Abstract
BackgroundNeisseria 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.ResultsWe 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.ConclusionsFlow 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.
Highlights
Neisseria gonorrhoeae is an obligate human pathogen and its adherence to host cells is essential for its pathogenesis
Viable bacteria are shown in Gate A which confirms that N. gonorrhoeae are sufficiently large and complex for them to be detected and quantified using the flow cytometer and that carboxyfluoroscein succidyl ester (CFSE) staining did not impact bacteria size or complexity (Fig. 1b).In Fig. 1c, we show flow cytometric analysis of unstained N. gonorrhoeae F62 and Gate B is drawn above the background fluorescence recorded in FL1
Negligible fluorescent events are detected in the unstained bacteria (0.2%; Fig. 1c).When Gate B was applied to the CFSE-stained N. gonorrhoeae, 99.9% of the bacteria were fluorescent (Fig. 1d)
Summary
Neisseria gonorrhoeae is an obligate human pathogen and its adherence to host cells is essential for its pathogenesis. 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. Human cervical ME-180 cells were infected with CFSE-stained N. gonorrhoeae (multiplicity of the infection 100:1) for 2 h. Bacteria are allowed to attach to solid-phase immobilized cells, they are washed and enumerated either by viable counts or by counting the bacteria visible under a microscope. These methods tend to be time consuming, are not always highly repeatable, and are prone to observer bias [1]. Gonococcal infection is a major public health threat because it facilitates the transmission and acquisition of Human Immunodeficiency Virus [6] and because gonorrhea may become untreatable as N. gonorrhoeae has developed resistance against all classes of antimicrobials [7]
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