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Expression of Opacity Proteins Interferes with the Transmigration of Neisseria gonorrhoeae across Polarized Epithelial Cells.

Stein DC, LeVan A, Hardy B, Wang LC, Zimmerman L, Song W - PLoS ONE (2015)

Bottom Line: When MS11Opa+, able to phase vary Opa expression, was inoculated, only those bacteria that turn off Opa expression transmigrate across the polarized epithelial monolayer.Apical inoculation of MS11Opa+, but not MS11∆Opa, induces the recruitment of the Opa host-cell receptor carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) to the apical junction and the vicinity of bacterial adherent sites.Our results suggest that Opa expression limits gonococcal ability to invade into subepithelial tissues by forming tight interactions with neighboring bacteria and by inducing CEACAM redistribution to cell junctions.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology & Molecular Genetics, University of Maryland, College Park, Maryland, United States of America.

ABSTRACT
Neisseria gonorrhoeae (GC) establishes infection at the mucosal surface of the human genital tract, most of which is lined with polarized epithelial cells. GC can cause localized as well as disseminated infections, leading to various complications. GC constantly change their surface structures via phase and antigenic variation, which has been implicated as a means for GC to establish infection at various anatomic locations of male and female genital tracks. However, the exact contribution of each surface molecule to bacterial infectivity remains elusive due to their phase variation. Using a GC derivative that is genetically devoid of all opa genes (MS11∆Opa), this study shows that Opa expression interferes with GC transmigration across polarized human epithelial cells. MS11∆Opa transmigrates across polarized epithelial cells much faster and to a greater extent than MS11Opa+, while adhering at a similar level as MS11Opa+. When MS11Opa+, able to phase vary Opa expression, was inoculated, only those bacteria that turn off Opa expression transmigrate across the polarized epithelial monolayer. Similar to bacteria alone or co-cultured with non-polarized epithelial cells, MS11∆Opa fails to form large microcolonies at the apical surface of polarized epithelial cells. Apical inoculation of MS11Opa+, but not MS11∆Opa, induces the recruitment of the Opa host-cell receptor carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) to the apical junction and the vicinity of bacterial adherent sites. Our results suggest that Opa expression limits gonococcal ability to invade into subepithelial tissues by forming tight interactions with neighboring bacteria and by inducing CEACAM redistribution to cell junctions.

No MeSH data available.


Related in: MedlinePlus

Opa expression alters GC-induced redistribution of CEACAM at the apical surface.Polarized T84 cells on transwells were incubated with GC MS11OpaH and MS11ΔOpa for 4 h. Cells were fixed, permeabilized, stained for the junctional protein ZO-1, gonococci and CEACAM1, and z-series images were acquired using a confocal microscope. Three z-series images at the apical surface were merged (A). Scale bar, 5 µm. Big arrows, big GC aggregates; and small arrows, small GC aggregates. The percentage of GC clusters with CEACAM patches in the vicinity was determined (B). The Pearson correlation coefficient between CEACAM and ZO1 staining was determined using NIH ImageJ software (C). Shown are representative images and the average values from three or four independent experiments. *p = 0.05. ***p = 0.001.
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pone.0134342.g002: Opa expression alters GC-induced redistribution of CEACAM at the apical surface.Polarized T84 cells on transwells were incubated with GC MS11OpaH and MS11ΔOpa for 4 h. Cells were fixed, permeabilized, stained for the junctional protein ZO-1, gonococci and CEACAM1, and z-series images were acquired using a confocal microscope. Three z-series images at the apical surface were merged (A). Scale bar, 5 µm. Big arrows, big GC aggregates; and small arrows, small GC aggregates. The percentage of GC clusters with CEACAM patches in the vicinity was determined (B). The Pearson correlation coefficient between CEACAM and ZO1 staining was determined using NIH ImageJ software (C). Shown are representative images and the average values from three or four independent experiments. *p = 0.05. ***p = 0.001.

