Vaccine and Infectious Disease Organization - International Vaccine Centre (VIDO-InterVac), University of Saskatchewan, Saskatoon, Saskatchewan, S7N 5E3, Canada.
School of Public Health, Vaccinology and Immunotherapeutics Program, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.
BMC Microbiol. 2019 Mar 25;19(1):67. doi: 10.1186/s12866-019-1438-2.
Neisseria gonorrhoeae is an obligate human pathogen and its adherence to host cells is essential for its pathogenesis. Gonococcal adherence assays are based on the enumeration of bacteria attached to human cells on solid media. Because conventional adherence assays are based on bacterial counts, they are often time consuming to perform and prone to observer bias. A flow cytometry based method, using the cell-permeable fluorescent dye 5'-carboxyfluoroscein succidyl ester (CFSE), was developed to dramatically increase the number of adherent N. gonorrhoeae quantified per assay while improving repeatability and removing observer bias. Piliated N. gonorrhoeae F62 were stained with CFSE then the staining reaction was quenched with foetal bovine serum. Human cervical ME-180 cells were infected with CFSE-stained N. gonorrhoeae (multiplicity of the infection 100:1) for 2 h. Infected cells were washed to remove loosely adhered bacteria. Flow cytometry was used to quantify the percentage of ME-180 cells associated with CFSE-stained N. gonorrhoeae and a minimum of 30,000 events were recorded. Real time-PCR analysis targeting opa gene (encoding N. gonorrhoeae opacity associated gonococcal outer membrane protein) was performed on infected ME-180 cells to confirm the flow cytometric adherence assay results. A rabbit was immunized with heat-killed N. gonorrhoeaeF62 to generate hyperimmune serum. The functional compatibility of the assay was confirmed by studying the effect of N. gonorrhoeae F62 antiserum on blocking adherence/invasion of CFSE-stained bacteria to ME-180 cells.
We observed that 20.3% (+/- 1.0) ME-180 cells were associated with CFSE-stained N. gonorrhoeae. Heat-inactivated hyperimmune serum, at 1:10 to 1:80 dilutions, significantly inhibited gonococcal adherence by 6 and 3 fold, respectively. Real time-PCR analysis targeting opa gene confirmed that hyperimmune serum blocked adherence/invasion of N. gonorrhoeae to the ME-180 cells in a dilution-dependent manner.
Flow cytometric analysis was amenable to quick, easy and high-throughput quantification of the association of N. gonorrhoeae with ME-180 cells and was functionally confirmed using PCR analysis. These approaches may be adapted for in vitro and in vivo adherence studies related to gonococcal pathogenesis.
淋病奈瑟菌是一种专性人类病原体，其对宿主细胞的附着对于其发病机制至关重要。淋病奈瑟菌的粘附测定基于在固体培养基上附着在人细胞上的细菌计数。由于传统的粘附测定基于细菌计数，因此通常需要花费很长时间才能完成，并且容易受到观察者偏见的影响。已经开发了一种基于流式细胞术的方法，该方法使用可渗透细胞的荧光染料 5'-羧基荧光素琥珀酰亚酯（CFSE），可极大地增加每个测定中定量的附着的淋病奈瑟菌的数量，同时提高重复性并消除观察者偏见。用 CFSE 染色有纤毛的淋病奈瑟菌 F62，然后用胎牛血清终止染色反应。将 CFSE 染色的淋病奈瑟菌（感染倍数为 100：1）以感染 ME-180 细胞 2 小时。用洗涤液洗涤感染的细胞以去除松散附着的细菌。使用流式细胞术定量与 CFSE 染色的淋病奈瑟菌相关联的 ME-180 细胞的百分比，并记录至少 30,000 个事件。对感染的 ME-180 细胞进行针对 opa 基因（编码淋病奈瑟菌不透明度相关的淋病奈瑟菌外膜蛋白）的实时 PCR 分析，以确认流式细胞术粘附测定结果。用热灭活的淋病奈瑟菌 F62 免疫兔子以产生高免疫血清。通过研究淋病奈瑟菌 F62 抗血清对阻止 CFSE 染色的细菌附着/入侵 ME-180 细胞的作用，确认了测定的功能兼容性。
我们观察到 20.3%（+/-1.0）ME-180 细胞与 CFSE 染色的淋病奈瑟菌相关联。在 1:10 至 1:80 的稀释度下，热失活的高免疫血清分别显著抑制淋球菌附着 6 倍和 3 倍。针对 opa 基因的实时 PCR 分析证实，高免疫血清以稀释依赖性方式阻断淋病奈瑟菌对 ME-180 细胞的附着/入侵。
流式细胞术分析适用于快速，简便和高通量定量淋病奈瑟菌与 ME-180 细胞的关联，并且通过 PCR 分析进行了功能验证。这些方法可以适应与淋病奈瑟菌发病机制相关的体外和体内粘附研究。
