改良的酶保护实验用于研究Staphylococcus aureus的内化作用及抗菌化合物的胞内活性

摘要

本方案旨在描述如何研究金黄色葡萄球菌（Staphylococcus aureus）在人宿主细胞内的内化程度及其在细胞内存活的能力，以及抗菌化合物在细胞内的有效性。

摘要

Staphylococcus aureus 表达毒力因子以触发其被真核细胞内吞，并在不同的亚细胞区室中存活。本文介绍一种酶保护实验，用于研究 S. aureus 在贴壁的非专职吞噬细胞（NPPCs）中的内化程度及其胞内存活情况，以及抗菌化合物在细胞内的效力。将 NPPCs 在多孔板中培养至完全汇合。S. aureus 菌种在细胞培养基中过夜培养。根据每孔细胞数量对细菌悬液进行稀释，以控制感染复数对细胞进行接种。接种后的细胞孵育 2 小时，使细菌被 NPPCs 内化，随后向培养基中加入溶葡萄球菌素，以选择性杀灭胞外细菌。在整个实验的其余阶段，培养基中均保留溶葡萄球菌素。

此时，可将感染的细胞与抗菌化合物共同孵育，以评估这些化合物对金黄色葡萄球菌（S. aureus）的胞内活性。随后，细胞需洗涤三次以去除药物，再通过在琼脂平板上培养来定量胞内金黄色葡萄球菌（S. aureus）的载量。或者，在研究参与胞内存活及细胞毒性的葡萄球菌毒力因子时，可使用蛋白酶K使溶葡萄球菌酶（lysostaphin）失活，从而省去洗涤步骤。该方法可提高胞内细菌载量定量的可靠性，尤其适用于因胞内金黄色葡萄球菌（S. aureus）大量增殖而导致细胞容易从培养板上脱落的情况。这些实验方案几乎适用于所有类型的贴壁原代人多能细胞（NPPCs），以及类器官等三维细胞培养模型。

引言

Staphylococcus aureus 是一种可危及生命的病原体，同时也是皮肤和黏膜的共生细菌，全球约有二十亿人携带该菌¹。在人类中，鼻腔携带 S. aureus 者更易感染自身携带的菌株；然而，S. aureus 黏膜定植的多因素决定机制仍不明确^1,2。除急性感染外，患者还可能发展为慢性 S. aureus 感染，此类感染通常难以治愈³。深入理解定植和感染过程中宿主与病原体之间的相互作用，对于开发新型治疗策略和改善患者管理至关重要。

体外, S. aureus 可触发其内化进入表达 α5β1 整合素的宿主细胞⁴金黄色葡萄球菌细胞壁锚定的纤连蛋白结合蛋白与宿主细胞及细胞外基质之间的三元相互作用 S. aureus，纤连蛋白以及宿主细胞表面表达的β1整合素被公认为主要通路 S. aureus 在角质形成细胞、成骨细胞、成纤维细胞以及上皮和内皮细胞等NPPC中的内化作用⁴近期研究表明 S. aureus 可在人类细胞内发现于鼻腔定植期间^5,6 和感染⁷然而，细胞内储存库在发病机制中的作用 S. aureus 感染机制仍不明确。宿主细胞可能为 S. aureus 可免受免疫系统的攻击⁸ 以及大多数抗菌化合物^6,9.

溶葡萄球菌素保护实验，由Proctor描述¹⁰ 20世纪80年代初期，可实现对细菌和宿主因子在细菌内化过程中作用的研究 S. aureus 分离株。溶葡萄球菌素是由 Staphylococcus simulans，这 对几乎所有病原体均表现出强效活性 S. aureus 分离株，包括耐药菌株¹¹溶葡萄球菌素已用于特异性破坏胞外菌 S. aureus 以实现仅对存活的胞内细菌进行计数¹²该技术已被广泛应用，并有助于发现多种病原体的毒力因子 S. aureus庆大霉素单独使用或与溶葡萄球菌素联合使用，也被广泛用于研究细胞内细菌。

