一种基于四唑的可溶性还原测定，用于评估抗体对 热带念珠菌 生物膜的影响
Summary
本文描述了一种基于96孔微量滴定板的方案，使用2，3-双（2-甲氧基-4-硝基-5-磺基苯基）-5-甲苯胺-2H-四唑（XTT）还原测定，以研究抗体对 热带梭菌形成的生物膜的影响。该 体外 方案可用于检查潜在的新抗真菌化合物对生物膜中 念珠菌 属细胞代谢活性的影响。
Abstract
念珠菌 属是系统性院内感染的第四大常见原因。全身性或侵袭性念珠菌病通常涉及植入装置或导管上的生物膜形成，这与毒力和死亡率增加有关。由不同 念珠菌 物种产生的生物膜对各种抗真菌药物表现出增强的抗性。因此，有必要开发针对 念珠菌 生物膜的有效免疫疗法或辅助治疗。虽然细胞免疫在抗念珠菌 保护中的作用已得到充分证实，但体液免疫的作用研究较少。
据推测，抑制生物膜形成和成熟是保护性抗体的主要功能之一，白色念珠菌胚管抗体（CAGTA）已被证明可以更早地抑制白色念珠菌的体外生长和生物膜形成。本文概述了评估抗体对热带梭菌形成的生物膜的作用的详细方案。该协议的方法涉及在96孔微量滴定板中形成热带梭菌生物膜，然后在存在或不存在抗原特异性抗体的情况下孵育，然后进行2，3-双（2-甲氧基-4-硝基-5-磺基苯基）-5-羧苯胺-2H-四唑（XTT）测定，用于测量生物膜中真菌细胞的代谢活性。
通过使用适当的血清对照（包括去除Sap2特异性抗体的血清）证实了特异性。结果表明，免疫动物血清中存在的抗体可以在体外抑制念珠菌生物膜成熟。总之，本文为抗体在开发侵袭性念珠菌病期间针对生物膜的新型免疫疗法和协同或辅助治疗的潜力提供了重要的见解。该体外方案可用于检查潜在的新抗真菌化合物对生物膜中念珠菌属细胞代谢活性的影响。
Introduction
系统性念珠菌病是院内感染的第四大原因，与全世界的高发病率和死亡率有关。在全球范围内，系统性念珠菌病影响约70万人1。念珠菌属，即白色念珠菌、热带念珠菌、副念珠菌、光滑念珠菌和耳念珠菌，是侵袭性 念珠菌感染的最常见原因2。念珠菌属是产生生物膜的机会性病原体3.生物膜主要与念珠菌毒力有关，念珠菌可以通过诱导生物膜形成来承受氧化和渗透应激条件4。生物膜进一步调节毒力因子和细胞壁成分的表达，形成外聚物保护基质，帮助念珠菌适应不同的宿主生态位4。生物膜有助于酵母粘附在宿主组织和医疗器械上5.因此，生物膜的形成与酵母的优势有关，因为生物膜内的酵母细胞可以逃避宿主免疫反应6。生物膜的形成还可以保护致病酵母免受抗真菌药物的作用5。Pierce等人已经证明了白色念珠菌生物膜对两性霉素B的敏感性降低7，8。此外，生物膜显示出对氟康唑的抗真菌药物耐药性，这损害了系统性念珠菌病的有效管理9，10。
微生物具有粘附在各种生物和非生物表面的内在倾向，从而导致生物膜的形成。 白色念珠菌是一种二态性真菌，以酵母和菌丝形式存在，其生物膜形成已在各种体外和体内模型系统中表征11。生物膜形成的步骤包括念珠菌细胞与底物的粘附、细丝化、增殖和生物膜成熟11。最初，白色念珠菌的酵母形式粘附在基质上，包括医疗器械和人体组织，然后是白色念珠菌的丝化和增殖成菌丝和假菌丝形式，最后是嵌入细胞外基质的生物膜成熟11。生物膜的形成在很大程度上有助于白色念珠菌的发病机制12。念珠菌物种形成耐药生物膜，这使得根除它们具有挑战性13。白色念珠菌生物膜产生人群的一小部分已被描述为对抗真菌药物两性霉素 B 和氯己定14 具有高度耐药性。值得注意的是，与浮游阶段和增殖阶段的酵母细胞相比，生物膜中的酵母细胞对多药治疗表现出高抗性14。有人提出，存在于生物膜中的酵母细胞对抗真菌药物具有高度耐受性，这有助于白色念珠菌在生物膜中的存活14。据报道，这些现有细胞是白色念珠菌的表型变体，而不是突变体14。此外，被称为“持久细胞”的念珠菌生物膜细胞可耐受高剂量的两性霉素-B治疗并有助于念珠菌存活，从而对高风险个体反复出现的全身性念珠菌感染构成巨大负担15。
念珠菌菌株中抗真菌耐药性的增加需要研究新的抗真菌药物和免疫疗法。从上述研究中可以明显看出，念珠菌生物膜显示出对抗真菌药物的敏感性降低。因此，需要改进免疫疗法来控制念珠菌生物膜的形成。早期的研究表明，CAGTA可以通过在体外抑制白色念珠菌生物膜的形成来提供有效的保护，以防止全身性念珠菌感染16。另一项研究报告说，用白色念珠菌rAls3-N蛋白免疫小鼠诱导高抗体滴度，干扰白色念珠菌生物膜的体外形成17。 抗Als3-N抗体也对白色念珠菌从生物膜中扩散产生抑制作用17。基于白色念珠菌的NDV-3A疫苗目前正在临床试验中，还发现抗NDV-3A血清可减少耳念珠菌生物膜的形成18。最近的一项研究发现，Sap2抗体抑制生物膜形成是系统性念珠菌病小鼠模型中的保护机制19。
本文概述了详细的 体外 方案，用于评估从不同组Sap2疫苗接种小鼠获得的多克隆血清中存在的抗原特异性抗体对预制热带 念珠菌 生物膜的影响。为了实现这一目标，在实验室中优化并开发了一种基于XTT还原测定的方法，该方法可以在存在或不存在抗体的情况下以快速，灵敏和高通量的方式测量生物膜活力。
