方法文章

一种优化的实验方案 Candida albicans 感染在 Schmidtea mediterranea 研究真菌致病机制与宿主防御

DOI：

10.3791/70500

⸱

2026年4月17日

 ,  ,  ,  ,  , 
1Department of Molecular and Cell Biology, University of California, Merced, 2Quantitative and Systems Biology Graduate Program, University of California, Merced, 3Health Sciences Research Institute, University of California, Merced, 4Center for Cellular and Biomolecular Machines, University of California, Merced

* These authors contributed equally

本文内容

摘要

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本方案提供了利用地中海涡虫（Schmidtea mediterranea）作为模式系统研究真菌感染过程中宿主-病原体相互作用的优化且详细的指南。该方法在先前使用人类真菌病原体感染涡虫的实验流程基础上进行了改进。 白色念珠菌，提供详细的指导以提高实验的可重复性和一致性。

摘要

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Candida albicans 是一种常见的人类机会性真菌病原体，可在无症状状态下定植于大多数人。尽管通常为良性共生菌，但因抗生素使用、免疫功能障碍或上皮屏障破坏所导致的菌群失调，可能引发真菌过度增殖和感染，其严重程度可从浅表黏膜疾病发展至危及生命的全身性念珠菌病。目前亟需新的临床前感染模型，以在不同感染阶段和可量化的宿主反应条件下，深入解析 C. albicans 在 体内 的致病机制。先前研究已确立扁形动物地中海涡虫 Schmidtea mediterranea 作为研究 C. albicans 感染过程中宿主-病原体相互作用的无脊椎动物宿主模型。S. mediterranea 完全依赖保守的先天免疫机制来抵御包括细菌和真菌在内的病原微生物感染。涡虫具有强大的再生能力以及易于研究的干细胞群体，使其成为在 体内 分析早期免疫应答、组织修复及病原体清除的可行模型。该模型支持对真菌毒力和宿主转录反应进行同步分析，为感染动态提供重要见解。本文介绍了一种经过更新的实验方案，包含详细的改进措施、标准化操作流程以及优化的 C. albicans 感染 S. mediterranea 步骤，旨在提高实验可重复性，并推动对真菌致病机制与宿主防御的系统性研究。

引言

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Candida albicans 是一种常见的机会性真菌病原体，可无症状定植于50-70%的人群1,2,3。通常作为一种无害的共生菌，C. albicans 在微生物群失衡、使用抗生素、免疫抑制、上皮屏障破坏或存在医疗装置等条件下可能发生过度增殖，从而引发从浅表黏膜感染到危及生命的全身性念珠菌病等一系列疾病4,5。在免疫功能受损个体中——包括HIV/AIDS患者、接受化疗或免疫抑制治疗的患者、慢性病患者以及重症监护病房中的患者——死亡率可超过70%，凸显了侵袭性念珠菌病对公共卫生造成的重大负担6,7,8。全球范围内，侵袭性真菌感染每年影响超过650万人，仅美国与Candida相关的感染每年直接医疗费用就超过80亿美元9,10。尽管已有抗真菌治疗方法，但数十年来由C. albicans感染引起的发病率和死亡率基本未变，突显了深入理解真菌疾病中宿主-病原体相互作用的迫切需求。

C. albicans 采用多种毒力因子，以支持其在多种宿主环境中的定植、持续存在和致病过程。这些因子包括对宿主组织的黏附、免疫逃逸、细胞壁重塑、水解酶的分泌、形态可塑性以及生物膜的形成11,12,13,14,15,16。这些特性共同使 C. albicans 能够适应不同的宿主微环境，调节免疫反应，并抵抗抗真菌治疗16,17,18,19,20,21。阐明这些过程如何影响感染结局——包括宿主的发病率、死亡率以及病原体清除情况——对于开发更有效的预防和治疗策略至关重要。

为了研究这些宿主-病原体相互作用，我们建立了一个以涡虫Schmidtea mediterranea为宿主的模型系统22,23,24。该模型为研究真菌感染过程中宿主的多系统反应提供了一个多功能平台，利用基于浸泡的感染方法，使动物能够同步暴露于C. albicans。下文介绍了对该方案的更新版分步操作流程，旨在优化实验步骤、提高可重复性，并促进不同实验室之间的一致性实施。

