Journal: The Journal of Infectious Diseases

Article Title: Desialylation of Neisseria gonorrhoeae Lipooligosaccharide by Cervicovaginal Microbiome Sialidases: The Potential for Enhancing Infectivity in Men

doi: 10.1093/infdis/jiw329

Figure Lengend Snippet: Sialidase activity (fluorescent changes based on the hydrolysis of 4-methyl-umbelliferyl-N-acetylneuraminic acid; Toronto Research Chemicals) [28]) in genital secretions; threshold for detection, <0.1 mU/µL (baseline). Group A denotes fluorescent readings for 84 cervicovaginal samples from women attending sexually transmitted diseases or adolescent health clinics. Group B denotes fluorescent readings in urethral exudates from 7 men, 5 of whom were sex partners of women represented in group B. Group C denotes fluorescent readings in samples from 11 gonococci-uninfected women attending a gynecology clinic for cervical cancer screening. Statistical analysis was performed comparing groups A and C was performed using the Mann–Whitney test.

Article Snippet: Statistical comparisons between the 3 groups of subjects in Figure were performed using the Mann–Whitney test, assuming a nonparametric distribution. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 1. caption a7 Sialidase activity (fluorescent changes based on the hydrolysis of 4-methyl-umbelliferyl- N -acetylneuraminic acid; Toronto Research Chemicals) [ 28 ]) in genital secretions; threshold for detection, <0.1 mU/µL (baseline).

Techniques: Activity Assay, MANN-WHITNEY

Journal: The Journal of Infectious Diseases

Article Title: Desialylation of Neisseria gonorrhoeae Lipooligosaccharide by Cervicovaginal Microbiome Sialidases: The Potential for Enhancing Infectivity in Men

doi: 10.1093/infdis/jiw329

Figure Lengend Snippet: Sodium dodecyl sulfate polyacrylamide gel electrophoresis of lipooligosaccharide (LOS) from Neisseria gonorrhoeae strain 1291 after incubation with sialidase containing secretions. LOS migration patterns: 4.8 k, fully sialylated; 4.5 k, desialylated. A–G, Heat-killed sialylated N. gonorrhoeae strain 1291 incubated with unheated or boiled (Δ) secretion samples, extracted with protease, and electrophoresed. LOS in unboiled secretion samples present in lanes A, E, F, and G (left-side of brackets) that migrated to 4.5 k indicated complete desialylation of LOS; C showed partial desialylation and B and D minimal desialylation. Controls included LOS in boiled secretion samples (A–G; right-side of brackets [Δ]) where sialylated LOS migrated to 4.8 k. Controls: C+, positive control demonstrating sialylated1291 LOS desialylated using Vibrio cholera sialidase. The respective sialidase assay values obtained for the corresponding sample (taken from Figure ​Figure2)2) are indicated above the gel image.

Article Snippet: Statistical comparisons between the 3 groups of subjects in Figure were performed using the Mann–Whitney test, assuming a nonparametric distribution. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 1. caption a7 Sialidase activity (fluorescent changes based on the hydrolysis of 4-methyl-umbelliferyl- N -acetylneuraminic acid; Toronto Research Chemicals) [ 28 ]) in genital secretions; threshold for detection, <0.1 mU/µL (baseline).

Techniques: Polyacrylamide Gel Electrophoresis, Incubation, Migration, Positive Control

Journal: The Journal of Infectious Diseases

Article Title: Desialylation of Neisseria gonorrhoeae Lipooligosaccharide by Cervicovaginal Microbiome Sialidases: The Potential for Enhancing Infectivity in Men

doi: 10.1093/infdis/jiw329

Figure Lengend Snippet: Immunodot assays using monoclonal antibody (MAb) 6B4, which recognizes the unsialylated N-acetyllactosamine terminus of Neisseria gonorrhoeae strain 1291 lipooligosaccharide (LOS). A, Cervicovaginal secretions used to treat sialylated N. gonorrhoeae 1291. B, Binding of MAb 6B4 (or lack thereof) to N. gonorrhoeae 1291 LOS following treatment with unboiled cervicovaginal secretions as a potential source of sialidase; samples 543-113 and 674-148 showed no/minimal sialidase assay values (indicated in lane D [see below also]). C, Binding (or lack thereof) of MAb 6B4 to sialylated N. gonorrhoeae 1291 LOS following treatment with boiled cervicovaginal secretions; sample 455-096, which contained blood, was positive following treatment with both boiled and unboiled secretions (see text). Lane D shows the respective sialidase assay values in milliunits per microliter obtained for the corresponding samples (taken from Figure ​Figure2).2). The immunodots labeled “positive control” demonstrate the effect of V. cholerae sialidase treatment (0.05 mU/µL) on sialylated N. gonorrhoeae 1291.