XTT 测定用于测量细胞代谢活性，作为细胞活力、细胞增殖和细胞毒性的指标20。该比色测定基于通过代谢活性细胞将黄色四唑盐钠 3′-[1-（苯氨基羰基）-3，4-四唑]-双（4-甲氧基-6-硝基）苯磺酸水合物 （XTT） 还原为橙色甲臜染料。由于只有活细胞才能降低XTT，因此还原的XTT甲臜的量与颜色强度和细胞活力成正比。形成的甲臜染料是水溶性的，使用酶标仪直接定量。由于其水溶性，XTT测定允许研究完整的生物膜，以及检查生物膜药物敏感性，而不会破坏生物膜结构21。此外，该方法由于其易用性、速度、准确性、高通量和高重现性而用于念珠菌真菌活力评估7，22。
除了 XTT 还原测定外，还确定了用于测量生物膜数量的许多替代技术。其中一些包括使用 MTT 还原测定、结晶紫染色、DNA 定量、定量 PCR、蛋白质定量、干细胞重量测量和活菌落计数。这些程序在时间和成本要求方面差异很大。Taff 等人对七种不同的 念珠 菌生物膜定量测定进行了比较分析，发现 XTT 测定为 定量估计白色念珠菌 生物膜提供了最可重复、最准确和最有效的方法23。结晶紫等染色技术有一定的局限性;结晶紫测试通过测量结晶紫染色的生物膜基质和细胞的光密度间接确定生物膜的量。尽管结晶紫测定提供了生物膜质量的良好测量，但它不能测量生物膜活力，因为它会染色微生物细胞和细胞外基质24。Dhale等人进一步报告说，与结晶紫测定25相比，XTT还原测定是检测生物膜产生的最灵敏，可重复，准确，有效和特异性的方法。文献报告表明，XTT 测定与 CFU 计数方法中的 CFU/mL 参数密切相关。然而，与XTT测定相比，CFU方法是劳动密集型且缓慢的26。此外，分离的活细胞的比例可能不代表初始生物膜群27。尽管 XTT 还原测定似乎是量化活力的最佳选择，但该技术存在一些局限性。虽然XTT方法对于涉及一种真菌菌株的比较很有用，但在比较不同的真菌菌株和物种时，其使用可能会受到限制。在没有详细标准化的情况下，菌株间比较可能很困难，因为不同的菌株代谢具有不同能力的底物21。
Protocol
Representative Results
Discussion
由念珠菌属引起的真菌感染与全世界的高发病率和死亡率有关。侵袭性真菌感染的威胁日益严重，因此需要及早管理这种危及生命的疾病。大多数念珠菌感染涉及生物膜的形成，生物膜粘附在各种医疗设备上，并导致医院环境中真菌感染的持续和复发31。生物膜由酵母或菌丝细胞组成，它们对大多数常规抗真菌药物表现出相当大的抗性32。念珠?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
这项工作得到了Ramalingaswami赠款DBT-843-BIO（印度政府生物技术部）和早期职业研究奖SER-1058-BIO（印度政府科学与工程研究委员会）的支持。作者承认ICMR-JRF向P.C.提供赠款，DBT-JRF向P.S.提供赠款。作者感谢Ravikant Ranjan博士在SEM期间对手稿和Pradeep Singh Thakur先生的手稿和技术援助提出的建议。
Materials
| 15 mL conical centrifuge tubes | BD Falcon | 546021 | |
| 1x PBS | – | Prepared in lab | NaCl : 4 g KCl : 0.1 g Na2HPO4: 0.72 g KH2PO4 : 0.12 g Water 500 mL. Adjust pH to 7.4 |
| 50 mL conical centrifuge tubes | BD Falcon | 546041 | |
| 96-well microtiter plates | Nunc | 442404 | |
| Incubator | Generic | ||
| Menadione | Sigma | M5625 | |
| Microtiter Plate Reader | Generic | ||
| Multichannel pipette and tips | Generic | ||
| Petri dishes | Tarson | 460090 | |
| Ringers Lactate | – | Prepared in lab | sodium chloride 0.6 g sodium lactate 0.312 g potassium chloride 0.035 g calcium chloride 0.027 g Water 100 mL. Adjust to pH 7.0 |
| RPMI 1640 MOPS | Himedia | AT180 | |
| Sabouraud dextrose Agar | SRL | 24613 | |
| Sabouraud dextrose Broth | SRL | 24835 | |
| XTT | Invitrogen | X6493 |
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