涡虫是一类自由生活的无脊椎动物，具有卓越的再生能力，能够快速替换因损伤或感染而丢失的组织25。它们缺乏适应性免疫系统，完全依赖保守的先天防御机制——包括模式识别受体、抗菌肽、黏液分泌以及吞噬细胞——在数天内清除细菌和真菌病原体26,27,28,29。由于先天免疫是真核生物普遍存在的第一道防线，且在哺乳动物体系之外的研究仍不充分27，涡虫为在活体（in vivo）条件下以细胞和分子分辨率研究这些过程提供了理想模型。其再生能力由成体多能干细胞（称为新胚细胞）驱动，这类细胞约占成体细胞总数的30%，并在感染后的恢复过程中发挥重要作用23,30。这些特性使得S. mediterranea成为在基因、细胞、组织和个体等多个尺度上解析病原体毒力与宿主防御机制的强有力模型，使我们能够探究传统哺乳动物系统中难以研究的诸多科学问题。

此外，涡虫体型微小（通常体长为几毫米）、成本低廉且易于培养31，适合开展大规模实验，同时避免了脊椎动物模型所固有的经费、伦理和监管限制。与C. albicans类似，S. mediterranea具有可操作的基因组，其基因组已实现全序列测定和注释，并支持多种先进的分子与细胞技术——包括转录谱分析、高分辨率免疫组织化学和组织学染色，以及高效的RNA干扰（RNAi）技术32,33,34,35,36,37,38。这些工具可实现对感染过程中宿主与病原体反应的并行分析。

已有多种替代性临床前模型被用于研究真菌感染。无脊椎动物宿主如大蜡螟（Galleria mellonella）、秀丽隐杆线虫（Caenorhabditis elegans）和黑腹果蝇（Drosophila melanogaster），以及脊椎动物斑马鱼（Danio rerio），各自具有独特的实验优势，但也存在明显局限性。这些系统通常依赖存活率作为主要终点指标，因为感染会迅速导致宿主死亡，从而无法对疾病严重程度、恢复过程及多系统反应进行详细评估——而这些特征恰恰可被涡虫模型有效捕捉。

哺乳动物模型，如Mus musculus（小鼠）、Rattus norvegicus（大鼠）、Oryctolagus cuniculus（兔）和Cavia porcellus（豚鼠），在模拟人类生理方面更为接近，但受限于高昂的成本、较小的群体规模、伦理与监管要求，以及难以区分固有免疫与适应性免疫应答。此外，数十年来针对小鼠的真菌致病机制研究主要集中于晚期全身性感染39,40,41,42,43，常常忽视了早期事件，如上皮屏障破坏和黏膜过度增殖——这些正是人类真菌疾病最常见的起始途径44,45,46。

因此，研究宿主-病原体相互作用对于理解真菌毒力、宿主防御及疾病进展背后的生物学过程至关重要。侵袭性真菌感染正成为日益严重的全球性威胁，多种真菌已对所有主要类别的抗真菌药物产生耐药性。据估计，超过10亿人受到真菌感染的影响，而气候变化、抗菌药物的广泛使用以及免疫功能低下人群的不断增加，正在加速抗真菌耐药病原体的出现47,48,49,50。深入了解致病性真菌如何定植宿主并与其相互作用，对于开发新的预防和治疗策略至关重要。

以下介绍一种经过修订和标准化的系统性感染方案，用于研究S. mediterranea与C. albicans之间的相互作用，旨在提高实验可重复性，并系统性地探究真菌致病机制及宿主防御机制。此前的研究出版物中采用的是原始方案22,23,24。表1总结了本修订版中纳入的关键更新内容，包括真菌接种程序的改进、孔板体积的减少、明确的动物纳入标准、感染剂量与致死剂量的确定，以及动物饲养建议的更新。该实验流程为C. albicans的培养、通过浸泡暴露法感染涡虫，以及在症状性和致死性剂量条件下，利用定性和定量指标评估真菌毒力和宿主反应提供了详细指导（图1）。此处优化的实验步骤整合了经实验证实为影响感染动态和宿主结局的关键变量，从而提高了实验的可重复性，并支持对宿主–病原体相互作用进行系统性评估。

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方案

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This protocol builds on the previous methods22,23,24 and establishes clear, standardized procedures to enhance reproducibility and enable systematic investigation of fungal pathogenesis and host responses in S. mediterranea. The key protocol refinements include prespecified animal inclusion and exclusion criteria, standardized microbial preparation, and comprehensive assessment of host endpoints.