然而，最近一项研究表明，庆大霉素以时间和浓度依赖的方式进入真核细胞，并到达细胞内化的细菌¹³本研究还表明，溶葡萄球菌素不会进入真核细胞，证实基于溶葡萄球菌素的酶保护实验（EPA）是定量胞内细菌的最准确方法 S. aureus 通过培养接种¹³无论使用何种化合物（如溶葡球菌素或庆大霉素）来杀灭细胞外细菌，在进行细胞内细菌铺板前，均应通过洗涤细胞的方式去除该化合物。 S. aureus 在琼脂平板上。连续洗涤可能导致细胞脱落，尤其是贴壁性较差的细胞（如重度感染的细胞），从而导致对胞内菌数量的低估 S. aureus 负载。本文详细描述了如何利用EPA来定量细胞内 S. aureus 加载 并利用细胞内抗菌化合物功效测定方法 体外 模型。值得注意的是，已有研究提出一种简单方法，通过避免剧烈洗涤来提高细胞内载荷定量的可靠性。

方案

1. Culture of human epithelial cells

1. Prepare complete culture medium with Dulbecco's modified Eagle medium (DMEM) high glucose with phenol red, supplemented with 10% fetal bovine serum (FBS) without antibiotics.
2. Grow A549 epithelial cells in complete culture medium at 36 ± 1 °C in 5% CO₂. Ensure the use of an appropriately sized culture vessel to have enough cells for subsequent steps (see step 1.10).
   NOTE: One 75 cm² (T-75) flask is sufficient to seed two 24-well plates and subculture the cells.
3. Two days before infection, prepare a single 24-well plate.
4. Remove and discard the spent culture medium from the T-75 flask and wash the cells once with 10 mL of Dulbecco′s phosphate-buffered saline (DPBS).
5. Add 5 mL of trypsin-EDTA and incubate the cells for 5 min at 36 ± 1 °C in 5% CO₂.
6. Add 5 mL of complete culture medium and transfer the cells into a tube.
7. Centrifuge the cells for 5 min at 300 × g.
8. Discard the supernatant and resuspend the cells in 10 mL of fresh complete culture medium.
9. Count the cells with an automatic cell counter (or a counting chamber).
10. Dilute the cells in complete culture medium to prepare 30 mL of cell suspension at a concentration of 2.0 × 10⁵ cells/mL.
11. Add 1 mL of the cell suspension to each well of a 24-well plate, which corresponds to a cell density of approximately 1.0 × 10⁵ cell/cm² for a well area of 2 cm².
12. Incubate the cells for 48 h at 36 ± 1 °C in 5% CO₂ until they reach 100% confluence.
    ​NOTE: In addition to the conditions to be tested, three wells should be reserved for cell counting on the day of infection (see step 3.1.4). According to the number of conditions to be tested, Up to two 24-well plates can be prepared simultaneously. Volumes indicated in the protocol should be increased accordingly.

2. Culture of S. aureus strains

1. Two days before infection, prepare complete infection medium with DMEM high glucose without phenol red, supplemented with 10% FBS without antibiotics.
2. Thaw S. aureus strains to be tested on agar plates.
3. Incubate the agar plates for 18-24 h at 36 ± 1 °C.
4. The day before inoculation, inoculate one colony of the S. aureus strain to be tested in 10 mL of complete infection medium.
5. Incubate the bacteria for 18-24 h at 36 ± 1 °C with shaking at 160 rpm. Use 50 mL tubes held at 45° to avoid the bacteria settling.
   ​NOTE: Before starting with a new strain, it is recommended to verify its lysostaphin susceptibility in the same conditions of culture that will be used for further experiments (media, bacterial loads, and lysostaphin concentration and incubation time). It is also important to determine the bacterial load corresponding to an OD_600nm of 0.5 because it could vary slightly from one strain to another. Culture conditions of bacterial strains could be adapted according to the experimental aim.

