对作者的支持包括马萨诸塞州总医院儿科、研究临时支持基金执行委员会 （ISF） 奖 2022A009041、美国国家过敏和传染病研究所资助 R21AI146405 以及美国国家糖尿病、消化和肾脏疾病研究所资助哈佛大学营养肥胖研究中心 （NORCH） 2P30DK040561-26。资助者在研究设计、数据收集和分析、发表决定或手稿准备方面没有任何作用。

分析结肠上皮细胞中 志贺氏菌 感染的方案

作者声明没有利益冲突。

该方案描述了一组三种标准化测定法，用于研究志贺氏菌的粘附，侵袭和肠上皮细胞的细胞内复制。尽管这些方法只是用于研究宿主细胞内各种细菌病原体的侵袭和细胞内复制的经典庆大霉素测定法的改进版本49,50,51，但在研究志贺氏菌时必须特别考虑。
志贺氏菌是兼性厌氧菌，在37°C...

由肠道细菌病原体引起的腹泻病是全球卫生的重大负担。2016 年，腹泻病导致全球 130 万人死亡，是 5 岁以下儿童的第四大死因 1,2。革兰氏阴性肠道细菌病原体志贺氏菌是志贺氏菌病的病原体，志贺氏菌病是全球腹泻死亡的主要原因3。志贺菌病每年在低收入和中等收入国家的儿童中造成严重的发病率和死亡率4,5，而高收入国家的感染与日托中心、食源性和水源性疫情有关6,7,8,9。无效的疫苗开发10和抗微生物药物耐药性（AMR）11,12的上升使大规模志贺氏菌疫情的管理变得复杂。美国疾病控制与预防中心最近的数据显示，2020 年美国近 46% 的志贺氏菌感染表现出耐药性13,14，而世界卫生组织已宣布志贺氏菌为 AMR 优先病原体，迫切需要新疗法15。
志贺氏菌感染在摄入受污染的食物或水后，或通过直接与人接触，很容易通过粪口途径传播。志贺氏菌已进化为一种有效的、适应人类的病原体，其感染剂量为 10-100 种细菌，足以引起疾病16。在小肠转运过程中，志贺氏菌暴露于环境信号，例如高温和胆汁17。检测这些信号可诱导转录变化以表达毒力因子，从而增强细菌感染人结肠的能力 17,18,19。志贺氏菌不会从顶端表面侵入结肠上皮，而是在被滤泡相关上皮细胞内特化的抗原呈递微折叠细胞（M 细胞）摄取后穿过上皮层20、21、22。转胞吞作用后，志贺氏菌细胞被驻留巨噬细胞吞噬。志贺氏菌迅速逃离吞噬体并触发巨噬细胞死亡，导致促炎细胞因子的释放 5,23,24。然后志贺氏菌从基底外侧侵入结肠上皮细胞，裂解巨细胞液泡，并在细胞质中建立复制生态位 5,25。促炎细胞因子，特别是白细胞介素-8 （IL-8），将多形核中性粒细胞白细胞 （PMN） 募集到感染部位，从而削弱上皮紧密连接，并使细菌浸润上皮内膜以加剧基底外侧感染5。PMN 破坏受感染的上皮内膜以控制感染，从而导致细菌性（血性）痢疾的特征性症状5。尽管侵袭和细胞内复制机制已被彻底表征，但新的研究正在证明志贺氏菌感染的重要新概念，包括胃肠道 （GI） 转运期间的毒力调节17、依从性19、通过屏障通透性改善基底外侧通路26 和营养不良儿童的无症状携带27。
志贺氏菌属引起腹泻病的能力仅限于人类和非人灵长类动物 （NHP）28。已经为斑马鱼29、小鼠30、豚鼠31、兔子21、32、33 和猪34、35 开发了志贺氏菌肠道感染模型。然而，这些模型系统都不能准确复制人类感染期间观察到的疾病特征36。尽管已经建立了志贺氏菌病的非人灵长类模型来研究志贺氏菌的发病机制，但这些模型系统的实施成本高昂，并且需要人为的高感染剂量，比人类的感染剂量高出9个数量级37,38,39,40,41,42。因此，志贺氏菌对人类宿主感染的显着适应需要使用人源性细胞培养物来重建生理相关模型，以准确询问志贺氏菌的发病机制。
在这里，描述了详细的程序，以测量 志贺氏菌 对 HT-29 结肠上皮细胞的粘附、侵袭和复制率。使用这些标准化方案，可以研究细菌毒力基因和环境信号影响 志贺氏菌 感染每一步的分子机制，以更好地了解动态宿主-病原体相互作用关系。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22 &#956;m PES filter</td>
			<td>Millipore-Sigma</td>
			<td>SCGP00525</td>
			<td>Sterile, polyethersulfone filter for sterilizing up to 50 mL media</td>
		</tr>
		<tr>
			<td>14 mL culture tubes</td>
			<td>Corning</td>
			<td>352059</td>
			<td>17 mm x 100 mm polypropylene test tubes with cap</td>
		</tr>
		<tr>
			<td>50 mL conical tubes</td>
			<td>Corning</td>
			<td>430829</td>
			<td>50 mL clear polypropylene conical bottom centrifuge tubes with leak-proof cap</td>
		</tr>
		<tr>
			<td>6-well tissue culture plates</td>
			<td>Corning</td>
			<td>3516</td>
			<td>Plates are treated for optimal cell attachment</td>
		</tr>
		<tr>
			<td>Bile salts</td>