Mentions: The proteins of the CEACAM family are binding targets of Opa on the surface of epithelial cells. The interaction of Opa with CEACAMs mediates GC adherence to host cells and leads to GC invasion [13, 40]. Therefore, we postulated that Opa-expressing GC but not those without Opa would recruit CEACAMs to adherent sites on polarized epithelial cells. To test this hypothesis, we examined the distribution of CEACAMs at the apical surface of polarized epithelial cells inoculated with or without piliated MS11Opa+, MS11ΔOpa and MS11OpaH using 3D immunofluorescence microscopy. We utilized the immunostaining of ZO1 to mark the apical junction, which divides the apical and basolateral surfaces. In uninoculated T84 cells, CEACAM staining concentrated primarily and distributed evenly at the apical surface (Fig 2Aa). Apical inoculation of either MS11OpaH or MS11ΔOpa induced the redistribution of CEACAMs at the apical surface into irregular patches; however, only in MS11OpaH-inoculated epithelial cells, CEACAM patches were located in the vicinity of bacteria, no matter if they were in big (big arrows) or small (small arrows) aggregates (Fig 2Ab and 2Ac). To evaluate the relationship between CEACAM patches and GC clusters, we determined the percentage of GC clusters with CEACAM patches in the vicinity (Fig 2B). Our results showed that there was a significant higher percentage (71%) of OpaH-expressing GC clusters with CEACAM patches in their vicinity than MS11ΔOpa (28%) (Fig 2B). Furthermore, in MS11OpaH inoculated but not MS11ΔOpa inoculated epithelial cells, CEACAM staining also appeared at the apical junction marked by ZO1 staining (Fig 2Ab and 2Ac). We quantified colocalization of CEACAMs with the apical junction by measuring the Pearson correlation coefficients between CEACAM and ZO1 staining (Fig 2C). We found that there was a significant colocalization between CEACAM and ZO1 staining in MS11OpaH-inoculated cells, compared to no significant CEACAM and ZO1 colocalization in MS11ΔOpa-inoculated and uninoculated epithelial cells (Fig 2C). These results indicate that Opa expression is required for recruitment of CEACAMs to the vicinity of GC adherent sites and the apical junction, but is not essential for the induction of CEACAM redistribution at the apical surface of polarized epithelial cells.


Expression of Opacity Proteins Interferes with the Transmigration of Neisseria gonorrhoeae across Polarized Epithelial Cells.

Stein DC, LeVan A, Hardy B, Wang LC, Zimmerman L, Song W - PLoS ONE (2015)

Opa expression alters GC-induced redistribution of CEACAM at the apical surface.Polarized T84 cells on transwells were incubated with GC MS11OpaH and MS11ΔOpa for 4 h. Cells were fixed, permeabilized, stained for the junctional protein ZO-1, gonococci and CEACAM1, and z-series images were acquired using a confocal microscope. Three z-series images at the apical surface were merged (A). Scale bar, 5 µm. Big arrows, big GC aggregates; and small arrows, small GC aggregates. The percentage of GC clusters with CEACAM patches in the vicinity was determined (B). The Pearson correlation coefficient between CEACAM and ZO1 staining was determined using NIH ImageJ software (C). Shown are representative images and the average values from three or four independent experiments. *p = 0.05. ***p = 0.001.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4526573&req=5