3. Infection assay with S. aureus

1. Determination of cell density and viability
   1. Remove and discard the spent culture medium from the three wells dedicated for counting A549 cells.
   2. Add 1 mL of complete infection medium containing 5 µg/mL of Hoechst 33342 and 1 µg/mL of propidium iodide.
      NOTE: Hoechst 33342 is a known mutagen and should be handled with care. Propidium iodide, a potential mutagen, must be handled with care and disposed of safely according to applicable regulations.
   3. Incubate the cells for 30 min at 36 ± 1 °C in 5% CO₂.
   4. Count the cell number and calculate the cell viability using a wield-field fluorescence microscope.
      NOTE: If a fluorescence microscope is not available, the cell density and viability can be calculated with trypan blue staining by using a cell counting chamber.
2. Preparation of the bacterial suspension
   1. Dispense 25 mL of complete infection medium in a tube and pre-warm at 36 ± 1 °C.
   2. Adjust the S. aureus suspension to anOD_600nm of 0.5 in complete infection medium using a cell density meter.
   3. Prepare 20 mL of bacterial suspension for cell inoculation by diluting the 0.5 OD_600nm in complete infection medium to achieve a multiplicity of infection (MOI) of 1 according to the number of cells per well.
      NOTE: The MOI corresponds to the number of bacteria added per cell in each well. For example, to achieve an MOI of 1 with 1.0 × 10⁶ cells per well, prepare a bacterial suspension at 2.0 × 10⁶ CFU/mL so that 10⁶ CFU can be added in a volume of 500 µL (see step 3.3.3). The MOI can be adjusted according to the cell types and bacterial strains to be tested.
   4. Use an automatic spiral plater to determine the S. aureus load of the diluted bacterial suspension to be used for the cell inoculation step.
   5. Incubate the agar plates for 18-24 h at 36 ± 1 °C.
   6. The next day, count the number of colonies with a colony counter to calculate the accurate MOI for each strain tested.
      NOTE: If no automatic spiral plater is available, the bacterial load could be determined by serial dilution on an agar plate. See the bacteriological analytical manual for details¹⁴.
3. Cell inoculation
   1. Observe every well of the 24-well plate by low magnification microscopy to ensure that the cells are healthy and growing as expected.
   2. Remove and discard the spent cell culture medium from the 24-well plate.
   3. Add 500 µL of the bacterial suspension for inoculation to each well with 100% confluent cells.
   4. Incubate the cells for 2 h at 36 ± 1 °C and 5% CO₂.
      NOTE: it is recommended to use three wells of the plate for each condition to be tested (triplicate) and to perform at least three independent experiments. The delay of incubation can be adapted according to the experimental aim.
4. Quantification of intracellular bacteria with improved enzyme protection assay (iEPA)
   1. Prepare 7 mL of 4x lysis buffer with 3.5 mL of 2% Triton X-100 in sterile water and 3.5 mL of trypsin-EDTA.
   2. Prepare a lysostaphin stock solution at 10 mg/mL in acetate buffer and aliquot 25 µL into cryovials. Store at -80 °C for up to 6 months.
   3. Prepare 250 µL of a fresh lysostaphin working solution at 1 mg/mL by mixing 25 µL of the lysostaphin stock solution (10 mg/mL) and 225 µL of 0.1 M Tris-HCl. Store at 4 °C for up to 48 h.
   4. Prepare 6.25 mL of complete infection medium supplemented with lysostaphin by adding 6 mL of complete infection medium to 250 µL of the lysostaphin working solution.
   5. Add 250 µL of complete infection medium supplemented with lysostaphin into each well and gently agitate the plate by swiveling the plate by hand.
   6. Incubate the cells for 1 h at 36 ± 1 °C in 5% CO₂ to let the lysostaphin kill the extracellular bacteria.
   7. At the end of the incubation time, add 10 µL of proteinase K at 20 mg/mL into each well to inactivate the lysostaphin.
   8. Incubate the cells for 2 min at room temperature.
   9. Add 250 µL of 4x lysis buffer to lyse the cells by osmotic shock.
   10. Incubate the cells for 10 min at 36 ± 1 °C.
   11. Mix thoroughly by pipetting up and down ten times all over the bottom of the well to ensure that the cells are fully lysed and homogenized.
   12. Use an automatic spiral plater to determine the S. aureus load of each well.
   13. Incubate the agar plates for 18-24 h at 36 ± 1 °C.
   14. The next day, count the number of colonies with a colony counter to calculate the intracellular S. aureus load of each well.
5. Measurement of intracellular efficacy of antimicrobial compounds with enzyme protection assay (EPA)
   1. Prepare 25 mL of 1x lysis buffer with 3.125 mL of 2% Triton X-100 in sterile water, 6.25 mL of trypsin-EDTA, and 15.625 mL of sterile water.
   2. Prepare 250 µL of a fresh lysostaphin working solution at 1 mg/mL by mixing 25 µL of a lysostaphin stock solution (10 mg/mL) and 225 µL of 0.1 M Tris-HCl.
   3. Prepare 25 mL of complete infection medium supplemented with lysostaphin by adding 24.75 mL of complete infection medium to 250 µL of the lysostaphin working solution.
   4. For each antimicrobial compound to be tested, prepare 3.1 mL of complete infection medium supplemented with lysostaphin and the antimicrobial compound at the concentration to be studied.
   5. Remove and discard the spent cell culture medium from the 24-well plate.
   6. Add 1 mL of complete infection medium supplemented with lysostaphin.
   7. Incubate the cells for 1 h at 36 ± 1 °C in 5% CO₂ to let the lysostaphin kill the extracellular bacteria.
   8. Remove and discard the medium supplemented with lysostaphin from the 24-well plate.
   9. Fill three wells with 1 mL of medium supplemented with lysostaphin plus the antimicrobial compound to be tested.
   10. Repeat step 3.5.9 for each antimicrobial compound to be tested.
   11. For the control condition, fill three wells with 1 mL of medium supplemented with lysostaphin without any antimicrobial compound.
   12. Incubate the cells for 24 h at 36 ± 1 °C in 5% CO₂.
   13. At the end of the incubation period, remove and discard the spent medium and gently wash each well three times with sterile DPBS with CaCl₂ and MgCl₂.
   14. Add 1 mL of 1x lysis buffer to each well to detach and lyse the cells by osmotic shock.
   15. Incubate the cells for 10 min at 36 ± 1 °C.
   16. Mix thoroughly by pipetting up and down ten times all over the well to ensure that the cells are fully lysed and homogenized.
   17. Use an automatic spiral plater to determine the S. aureus load of each well.
   18. Incubate the agar plates for 18-24 h at 36 ± 1 °C.
   19. The next day, count the number of colonies with a colony counter to calculate the intracellular S. aureus load of each well.
       NOTE: The intracellular activity of each antimicrobial compound should be calculated according to the bacterial load of the control condition. It is also important to check the cytotoxicity of all antimicrobial compounds to prove that the differences observed between the control and the compounds are not due to cell death.