All procedures must be performed using appropriate sterile and aseptic biosafety level 2 techniques. Standard laboratory personal protective equipment (PPE), including gloves, a lab coat, and closed-toe shoes, should be worn at all times. Procedures involving the planarian S. mediterranea were conducted in accordance with institutional guidelines for the ethical use of invertebrates.

All protocol refinements are summarized in Table 1. All reagents, critical equipment, and media, buffers, and immunostaining recipes are listed in the Table of Materials and Table 2.
NOTE: All procedures involving the invertebrate animal model S. mediterranea should be conducted to ensure the highest standards of animal welfare. S. mediterranea should be housed in optimized conditions simulating natural habitats. Experiments should be designed to minimize potential pain or distress to ensure ethical treatment throughout the study.

1. Fungal culturing

  1. C. albicans strain information
    1. The standard C. albicans lab strain SN25051 was used as the wild-type strain for all experiments, and is publicly available at the Fungal Genetics Stock Center (http://www.fgsc.net/). SN250 is derived from the clinical isolate SC5314, originally collected from a blood culture of a patient with disseminated candidiasis52.
    2. C. albicans strains are maintained as 25% glycerol stocks at −80°C.
  2.  Culturing C. albicans strains for infection
    1. Streaking from cryogenic stock
      1. Using a sterile applicator, streak the desired strain from −80°C stock onto yeast peptone extract (YPD) (1% yeast extract, 2% peptone, 2% glucose; pH 6.8) agar plates.
      2. Incubate statically at 30°C for 72 h.
  3. Preparing overnight cultures
    1. Inoculate a single colony into 4 mL liquid YPD in sterile borosilicate glass culture tubes (18 mm × 150 mm) using a sterile applicator stick.
    2. Include a negative control tube containing uninoculated YPD under identical conditions.
    3. Loosely cap the tubes to allow aeration and incubate overnight (16 h) at 30 °C with shaking at 220 rpm (orbital) in a shaking incubator.
      NOTE: If microbial growth occurs in the negative control, abort the experiment and restart with new media and sterile technique.
  4. Harvesting and washing cells
    1. Using aseptic technique, transfer cultures to labeled 15 mL centrifuge tubes.
    2. Centrifuge cultures for 5 min at ~3,000 × g to pellet the cells.
    3. Carefully remove the YPD supernatant without disturbing the pellet.
    4. Resuspend the pellet in planarian water (refer to Table 2 for composition) in a volume equal to that of the YPD used.
    5. Mix thoroughly by vortexing.
    6. Repeat steps 1.4.2-1.4.4 twice more to remove the growth media entirely.
    7. After the final wash, resuspend the cell pellet in fresh planarian water by vortexing.
  5. Quantifying C. albicans cell concentration
    1. Dilution for OD measurement
      1. Prepare three sterile 1.7 mL microcentrifuge tubes with a 1:20 dilution (50 µL culture + 950 µL planarian water) or an alternative chosen dilution factor. Mix thoroughly.
      2. Prepare a blank tube containing 1000 µL planarian water.
    2. Spectrophotometry
      1. Set the spectrophotometer to measure optical density at 600 nm (OD600).
      2. Blank the spectrophotometer with planarian water.
      3. Measure OD600 for each diluted sample and record values.
      4. Average the three readings and multiply by the dilution factor to determine the OD600 of the stock culture. Convert OD600 to cell concentration.
        ​NOTE: The optical density conversion factor should be determined empirically for each spectrophotometer. Based on the specific instrumentation used, an OD600 of 1 corresponds to approximately 2 ×107 C. albicans cells/mL53.
  6. Determining experimental inoculum concentrations
    1. Prepare the desired inoculum concentration for each experimental condition.
      NOTE: Perform at least three biological replicates to determine the effective dose for the planarian colony, particularly for infectious or lethal endpoints. A representative calculation for preparing infectious and lethal inoculum concentrations is provided in Supplementary File 1.