			<td>Sigma-Aldrich</td>
			<td>B8756</td>
			<td>1:1 ratio of cholate to deoxycholate</td>
		</tr>
		<tr>
			<td>Congo red dye</td>
			<td>Sigma-Aldrich</td>
			<td>C6277</td>
			<td>A benzidine-based anionic diazo dye, &#62;85% purity</td>
		</tr>
		<tr>
			<td>Countess cell counting chamber slide</td>
			<td>Invitrogen</td>
			<td>C10283</td>
			<td>To be used with the Countess Automated Cell Counter</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D8418</td>
			<td>A a highly polar organic reagent</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle Medium (DMEM)</td>
			<td>Gibco</td>
			<td>10569-010</td>
			<td>DMEM is supplemented with high glucose, sodium pyruvate, GlutaMAX, and Phenol Red</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Sigma-Aldrich</td>
			<td>F4135</td>
			<td>Heat-inactivated, sterile</td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Sigma-Aldrich</td>
			<td>G3632</td>
			<td>Stock concentration is 50 mg/mL</td>
		</tr>
		<tr>
			<td>HT-29 cell line</td>
			<td>ATCC</td>
			<td>HTB-38</td>
			<td>Adenocarcinoma cell line; colorectal in origin</td>
		</tr>
		<tr>
			<td>Paraffin film</td>
			<td>Bemis</td>
			<td>PM999</td>
			<td>Laboratory sealing film</td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>Thermo Fisher Scientific</td>
			<td>FB0875713</td>
			<td>100 mm x 15 mm Petri dishes for solid media</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>10010049</td>
			<td>1x concentration; pH 7.4</td>
		</tr>
		<tr>
			<td>Select agar</td>
			<td>Invitrogen</td>
			<td>30391023</td>
			<td>A mixture of polysaccharides extracted from red seaweed cell walls to make bacterial plating media</td>
		</tr>
		<tr>
			<td>T75 flasks</td>
			<td>Corning</td>
			<td>430641U</td>
			<td>Tissue culture flasks</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td>A common non-ionic surfactant and emulsifier&#160;</td>
		</tr>
		<tr>
			<td>Trypan blue stain</td>
			<td>Invitrogen</td>
			<td>T10282</td>
			<td>A dye to detect dead tissue culture cells; only live cells can exclude the dye</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td>Reagent for cell dissociation for cell line maintenance and passaging</td>
		</tr>
		<tr>
			<td>Tryptic Soy Broth (TSB)</td>
			<td>Sigma-Aldrich</td>
			<td>T8907</td>
			<td>Bacterial growth media</td>
		</tr>
	</tbody>
</table>