pone.0134342.g002: Opa expression alters GC-induced redistribution of CEACAM at the apical surface.Polarized T84 cells on transwells were incubated with GC MS11OpaH and MS11ΔOpa for 4 h. Cells were fixed, permeabilized, stained for the junctional protein ZO-1, gonococci and CEACAM1, and z-series images were acquired using a confocal microscope. Three z-series images at the apical surface were merged (A). Scale bar, 5 µm. Big arrows, big GC aggregates; and small arrows, small GC aggregates. The percentage of GC clusters with CEACAM patches in the vicinity was determined (B). The Pearson correlation coefficient between CEACAM and ZO1 staining was determined using NIH ImageJ software (C). Shown are representative images and the average values from three or four independent experiments. *p = 0.05. ***p = 0.001.
Mentions: The proteins of the CEACAM family are binding targets of Opa on the surface of epithelial cells. The interaction of Opa with CEACAMs mediates GC adherence to host cells and leads to GC invasion [13, 40]. Therefore, we postulated that Opa-expressing GC but not those without Opa would recruit CEACAMs to adherent sites on polarized epithelial cells. To test this hypothesis, we examined the distribution of CEACAMs at the apical surface of polarized epithelial cells inoculated with or without piliated MS11Opa+, MS11ΔOpa and MS11OpaH using 3D immunofluorescence microscopy. We utilized the immunostaining of ZO1 to mark the apical junction, which divides the apical and basolateral surfaces. In uninoculated T84 cells, CEACAM staining concentrated primarily and distributed evenly at the apical surface (Fig 2Aa). Apical inoculation of either MS11OpaH or MS11ΔOpa induced the redistribution of CEACAMs at the apical surface into irregular patches; however, only in MS11OpaH-inoculated epithelial cells, CEACAM patches were located in the vicinity of bacteria, no matter if they were in big (big arrows) or small (small arrows) aggregates (Fig 2Ab and 2Ac). To evaluate the relationship between CEACAM patches and GC clusters, we determined the percentage of GC clusters with CEACAM patches in the vicinity (Fig 2B). Our results showed that there was a significant higher percentage (71%) of OpaH-expressing GC clusters with CEACAM patches in their vicinity than MS11ΔOpa (28%) (Fig 2B). Furthermore, in MS11OpaH inoculated but not MS11ΔOpa inoculated epithelial cells, CEACAM staining also appeared at the apical junction marked by ZO1 staining (Fig 2Ab and 2Ac). We quantified colocalization of CEACAMs with the apical junction by measuring the Pearson correlation coefficients between CEACAM and ZO1 staining (Fig 2C). We found that there was a significant colocalization between CEACAM and ZO1 staining in MS11OpaH-inoculated cells, compared to no significant CEACAM and ZO1 colocalization in MS11ΔOpa-inoculated and uninoculated epithelial cells (Fig 2C). These results indicate that Opa expression is required for recruitment of CEACAMs to the vicinity of GC adherent sites and the apical junction, but is not essential for the induction of CEACAM redistribution at the apical surface of polarized epithelial cells.

Bottom Line: When MS11Opa+, able to phase vary Opa expression, was inoculated, only those bacteria that turn off Opa expression transmigrate across the polarized epithelial monolayer.Apical inoculation of MS11Opa+, but not MS11∆Opa, induces the recruitment of the Opa host-cell receptor carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) to the apical junction and the vicinity of bacterial adherent sites.Our results suggest that Opa expression limits gonococcal ability to invade into subepithelial tissues by forming tight interactions with neighboring bacteria and by inducing CEACAM redistribution to cell junctions.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology & Molecular Genetics, University of Maryland, College Park, Maryland, United States of America.

ABSTRACT
Neisseria gonorrhoeae (GC) establishes infection at the mucosal surface of the human genital tract, most of which is lined with polarized epithelial cells. GC can cause localized as well as disseminated infections, leading to various complications. GC constantly change their surface structures via phase and antigenic variation, which has been implicated as a means for GC to establish infection at various anatomic locations of male and female genital tracks. However, the exact contribution of each surface molecule to bacterial infectivity remains elusive due to their phase variation. Using a GC derivative that is genetically devoid of all opa genes (MS11∆Opa), this study shows that Opa expression interferes with GC transmigration across polarized human epithelial cells. MS11∆Opa transmigrates across polarized epithelial cells much faster and to a greater extent than MS11Opa+, while adhering at a similar level as MS11Opa+. When MS11Opa+, able to phase vary Opa expression, was inoculated, only those bacteria that turn off Opa expression transmigrate across the polarized epithelial monolayer. Similar to bacteria alone or co-cultured with non-polarized epithelial cells, MS11∆Opa fails to form large microcolonies at the apical surface of polarized epithelial cells. Apical inoculation of MS11Opa+, but not MS11∆Opa, induces the recruitment of the Opa host-cell receptor carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) to the apical junction and the vicinity of bacterial adherent sites. Our results suggest that Opa expression limits gonococcal ability to invade into subepithelial tissues by forming tight interactions with neighboring bacteria and by inducing CEACAM redistribution to cell junctions.

No MeSH data available.


Related in: MedlinePlus