2. Planarian species and maintenance

  1. Planarian species and maintenance
    1. Use the CIW4 asexual clonal line of the planarian S. mediterranea for all experiments.
    2. Maintain planarian colonies in clear food-grade plastic containers containing 1,500–2,000 mL of 1× planarian water.
    3. Keep containers in darkness at 20 °C with lids loosely closed to allow gas exchange.
    4. Do not treat planarians with antibiotics.
  2. Colony maintenance and feeding
    1. Feed colonies fresh strained beef liver purée once per week.
    2. Clean containers twice per week.
    3. Maintain planarian density at 200–400 animals per container. Previous work demonstrates detailed procedures on colony preparation and long-term maintenance31.
  3. Pre-experiment conditioning
    1. Prior to experimental infection, starve planarians for 7–12 days to standardize metabolic and size conditions.
    2. Use groups of 5–10 animals per well for each experimental condition. Ensure equal numbers of animals across all conditions, including positive and negative controls.
  4. Animal size selection
    1. Select planarians approximately 5 mm in length, corresponding to ~5 × 105 host cells per animal54.
    2. Avoid using planarians smaller than 5 mm, as animals of this size may include individuals with blastemas or incomplete development that can produce inconsistent results.
    3. Avoid using planarians larger than 5 mm, as larger animals are less suitable for fluorescence immunostaining.
    4. Use planarians approximately 5 mm in length when establishing infectious dose (ID50) and lethal dose (LD50) conditions.
      NOTE: To facilitate consistent size selection, print a 5 mm × 5 mm reference grid and place it inside a plastic binder sleeve beneath the transparent planarian container. Ensure planarians are fully extended before measuring length.
  5. Health and inclusion criteria
    1. CRITICAL STEP: Examine all planarians under a stereomicroscope prior to use.
    2. Exclude animals with visible damage or injury, including the following conditions:
      1. Presence of blastemas (tissue lacking pigmentation) or incomplete pigmentation development.
      2. Abnormal pigmentation, such as significantly darker regions.
      3. Missing or damaged tissue.
      4. Lesions or scars.
      5. Open wounds.
      6. Morphological abnormalities or deformities.
      7. Any chronic or non-healing condition.
    3. Exclude animals with developmental abnormalities.
      1. Bifurcated or duplicated head or tail regions.
      2. Misshapen or asymmetric body morphology.
      3. Incorrect number of photoreceptors (only one bilateral pair is acceptable).
      4. Abnormally positioned pharynx.
      5. Defective ciliation resulting in impaired motility or irregular gliding behavior.
    4. Include only animals that are approximately 5 mm in length, fully developed, and free of visible injuries or abnormalities.
  6. Plate preparation
    1. Transfer healthy planarians meeting all inclusion criteria into 2 mL of fresh planarian water per well in a non–tissue culture-treated 6-well polystyrene plate.
      NOTE: Transfer animals one day prior to infection to allow acclimation to the new environment and population density.
    2. Assign wells for the following experimental conditions:
      1. Negative controls: uninfected or mock-treated animals.
      2. Positive controls: animals infected with wild-type C. albicans.
    3. Remove and replace all water with 2 mL of fresh planarian water to ensure uniformity across wells.

3. Infection setup 

  1. Prepare inoculation (see Supplementary File 1 for sample calculation)
    1. Using aseptic technique, gently remove all water from the 6-well plate by tilting the plate toward the user.
    2. Add the calculated volume of planarian water to each well.
      NOTE: Proceed one well at a time to prevent animals from drying out.
    3. Add the calculated volume of C. albicans culture to each well.
      NOTE: Avoid exposing animals to air for prolonged periods, as drying can cause stress and alter infection outcomes.
  2. Experimental plate incubation conditions
    1. Place the inoculated 6-well plate in a dark, static location at room temperature.
    2. Maintain experimental plates in a separate area from uninfected planarian colonies to prevent cross-contamination.
    3. Record the date and time of inoculation (designated as time 0 h).
      NOTE: Avoid high-traffic or vibration-prone areas (e.g., drawers, instrument surfaces, or busy benchtops), as movement can stress animals and confound results.
    4. Incubate planarians with C. albicans for up to 72 h, depending on the desired endpoint.
  3. Resuspension of fungal cells
    1. At each time point, gently resuspend settled C. albicans cells without disturbing the planarians.
      NOTE: Time (hours [h] or days [d] post-infection) refers to the elapsed time since the initial exposure (time 0).
    2. At 24 h post-infection (hpi)
      1. Using a sterile 3 mL transfer pipette, tilt the plate slightly toward the user.
      2. Aspirate the planarian water and gently dispense it into an area of the well free of animals to mix the settled fungal layer.
      3. Repeat this step 5–10 times per well to break up the settled fungal layer, avoiding direct contact with planarians.
      4. Observe fungal settlement against a dark background for best visibility before and after the resuspending steps.
      5. Resuspend uninfected control wells under identical conditions.
    3. At 48 hpi
      1. Repeat the resuspension procedure described above.
      2. Perform all resuspension steps within ± 1 h of the designated timepoint.
  4. End of exposure (72 hpi)
    1. At 72 hpi, gently transfer planarians to a new 6-well plate containing 2 mL of fresh planarian water per well.
    2. Minimize transfer of fungal culture by using the smallest possible volume of liquid.
    3. Once animals are transferred, remove all water and replace it with fresh planarian water.
      ​NOTE: Handle animals and any tissue fragments gently to avoid mechanical injury or additional stress.