epithelial cell infection analyses with shigella

人类适应的肠道细菌病原体志贺氏菌每年引起数百万例感染，在儿科患者中产生长期生长效应，并且是全球腹泻死亡的主要原因。由于病原体通过胃肠道并感染结肠内壁的上皮细胞，感染会引起水样或血性腹泻。随着抗生素耐药性的惊人增加和目前缺乏批准的疫苗，标准化的研究方案对于研究这种可怕的病原体至关重要。在这里，提出了使用结肠上皮细胞中细菌粘附、侵袭和细胞内复制的体外分析来检查志贺氏菌分子发病机制的方法。在感染分析之前，志贺氏菌菌落的毒力表型通过在琼脂平板上摄取刚果红染料来验证。在细菌培养过程中，也可以考虑使用补充的实验室培养基来模拟体内条件。然后，在标准化方案中使用细菌细胞，以已建立的感染多重感染感染组织培养板中的结肠上皮细胞，并适应分析感染的每个阶段。对于依从性测定，志贺氏菌细胞与降低的培养基水平一起孵育，以促进细菌与上皮细胞的接触。对于侵袭和细胞内复制测定，庆大霉素应用于不同的时间间隔，以消除细胞外细菌并能够评估侵袭和/或细胞内复制速率的定量。所有感染方案都通过连续稀释受感染的上皮细胞裂解物并相对于感染滴度在刚果红琼脂平板上铺板细菌集落形成单位来枚举贴壁、侵袭和/或细胞内细菌。总之，这些方案能够对上皮细胞志贺氏菌感染的每个阶段进行独立的表征和比较，以成功研究这种病原体。

Diarrheal diseases caused by enteric bacterial pathogens are a significant global health burden. In 2016, diarrheal diseases were responsible for 1.3 million deaths worldwide and were the fourth leading cause of death in children younger than five years of age1,2. The Gram-negative, enteric bacterial pathogen Shigella is the causative agent of shigellosis, a major cause of diarrheal deaths worldwide3. Shigellosis causes significant morbidity and mortality each year in children from lower- and middle-income countries4,5, while infections in high-income countries are linked to daycare center, foodborne, and waterborne outbreaks6,7,8,9. Ineffective vaccine development10 and rising rates of antimicrobial resistance (AMR)11,12 have complicated the management of large-scale Shigella outbreaks. Recent Centers for Disease Control and Prevention data show that nearly 46% of Shigella infections in the United States displayed drug resistance in 202013,14, while the World Health Organization has declared Shigella as an AMR priority pathogen for which new therapies are urgently needed15.
Shigella infections are easily transmitted via the fecal-oral route upon ingestion of contaminated food or water, or through direct human contact. Shigella has evolved to be an efficient, human-adapted pathogen, with an infectious dose of 10-100 bacteria sufficient to cause disease16. During small intestinal transit, Shigella is exposed to environmental signals, such as elevated temperature and bile17. Detection of these signals induces transcriptional changes to express virulence factors that enhance the ability of the bacteria to infect the human colon17,18,19. Shigella does not invade the colonic epithelium from the apical surface, but rather transits across the epithelial layer following uptake into specialized antigen-presenting microfold cells (M cells) within the follicle-associated epithelium20,21,22. Following transcytosis, Shigella cells are phagocytosed by resident macrophages. Shigella rapidly escapes the phagosome and triggers macrophage cell death, resulting in the release of pro-inflammatory cytokines5,23,24. Shigella then invades colonic epithelial cells from the basolateral side, lyses the macropinocytic vacuole, and establishes a replicative niche in the cytoplasm5,25. Pro-inflammatory cytokines, particularly interleukin-8 (IL-8), recruit polymorphonuclear neutrophil leukocytes (PMNs) to the site of infection, which weakens epithelial tight junctions, and enables bacterial infiltration of the epithelial lining to exacerbate basolateral infection5. The PMNs destroy the infected epithelial lining to contain the infection, which results in the characteristic symptoms of bacillary (bloody) dysentery5. Although invasion and intracellular replication mechanisms have been thoroughly characterized, new research is demonstrating important new concepts in Shigella infection, including virulence regulation during gastrointestinal (GI) transit17, adherence19, improved basolateral access through barrier permeability26, and asymptomatic carriage in malnourished children27.
The ability of Shigella spp. to cause diarrheal disease is restricted to humans and non-human primates (NHP)28. Shigella intestinal infection models have been developed for zebrafish29, mice30, guinea pigs31, rabbits21,32,33, and pigs34,35. However, none of these model systems can accurately replicate the disease characteristics observed during human infection36. Although NHP models of shigellosis have been established to study Shigella pathogenesis, these model systems are expensive to implement and require artificially high infectious doses, up to nine orders of magnitude higher than the infectious dose of humans37,38,39,40,41,42. Thus, the remarkable adaptation of Shigella for infection of human hosts necessitates the use of human-derived cell cultures to recreate physiologically relevant models for accurate interrogation of Shigella pathogenesis.
Here, detailed procedures are described to measure the rates of Shigella adherence to, invasion of, and replication within HT-29 colonic epithelial cells. Using these standardized protocols, the molecular mechanisms by which bacterial virulence genes and environmental signals impact each step of Shigella infection can be interrogated to better understand the dynamic host-pathogen interaction relationship.