结果

S. aureus 被 A549 上皮细胞内化的实验结果如图1A所示。A549 细胞以感染复数（MOI）为 1 的条件分别接种 S. aureus SF8300 WT 和缺失纤维连接蛋白结合蛋白 A 与 B 的突变株 SF8300 ΔfnbA/B，孵育 2 小时。为清除胞外 S. aureus，向培养基中加入溶葡萄球菌素（lysostaphin），继续孵育 1 小时。随后，通过洗涤去除溶葡萄球菌素以进行 EPA 检测，或使用蛋白酶 K 灭活溶葡萄球菌素以进行 iEPA 检测。接着，用裂解缓冲液裂解细胞，并通过培养法对细菌载量进行定量。采用 EPA 方法时，SF8300 WT 和 SF8300 ΔfnbA/B 的平均胞内载量分别为 4.46 和 0.49 Log CFU/mL（图1A，绿色柱状图）。采用 iEPA 方法时，SF8300 WT 和 SF8300 ΔfnbA/B 的平均胞内载量分别为 4.53 和 0.56 Log CFU/mL（图1A，红色柱状图）。值得注意的是，EPA 与 iEPA 两种方法所得结果相似，这可归因于当细胞状态良好时洗涤操作易于完成，且在本实验条件下 S. aureus 诱导的细胞毒性极低（数据未显示）。