4. Host endpoint assessment

  1. Recording host outcomes
    1. Begin assessing host outcomes at 1-day post-infection (dpi) and continue daily until the desired experimental endpoint.
    2. Quantify host damage using the standardized 0–3 scoring system (Figure 2).
      Score 0: No change from uninfected controls (healthy, asymptomatic).
      Score 1: Mild symptom(s).
      Score 2: Severe symptom(s).
      Score 3: Death.
  2. Criteria for host symptom evaluation
    1. Score 0 (Asymptomatic):
      1. Identify animals that display normal morphology, pigmentation, behavior, and responsiveness with no observable changes relative to uninfected controls.
    2. Score 1 (Mild):
      Assign a mild score when at least one of the following symptoms is observed relative to asymptomatic controls.
      1. Reduced movement speed or abnormal gliding behavior.
      2. Partial or complete loss of phototactic response.
      3. Sustained or repetitive body contractions.
      4. Abnormal or non-uniform pigmentation (lighter or darker regions).
      5. Regression of head or tail tissue.
    3. Score 2 (Severe):
      Assign a severe score when one or more of the following symptoms are present while animals remain alive.
      1. Eye film or loss of one or both eyes.
      2. Complete head loss or notable anterior tissue damage.
      3. Major tissue loss in the pre-pharyngeal region.
      4. Fragmentation into multiple body regions (cephalic, pharyngeal, or posterior segments).
      5. Open wounds with or without mucus secretion or internal leakage.
      6. Partial tissue lysis resulting in loss of structural integrity.
      7. “C-shaped” body posture, paralysis, or severe motility impairment.
    4. Score 3 (Death):
      Identify death based on one or more of the following criteria.
      1. Complete tissue or whole-animal lysis.
      2. Total loss of response to stimuli.
      3. Failure to recover or regenerate.
        NOTE: Figure 2 provides representative images and defining features for each score to ensure consistent host phenotype classification.

5. Determining host survival after fungal exposure

  1. At 3 dpi, gently transfer planarians to a new 6-well plate containing 2 mL of fresh planarian water per well, avoiding transfer of residual fungal culture.
  2. Observe animals using light microscopy (≥ 10x objective).
  3. Record host outcomes using the standardized scoring sheet provided in Supplementary File 2.
  4. Determining live animals
    1. Identify living animals based on one or more of the following criteria.
      1. Negative phototaxis.
      2. Response to gentle transfer pipette perturbation.
      3. Intact body structure.
      4. Stable attachment to the plate.
  5. Determining dead animals
    1. Identify dead animals based on one or more of the following criteria.
      1. Complete lysis or disintegration (residual debris only).
      2. Transparent or eviscerated body.
      3. Absence of movement or response to stimuli.
      4. Failure to regenerate (absence of blastema formation).
  6. Removal of dead animals
    1. Remove dead animals and debris only after confirming non-viability under microscopy.
    2. CRITICAL STEP: Remove dead animals immediately to prevent cross-contamination or altered survival measurements.
    3. If debris or fungal carryover remains, transfer surviving animals again to a fresh plate containing clean planarian water to prevent C. albicans reseeding or biofilm formation.
      NOTE: Place 6-well plates over a dark background during visual inspection to improve contrast and facilitate phenotype assessment.