Article Snippet: Statistical comparisons between the 3 groups of subjects in Figure were performed using the Mann–Whitney test, assuming a nonparametric distribution. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 1. caption a7 Sialidase activity (fluorescent changes based on the hydrolysis of 4-methyl-umbelliferyl- N -acetylneuraminic acid; Toronto Research Chemicals) [ 28 ]) in genital secretions; threshold for detection, <0.1 mU/µL (baseline).

Techniques: Binding Assay, Labeling, Positive Control

Journal: The Journal of Infectious Diseases

Article Title: Desialylation of Neisseria gonorrhoeae Lipooligosaccharide by Cervicovaginal Microbiome Sialidases: The Potential for Enhancing Infectivity in Men

doi: 10.1093/infdis/jiw329

Figure Lengend Snippet: Representative confocal microscopic image of a Neisseria gonorrhoeae–infected genital (vaginal) secretion. A and B, Confocal images at 400× original magnification with phase contrast and stained with monoclonal antibody (MAb) 6B4 and FITC. A, A secretion from an individual with a sialidase level of 1.20 mU/µL in her secretion sample. MAb 6B4–stained organisms (white arrows) can be seen associated with the cells in the secretion, indicating the presence of the N-acetyllactosamine epitope on the surface. In the lower left hand corner, the dotted box is enlarged 2-fold to demonstrate the biscuit-like diplococcic characteristic of N. gonorrhoeae. B, Findings from a similar study of another subject also infected with N. gonorrhoeae but with a sialidase level of approximately 0.10 mU/µL. No organisms with a free N-acetyllactosamine epitope on the surface were observed upon careful scanning of the samples from 2 subjects. These studies suggest that, as we show in the laboratory studies, the level of sialidase in the secretion is critical with regard to whether desialylation of the gonococcus occurs. C, Results of confocal studies from another subject in which the eukaryotic cell nuclei (PMN and epithelial cell) were stained with ethidium bromide (red) and then counterstained with MAb 6B4 (murine immunoglobulin M [IgM]) that recognizes the gonococcal N-acetyllactosamine epitope; fluorescein-labeled goat anti-mouse IgM (green) was used as secondary antibody. White arrows designate some of the N. gonorrhoeae in the field. D, An infected vaginal-secretion specimen showing colocalization of gonococcal MAb 6B4 and MAb 2C3 (murine immunoglobulin G [IgG]) specific for the gonococcal H.8 protein, to ensure that MAb 6B4 staining was specific for N. gonorrhoeae. Texas red–labeled goat anti-mouse IgG was used as secondary antibody for labeling of MAb 2C3, and fluorescein-labeled goat anti-mouse IgM (green) was used as secondary antibody for MAb 6B4. The merged label on some of the stained gonococci is yellow and designated by white arrows showing the MAbs 6B4 and 2C3 colocalization.

Article Snippet: Statistical comparisons between the 3 groups of subjects in Figure were performed using the Mann–Whitney test, assuming a nonparametric distribution. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 1. caption a7 Sialidase activity (fluorescent changes based on the hydrolysis of 4-methyl-umbelliferyl- N -acetylneuraminic acid; Toronto Research Chemicals) [ 28 ]) in genital secretions; threshold for detection, <0.1 mU/µL (baseline).

Techniques: Infection, Staining, Labeling

Journal: The Journal of Infectious Diseases

Article Title: Desialylation of Neisseria gonorrhoeae Lipooligosaccharide by Cervicovaginal Microbiome Sialidases: The Potential for Enhancing Infectivity in Men

doi: 10.1093/infdis/jiw329

Figure Lengend Snippet: Figure ​Figure55 shows results of a confocal study of a subject with sialidase levels in secretions (0.22 mU/µL) in whom the desialylation of the genital tract epithelial cells can be seen after staining with monoclonal antibody (MAb) 6B4. A, An image (400× original magnification) of a secretion sample stained with MAb 6B4 with phase contrast. B, An image (630× original magnification) of the area enclosed in the dashed box in panel A. As can be seen, the surface of a number of cells in both images is stained by MAb 6B4, indicating desialylation of the cell surface. White arrows designate the putative gonococci in the image.

Article Snippet: Statistical comparisons between the 3 groups of subjects in Figure were performed using the Mann–Whitney test, assuming a nonparametric distribution. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 1. caption a7 Sialidase activity (fluorescent changes based on the hydrolysis of 4-methyl-umbelliferyl- N -acetylneuraminic acid; Toronto Research Chemicals) [ 28 ]) in genital secretions; threshold for detection, <0.1 mU/µL (baseline).

Techniques: Staining