作者聚焦：了解和对抗 志贺氏菌 感染的进展

志贺氏菌上皮细胞感染分析

Shigella is a significant global pathogen that causes devastating infection rates every year. Shigella infects the gastrointestinal tract to cause diarrhea or dysentery. Our research aims to improve our understanding of infection to help develop more effective therapeutics like vaccines and other antimicrobial products.First, animal models and emerging tissue culture techniques allow us to better understand the physiology of Shigella infection and faithfully reproduce the complexity of the human GI tract. Second, characterization of clinical Shigella isolates has helped to capture the diversity of this genus, identify important virulence genes, and improve our understanding of infection. We have used gastrointestinal signals like bile salts and glucose to understand how Shigella survives transit in the small intestine and regulates virulence gene expression for infection in the large intestine.We have demonstrated how Shigella resists bile salts and have shown that Shigella produces adherence proteins to help initiate contact with epithelial cells to start infection. These protocols are designed to look at key aspects of epithelial infection like adherence, invasion, and intracellular survival of Shigella. We are performing these protocols using a standard epithelial cell line, but have also adapted the methods for sophisticated human GI models that are now available.These protocols consider each phase of infection independently, which is critical to understand the key steps in Shigella infection. They also highlight that combined analyses can facilitate a greater understanding of Shigella infection at a holistic level.

将 S. flexneri 2457T 野生型 （WT） 与 S. flexneri ΔVF （ΔVF） 进行比较，后者是一种假设负调节志贺氏菌毒力的突变体。由于志贺氏菌使用胆盐作为调节毒力的信号17,18,47，因此在TSB培养基中的细菌传代培养以及补充有0.4%（w/v）胆盐的TSB中进行实验18。在传代培养步骤中暴露胆汁盐...

Watch this Scientific Journal Video about Epithelial Cell Infection Analyses with Shigella at JoVE.com

本方案描述了使用体外上皮细胞系询问志贺氏菌粘附、侵袭和细胞内复制的感染测定。

The human-adapted enteric bacterial pathogen Shigella causes millions of infections each year, creates long-term growth effects among pediatric patients, ...

志贺氏菌是一种重要的全球病原体，每年都会造成毁灭性的感染率。志贺氏菌感染胃肠道引起腹泻或痢疾。我们的研究旨在提高我们对感染的理解，以帮助开发更有效的治疗方法，如疫苗和其他抗菌产品。首先，动物模型和新兴的组织培养技术使我们能够更好地了解志贺氏菌感染的生理学，并忠实地再现人类胃肠道的复杂性。其次，临床志贺氏菌分离株的表征有助于捕获该属的多样性，鉴定重要的毒力基因，并提高我们对感染的理解。我们使用胆汁盐和葡萄糖等胃肠道信号来了解志贺氏菌如何在小肠中存活并调节大肠感染的毒力基因表达。我们已经证明了志贺氏菌如何抵抗胆盐，并表明志贺氏菌产生粘附蛋白以帮助启动与上皮细胞的接触以开始感染。这些方案旨在研究上皮感染的关键方面，如志贺氏菌的粘附、侵袭和细胞内存活。我们正在使用标准的上皮细胞系执行这些方案，但也调整了现在可用的复杂人类胃肠道模型的方法。这些方案独立考虑感染的每个阶段，这对于了解志贺氏菌感染的关键步骤至关重要。他们还强调，综合分析可以促进从整体层面更好地了解志贺氏菌感染。

epithelial-cell-infection-analyses-with-shigella

Research

JoVE Journal

Immunology and Infection

Epithelial Cell Infection Analyses with Shigella

551 次观看.  Massachusetts General Hospital. 志贺氏菌是一种重要的全球病原体，每年都会造成毁灭性的感染率。志贺氏菌感染胃肠道引起腹泻或痢疾。我们的研究旨在提高我们对感染的理解，以帮助开发更有效的治疗方法，如疫苗和其他抗菌产品。首先，动物模型和新兴的组织培养技术使我们能够更好地了解志贺氏菌感染的生理学，并忠实地再现人类胃肠道的复杂性。其次，临床志贺氏菌分离株的表征有助于捕?...

作者聚焦：了解和对抗 志贺氏菌 感染的进展

作者聚焦：了解和对抗志贺氏菌感染的进展