6. Fungal burden assessment

  1. Colony-forming unit (CFU) quantification
    1. At the desired infection timepoints, collect planarians (5–10 animals per condition per day). Include matched uninfected/mock controls and wild-type infected controls.
    2. Gently rinse wells two times with 2 mL of fresh planarian water to remove background C. albicans cells.
    3. Transfer planarians into sterile and labeled 1.7 mL or 5.0 mL microcentrifuge tubes and remove all residual water.
    4. Add 300 µL of fresh planarian water to each tube.
    5. Homogenize animals using a sterile pestle until no large fragments remain (approximately 30–45 s).
    6. Perform serial dilutions of the homogenate (e.g., 1:10, 1:100, 1:1000).
    7. Plate 100–200 µL of each dilution onto YPD agar plates supplemented with 50 µg/mL each of ampicillin, rifampicin, streptomycin, and neomycin. Spread evenly using sterile glass beads or a sterile cell spreader.
    8. Allow plates to remain upright on the benchtop for 5–10 min after plating to permit absorption of liquid into the agar.
    9. Prepare triplicate plates for each dilution and condition.
    10. Incubate plates at 30 °C for 48 h.
    11. Count CFUs and multiply by the dilution factor.
    12. Normalize CFU values to the number of planarians to calculate average CFUs per planarian per condition. For example, if 100 colonies are observed at a 10× dilution from a homogenate of 5 animals, the calculated value is 200 CFUs per animal.

7. Immunofluorescence anti-Candida staining

  1. General staining considerations
    1. Perform all steps with gentle orbital rocking (100–140 rpm) at room temperature unless otherwise specified. See Supplemental File 1 for antibody details and reagent sources.
      CAUTION: Formaldehyde, sodium dodecyl sulfate (SDS), and hydrogen peroxide are hazardous chemicals and must be handled in accordance with institutional safety regulations. Formaldehyde is toxic and classified as a carcinogen; SDS is an irritant; and hydrogen peroxide is a strong oxidizer that may cause burns. Wear appropriate personal protective equipment (lab coat, nitrile gloves, closed-toe shoes, and safety goggles). Perform procedures involving volatile reagents in a certified chemical fume hood and dispose of waste according to institutional Environmental Health and Safety guidelines.
  2. Sample collection
    1. Collect animals from each experimental condition and time point.
    2. Transfer animals into labeled 20 mL scintillation vials containing 2 mL of planarian water.
    3. Remove all liquid, rinse once with 2 mL of fresh planarian water, then all liquid once more.
  3. Fixation and permeabilization
    1. Sacrifice animals in 7.5% N-acetylcysteine (NAC) in 1× PBS for 3 min.
    2. Remove the solution and fix animals in 4% formaldehyde in 0.3% PBSTx for 15 min.
    3. Rinse samples two times with 1× PBS.
    4. Permeabilize samples in 1% SDS for 15 min.
    5. Rinse samples three times with 1× PBS.
  4. Bleaching
    1. Bleach samples in 6% hydrogen peroxide in 1× PBS under a bright LED light for 4–12 h.
      NOTE: If bleaching cannot proceed immediately, store fixed samples in 1× PBS at 4 °C for up to 1 week or dehydrate samples in 100% methanol for long-term storage.
  5. Blocking and antibody incubation
    1. Transfer animals to 24-well plates containing 1× PBS.
    2. Block nonspecific binding in 2.5% PBS-TB (PBS containing Triton X-100 and bovine serum albumin) for 4 h at room temperature or 8 h at 4 °C.
    3. Incubate samples with primary anti-Candida antibody (1:500 in PBS-TB) for 4 h at room temperature or 8 h at 4 °C.
    4. Remove primary antibody and wash samples eight times every 20 min with 0.3% PBSTx (total ~2.5 h).
    5. Incubate samples with Alexa-568-conjugated anti-rabbit secondary antibody (1:800 in PBS-TB) for 4 h at room temperature or 8 h at 4 °C.
      NOTE: Protect samples from light to prevent photobleaching. Antibody solutions may be reused.
    6. Remove secondary antibody and wash samples eight times every 20 min with 0.3% PBSTx.
  6. Optional slide mounting
    1. Place animals onto a microscope slide with all animals oriented anterior side up.
    2. Remove excess water using a transfer pipette.
    3. Carefully absorb remaining liquid from the edges using the edge of a paper towel.
    4. Add 150–175 µL of Gelvatol mounting medium (Table 2 for composition) dropwise until animals are fully covered.
    5. Carefully place a coverslip over the sample, avoiding air bubbles.
    6. Allow Gelvatol to solidify for at least 2 h before imaging.
    7. Store slides in a cool, dark location for long-term preservation.
  7. Imaging
    1. Image samples using yellow-green excitation (~578 nm) and observe orange-red emission (~603 nm).
    2. Use a 2x objective lens to visualize whole-animal staining patterns.