万古霉素、利福平和左氧氟沙星对S. aureus的胞内活性结果如图1B所示。为测定这些抗生素的胞内活性，使用MOI为1的S. aureus ATCC 29213菌株感染HaCaT细胞，感染时间为2小时。随后，将细胞与溶葡萄球菌素（含或不含待测抗菌化合物）共同孵育24小时。通过洗涤去除溶葡萄球菌素和抗菌化合物后，用裂解缓冲液裂解细胞，并通过培养法定量细菌载量。对照组、万古霉素（50 µg/mL）、利福平（7 µg/mL）和左氧氟沙星（10 µg/mL）处理组的平均胞内载量分别为4.57、4.51、3.03和2.91 log CFU/mL（图1B）。

图1：上皮细胞内金黄色葡萄球菌（Staphylococcus aureus）的载量。（A）在感染了S. aureus SF8300 WT和ΔfnbA/B菌株的A549细胞中进行的酶保护实验（绿色柱）和改良酶保护实验（红色柱）。（B）在感染了S. aureus ATCC 29213的HaCaT细胞中抗菌化合物的细胞内活性。柱状图表示三次独立实验（每组实验设三个重复）的平均值，误差线表示标准差。**** p < 0.0001。缩写：Ctrl = 对照；cfu = 菌落形成单位。请点击此处查看该图的放大版本。

讨论

本文所述的检测方法对于研究金黄色葡萄球菌（S. aureus）在NPPCs中的内化程度、胞内存活情况以及抗菌化合物的胞内抗菌效果具有重要价值^6,15,16。两种检测方案中的某些步骤尤为关键。细胞的健康状态和密度必须在各次独立实验之间得到精确控制并保持一致。细菌接种物必须经过仔细标准化，以获得接近目标理论感染复数（MOI）的实际MOI。通常情况下，加样时需格外小心，避免细胞脱落。在EPA中，去除溶葡萄球菌素和抗生素的洗涤步骤至关重要。研究发现，在未使用抗生素的情况下，使用蛋白酶K可改善该步骤（见下文）。最后但同样重要的是，在裂解缓冲液孵育后，应确保每个孔中的细胞完全脱离并充分匀浆，以准确量化金黄色葡萄球菌（S. aureus）的胞内载量。

在某些情况下可能会遇到问题，需要首先检查几个要点。如果实验结果缺乏可重复性，必须注意S. aureus可能形成菌团，导致通过吸光度进行定量不准确。当更换培养基（例如为了去除分泌蛋白）而进行离心和洗涤步骤时，细菌的聚集现象可能加剧。细菌悬液应尽快使用，因为细菌在室温下会持续生长。溶葡球菌酶（lysostaphin）的效力可能因储存条件不当、培养基中酶活性的pH非最适、不同批次或供应商之间的酶活性差异，以及某些菌株在特定生长条件下对溶葡球菌酶不敏感而降低。酚红可能具有轻微的抑菌作用，尤其是在培养基营养成分相对于常规细菌培养肉汤较为贫乏的情况下。因此，建议使用不含酚红的细胞培养基，这也有助于减少背景噪声，从而改善荧光显微镜观察效果。

尽管该方法是研究不同菌株细胞内命运的有力工具，但仍需考虑其某些局限性。使用过高的感染复数（MOI）可能会超出NPPCs的内化能力，从而抹平不同测试菌株之间的差异。由于溶葡球菌酶（或抗生素）会迅速破坏受损细胞所释放的S. aureus，细胞毒性最强的菌株其内化程度可能被低估。因此，对于细胞毒性较低的菌株，更易于开展长时间孵育的实验（例如研究细胞内存活或抗生素的细胞内活性）。因此，孵育时间与MOI应根据菌株毒力、细胞类型及实验目的进行精确调整。

