Development of flow cytometry based adherence assay for Neisseria gonorrhoeae using 5′-carboxyfluorosceinsuccidyl ester
| Autor: | S D Thakur, Jo-Anne R. Dillon, Siew Hon Ng, Milan Obradovic, Heather L. Wilson |
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| Rok vydání: | 2019 |
| Předmět: |
Microbiology (medical)
lcsh:QR1-502 Succinimides Cervix Uteri Neutralizing antibodies medicine.disease_cause Microbiology lcsh:Microbiology Bacterial Adhesion Cell Line Flow cytometry 03 medical and health sciences In vivo medicine Humans 5′-carboxyfluoroscein succidyl ester Bovine serum albumin Fluorescent Dyes Antiserum 0303 health sciences biology medicine.diagnostic_test 030306 microbiology Methodology Article Epithelial Cells Flow Cytometry Fluoresceins biology.organism_classification Neisseria gonorrhoeae In vitro 3. Good health Staining biology.protein Female Bacteria Bacterial Outer Membrane Proteins |
| Zdroj: | BMC Microbiology BMC Microbiology, Vol 19, Iss 1, Pp 1-8 (2019) |
| ISSN: | 1471-2180 |
| Popis: | Background 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. Results 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. Conclusions 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. Electronic supplementary material The online version of this article (10.1186/s12866-019-1438-2) contains supplementary material, which is available to authorized users. |
| Databáze: | OpenAIRE |
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