8. Troubleshooting

  1. If infection outcomes are inconsistent, perform a dose-titration curve using wild-type SN250 or another baseline strain in increments of 5–10 × 107 cells/mL.
  2. Perform at least three biological replicates containing 5–10 animals per group.
  3. Include negative and positive control groups to identify potential issues such as cross-contamination, aseptic technique failure, strain viability problems, or poor baseline animal health.
  4. After completing the dose-titration experiment, pool replicates to determine median survival per group.
  5. Analyze survival differences using Mantel–Cox log-rank pairwise analysis.
  6. Use Kaplan–Meier survival analysis to evaluate time-to-death differences between strains or treatments.
  7. For sublethal or infectious dose conditions, analyze symptomatic host outcomes (e.g., mild or severe phenotypes or fungal colonization) instead of survival.
  8. Compare host responses across dose groups to determine the appropriate infectious or lethal dose range for subsequent experiments.

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结果

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本文介绍了一种更新的感染方案，用于用C. albicans感染涡虫（图1），并系统地量化宿主的感染结果（图2）。该优化后的实验流程可稳健评估多种感染参数，包括发病情况（症状性疾病的严重程度）、存活率（致死性）、真菌载量（菌落形成单位，CFUs）以及病原体定植的空间-时间动态（荧光免疫染色）。与大多数仅提供简单的存活/死亡二元读数的无脊椎动物感染模型不同，该涡虫模型能够捕捉更广泛的感染结果和宿主反应谱。涡虫可用于多种微生物攻击终点的研究，例如亚致死性感染引发的症状性疾病进展及后续恢复过程；也可用于生存率研究，包括致死剂量接种实验。这些特性共同凸显了涡虫作为一种灵活平台，在研究多种疾病表型下宿主-病原体相互作用中的广泛适用性。

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讨论

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

改变野生型 C. albicans 的感染剂量可在 S. mediterranea 中产生可重复的、剂量依赖性的发病率差异，突显该模型在定量评估真菌毒力方面的敏感性。本方案概述了在实验室维持的涡虫群体中确定基础感染剂量和致死剂量的策略，并提出了通过浸泡法建立系统性感染成功的判定标准，结合宿主健康评分与菌落形成单位（CFU）定量及免疫染色法测定真菌负荷。

通过系统性优化，确定了多个对实现稳定感染动态和可重复宿主结果至关重要的实验参数。除接种浓度外，涡虫体型、种群密度以及表面积与体积之比等因素也显著影响感染的进展和严重程度。本文引入的改进措施——包括标准化纳入标准、受控的感染条件以及详细的终点评分方法——显著提高了实验的可重复性，并支持在不同实验及不同实验室之间进行有意义的比较。

涡虫兼具实验易操作性与生物学复杂性的罕见优势，使得在多细胞生物体内开展宿主-病原体相互作用的in vivo研究成为可能。其体型大小便于快速进行疾病表型的宏观评估，包括运动能力缺陷、形态学变化及存活情况...

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披露

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

C.J.N. 是 BioSynesis 公司的联合创始人，该公司致力于开发生物膜感染的诊断和治疗方法。其他所有作者均声明无竞争利益。

致谢

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

作者感谢Nobile、Oviedo和Hernday实验室的所有成员就S. mediterranea-C. albicans感染模型进行的深入讨论，并感谢Edelweiss Pfister在实验室管理和涡虫维护方面提供的支持。本研究工作由美国国立卫生研究院（NIH）下属的国家普通医学科学研究所（NIGMS）资助，项目编号分别为C.J.N.获得的R35GM156045和R35GM124594，以及N.J.O.获得的R35GM158501和R01GM132753。本研究还得到了Kamangar家族对C.J.N.设立的捐赠讲席基金的支持。N.M.S.的研究得到了细胞与生物分子机器中心（CCBM）美国国家科学基金会（NSF）科学研究卓越中心（CREST）奖学金的支持，项目编号为NSF-HRD-1547848和NSF-EES-2112675。