本文所述使用溶葡萄球菌酶的方法比基于庆大霉素的方法更可靠，因为与溶葡萄球菌酶不同，庆大霉素容易被宿主细胞内化¹³。另一个优势在于溶葡萄球菌酶可被灭活。Kim 等人¹³曾报道使用 EDTA 螯合锌离子或使用 1,10-菲啰啉抑制溶葡萄球菌酶活性；然而，在细菌涂板前仍需进行多次洗涤以彻底去除酶。本方法中，蛋白酶 K 可快速灭活溶葡萄球菌酶。我们观察到，由于胞内金黄色葡萄球菌（S. aureus）的增殖，细胞在严重感染时容易从培养板上脱落。通过省略最后的洗涤步骤，iEPA 方法显著简化了操作流程，并能够回收那些松散贴壁或已脱落细胞中的内化细菌。

iEPA 中使用的更高浓度的试剂和缓冲液还有助于减少移液操作并最大限度降低细胞损失。此外，iEPA 可用于悬浮细胞，也可用于难以洗涤的类器官。综上所述，酶保护实验可用于研究内化程度及内化后分子的细胞内命运 S. aureus，以及不同抗菌药物的细胞内活性 体外 模型。应进一步改进以更准确地表征内化作用与细胞毒性的关系，从而更充分地认识开发能够到达靶点的药物的重要性 S. aureus 细胞内

材料

本文使用的材料清单

姓名	公司	目录编号	评论
24孔板	CORNING-FALCON	353047
A549细胞系	ATCC	CCL-185
乙酸盐缓冲液，pH 4.6	Fluka	31048	用于在20 mM醋酸钠溶液中配制10 mg/mL的溶葡萄球菌素储备液。
AMBICIN（重组溶葡萄球菌素）	AMBI	LSPN-50	冻干重组溶葡萄球菌素。长期保存需在-80 °C下冷冻。
COS - 哥伦比亚琼脂 + 5% 羊血	Biomerieux	43049	可使用任何适合培养葡萄球菌的琼脂平板替代。
密度计 WPA CO8000	Biochrom Ltd.	80-3000-45	细胞密度测定仪
Dulbecco’s 改良Eagle’s 培养基，高糖含酚红	Sigma-Aldrich	D6429
Dulbecco’s 改良Eagle’s 培养基，高糖不含酚红	Sigma-Aldrich	D1145
Dulbecco’s 磷酸盐缓冲液	Sigma-Aldrich	D8537
Dulbecco′s 含MgCl2和CaCl2的磷酸盐缓冲液，经无菌过滤	Sigma-Aldrich	D8662
Dulbecco′s 磷酸盐缓冲液，经无菌过滤	Sigma-Aldrich	D8537
Easyspiral 稀释仪	Interscience	414000	自动稀释仪和螺旋接种仪
胎牛血清	Gibco	10270-106
HaCaT细胞系	细胞系服务（CLS）	300493
Hoechst 33342，三盐酸盐，三水合物，水中10 mg/mL溶液	Fisher scientific	11534886
碘化丙啶，水中1.0 mg/mL溶液	Invitrogen	P3566
蛋白酶K，重组型，20 mg/mL	Eurobio	GEXPRK01-B5	> 30 U/mg，批号901727
Scan 4000	Interscience	438000	自动菌落计数器
无菌水	OTEC	600500
T-75培养瓶	CORNING-FALCON	353136
TC20自动细胞计数仪	Biorad	1450102	自动细胞计数仪
Tris 1 M，pH 8.0	Invitrogen	AM9855G	用于在0.1 M Tris-HCl中配制1 mg/mL的溶葡萄球菌素工作液。
Triton X-100	Sigma-Aldrich	T8787
胰蛋白酶-EDTA溶液	Sigma-Aldrich	T3924	含0.05%猪胰蛋白酶和0.02% EDTA的Hanks′平衡盐溶液，含酚红
宽场荧光显微镜	Nikon	Ti2