相同的细胞产生的示于图3和4 38。在这项研究中?…
Discussion
目前暂无信息可到跨越多个域分类， 即原核，真核和病毒宿主微生物的半常任理事国，常任之间复杂的相互作用。因此，我们开发了一种新的体外模型系统来研究由S.生物膜启动金黄色葡萄球菌和C.白色念珠菌对HSV-1和HSV-2感染的HeLa 229（HeLa细胞）细胞38。 HeLa细胞模型系统呈现出独特的优势。这是由于它们缺乏表面纤连蛋白表达的，作为两个S的受体…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
This project was supported by Midwestern University, IL Office of Research and Sponsored Programs (ORSP) and Midwestern University College of Dental Medicine-Illinois (CDMI).
Materials
| C.albicans | |||
| BBL Sabouraud Dextrose | BD | 211584 | |
| Fungisel Agar | Dot Scientific | 7205A | |
| S.aureus | |||
| Mannitol Salt Agar | Troy Biologicals | 7143B | |
| Sheep blood agar | Troy Biologicals | 221239 | |
| Hela cells | |||
| 1xDMEM (Dubelcco's Modified Eagle Medium, with 4.5 g/L glucose and L-glutamine, without sodium pyruvate | Corning | 10-017-CM | |
| Gentamicin 50mg/ml | Sigma | 1397 | 50µg/ml final concentration in the complete DMEM |
| Trypsin EDTA (0.05% Trypsin, 0.53M EDTA)Solution 1X | Corning | 25-052-CI | |
| Fetal Bovine Serum | Atlanta Biologicals | S11150 | 10% final concentration in the complete DMEM |
| Other medium and reagents | |||
| ONPG | Thermo Scientific | 34055 | |
| Ultra-Pure X gal | Invitrogen | 15520-018 | |
| 1x HBSS (Hanks' Balanced Salt Solution) | Corning | 20-021-CV | |
| 1XPBS | Dot Scientific | 30042-500 | |
| RIPA Lysis | Life Technologies | 89901 | |
| Staining | |||
| Methanol | Fisher Scientific | A433P-4 | |
| HSV 1&2, specific for gD | ViroStat | 196 | |
| DAPI | SIGMA | D8417-5MG | |
| Gram Crystal Violet | Troy Biologicals | 212527 | |
| Supplies | |||
| Petri dish 100X15 | Dot Scientific | 229693 | |
| Petri dish 150X15 | Kord Valmark | 2902 | |
| 96-Well plates | Evergreen Scientific | 222-8030-01F | |
| 24-well plates | Evergreen Scientific | 222-8044-01F | |
| Culture tubes 100×13 | Thomas Scientific | 9187L61 | |
| Cover slip circles, 12mm | Deckglaser | CB00120RA1 |
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Citazione di questo articolo
Plotkin, B. J., Sigar, I. M., Tiwari, V., Halkyard, S. Determination of Biofilm Initiation on Virus-infected Cells by Bacteria and Fungi. J. Vis. Exp. (113), e54162, doi:10.3791/54162 (2016).