资助方在研究设计、数据收集与分析、数据解释、论文撰写以及决定发表结果方面均无任何作用。N.M.S. 感谢使用 ChatGPT 辅助编辑论文（检查拼写、语法、格式和句子结构）。

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材料

本文使用的材料清单
姓名公司目录编号评论
1.7 mL 微量离心管 VWR/Genemate490004-436-BOXOF500透明，500片装盒
20 mL 闪烁瓶Fisher Scientific12-100-006用于免疫染色的透明玻璃瓶
24孔未处理聚苯乙烯板Fisher Scientific08-772-51免疫染色
5.0 mL 微型离心管 Eppendorf/Sigma AldrichEP0030119487-200EA透明，200 片装盒
6孔未经处理的聚苯乙烯板Fisher Scientific08-772-49感染实验
琼脂VWR89405-066YPD培养基用细菌学级
涂抹棒赛默飞世尔/普里敦22-029-491C. albicans 培养
BioRenderBioRender 公司N/A用于生成示意图的图形设计软件
氯化钙（CaCl2）Sigma-AldrichC5080-500G涡虫培养用水/1× Montjuïc 盐
Candida albicans 多克隆抗体 抗兔IgG赛默飞世尔科技PA1-27158免疫染色
离心管VWR89039-664C. albicans 收获
培养管，硼硅酸盐玻璃，18×150 mmVWR47729-583C. albicans 培养
比色皿Fisher Scientific14-955-127测量培养物的光密度
葡萄糖（D-葡萄糖）Fisher ScientificD16-3YPD培养基
甘油Fisher ScientificG33-4分子生物学级，用于低温储存的补充剂 C. albicans 
绘图与数据可视化工具GraphPad/Prism统计软件与可视化工具。版本 10.6.1。
绘图工具R统计软件。版本 4.4.3。
摇床培养箱EppendorfEPM1282-0004-1EA中国台湾新不伦瑞克 Innova 44/44R 用于 C. albicans 培养
氯化镁（MgCl2）SigmaM8266-100G用于涡虫的水  (1x Montjuïc 盐
硫酸镁（MgSO4）Sigma-AldrichM7506-500G用于涡虫的水  (1x Montjuïc 盐
显微镜载玻片VWR16004-368
多焦显微镜尼康Nikon AZ100 
N-乙酰-L-半胱氨酸Sigma AldrichA7250-50G黏液溶解剂与牺牲试剂  固定
对苯二甲醛，16% 溶液，分子生物学级电子显微镜科学公司15710固定剂试剂
研磨杵电动无绳马达Fisher Scientific12-141-361涡虫组织匀浆
蛋白胨，细菌学级HIMEDIA/VWR89129-480YPD培养基
研钵杵Fisher Scientific12-141-368无RNase一次性涡虫匀浆 pellet
培养皿，100 × 15 mmVWR25384-342
聚乙烯醇Sigma-AldrichP-8136 对于 Gelvatol
氯化钾（KCl）Sigma生命科学P9541-500G涡虫培养液/水  (1x Montjuïc 盐
二抗 山羊抗兔IgG，Alexa Fluor 568Fisher ScientificA-11011免疫染色
SN250真菌遗传学保藏中心野生型 C. albicans 菌株（https://www.fgsc.net/）
叠氮化钠Fisher ScientificS227-100对于 Gelvatol
碳酸氢钠（NaHCO₃）Sigma-AldrichS5761-1KG涡虫培养液/水  (1x Montjuïc 盐
氯化钠（NaCl）Sigma生命科学S3014-500G涡虫培养液/水  (1x Montjuïc 盐
分光光度计安捷伦26354使用 BioTek Epoch 2 微孔板读板仪测定光密度
静态培养箱Fisher Scientific15-015-2634C. albicans 培养
移液管Fisher Scientific13-711-9CM用于转移涡虫的无菌一次性刻度移液管
Tris碱Sigma-Aldrich252859对于 Gelvatol
酵母提取物赛默飞世尔科技212750YPD 培养基

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