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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 reduces the kinetics and magnitude of GC transmigration.(A-C) Polarized T84 cells grown on transwells were incubated with GC apically at a MOI of 10:1 for varying periods of times. The basolateral media were plated onto GCK to enumerate the number of GC that transmigrated into the basolateral media (A and C). Epithelial cells were lysed and plated to determine the number of adhered GC (B). Shown is the average CFU per transwell (A-B) or the average percentage of transmigrated GC over total epithelial-associated GC (C) from three independent experiments, each of which performed in triplicate. *p< 0.05. (D) The cells were fixed and stained for GC and ZO1. Images of z-series were acquired using a confocal microscope. Shown are xy images at the apical junction areas (top) and xz images from 3D reconstitution across both apical and basolateral surfaces (bottom) from three independent experiments. Scale bar, 10 μm. (E) The cells were processed for transmission electronic microscopy. Arrows, GC. Scale bar, 2 μm.
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pone.0134342.g003: Opa expression reduces the kinetics and magnitude of GC transmigration.(A-C) Polarized T84 cells grown on transwells were incubated with GC apically at a MOI of 10:1 for varying periods of times. The basolateral media were plated onto GCK to enumerate the number of GC that transmigrated into the basolateral media (A and C). Epithelial cells were lysed and plated to determine the number of adhered GC (B). Shown is the average CFU per transwell (A-B) or the average percentage of transmigrated GC over total epithelial-associated GC (C) from three independent experiments, each of which performed in triplicate. *p< 0.05. (D) The cells were fixed and stained for GC and ZO1. Images of z-series were acquired using a confocal microscope. Shown are xy images at the apical junction areas (top) and xz images from 3D reconstitution across both apical and basolateral surfaces (bottom) from three independent experiments. Scale bar, 10 μm. (E) The cells were processed for transmission electronic microscopy. Arrows, GC. Scale bar, 2 μm.

Mentions: As Opa expression alters the distribution of bacteria and CEACAM at the apical surface of polarized epithelial cells, we investigated whether Opa expression has any impact on the ability of GC to adhere and transmigrate across a polarized epithelial monolayer. Polarized T84 cells were incubated with piliated bacteria in the apical chamber for various times and the number of transmigrated bacteria in the basolateral medium and adhered bacteria in epithelial lysates were enumerated. We were able to collect ~100 CFU of MS11ΔOpa GC in the basolateral media as early as 4 h, compared to an occasional MS11Opa+ (Fig 3A). By 6 h, the basolateral MS11ΔOpa reached >1000 CFU, compared to only ~10 CFU of MS11Opa+ GC in the basolateral medium (Fig 3A). The transmigration efficiency of MS11OpaH was similar to that of MS11Opa+ (data not shown). In contrast, we did not detect a significant difference in the numbers of the two different strains of bacteria adhered to polarized epithelial cells after 6 h incubation (Fig 3B). Furthermore, non-piliated MS11ΔOpa showed a similar increase in the transmigration efficiency over non-piliated MS11Opa+ bacteria (Fig 3C). The similar levels of adherence between MS11Opa+ and MS11ΔOpa and similar increases in the transmigration efficiency of non-piliated MS11ΔOpa over non-piliated MS11Opa+ GC suggest that pili phase variation has no significant impact on GC adherence to and transmigration across polarized epithelial cells. Since Opa deletion does not change the level of GC adherence, this indicates that the different aggregative ability of the Opa+ and ΔOpa strains does not significantly affect bacterial quantification.


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 reduces the kinetics and magnitude of GC transmigration.(A-C) Polarized T84 cells grown on transwells were incubated with GC apically at a MOI of 10:1 for varying periods of times. The basolateral media were plated onto GCK to enumerate the number of GC that transmigrated into the basolateral media (A and C). Epithelial cells were lysed and plated to determine the number of adhered GC (B). Shown is the average CFU per transwell (A-B) or the average percentage of transmigrated GC over total epithelial-associated GC (C) from three independent experiments, each of which performed in triplicate. *p< 0.05. (D) The cells were fixed and stained for GC and ZO1. Images of z-series were acquired using a confocal microscope. Shown are xy images at the apical junction areas (top) and xz images from 3D reconstitution across both apical and basolateral surfaces (bottom) from three independent experiments. Scale bar, 10 μm. (E) The cells were processed for transmission electronic microscopy. Arrows, GC. Scale bar, 2 μm.
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Related In: Results  -  Collection

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pone.0134342.g003: Opa expression reduces the kinetics and magnitude of GC transmigration.(A-C) Polarized T84 cells grown on transwells were incubated with GC apically at a MOI of 10:1 for varying periods of times. The basolateral media were plated onto GCK to enumerate the number of GC that transmigrated into the basolateral media (A and C). Epithelial cells were lysed and plated to determine the number of adhered GC (B). Shown is the average CFU per transwell (A-B) or the average percentage of transmigrated GC over total epithelial-associated GC (C) from three independent experiments, each of which performed in triplicate. *p< 0.05. (D) The cells were fixed and stained for GC and ZO1. Images of z-series were acquired using a confocal microscope. Shown are xy images at the apical junction areas (top) and xz images from 3D reconstitution across both apical and basolateral surfaces (bottom) from three independent experiments. Scale bar, 10 μm. (E) The cells were processed for transmission electronic microscopy. Arrows, GC. Scale bar, 2 μm.
Mentions: As Opa expression alters the distribution of bacteria and CEACAM at the apical surface of polarized epithelial cells, we investigated whether Opa expression has any impact on the ability of GC to adhere and transmigrate across a polarized epithelial monolayer. Polarized T84 cells were incubated with piliated bacteria in the apical chamber for various times and the number of transmigrated bacteria in the basolateral medium and adhered bacteria in epithelial lysates were enumerated. We were able to collect ~100 CFU of MS11ΔOpa GC in the basolateral media as early as 4 h, compared to an occasional MS11Opa+ (Fig 3A). By 6 h, the basolateral MS11ΔOpa reached >1000 CFU, compared to only ~10 CFU of MS11Opa+ GC in the basolateral medium (Fig 3A). The transmigration efficiency of MS11OpaH was similar to that of MS11Opa+ (data not shown). In contrast, we did not detect a significant difference in the numbers of the two different strains of bacteria adhered to polarized epithelial cells after 6 h incubation (Fig 3B). Furthermore, non-piliated MS11ΔOpa showed a similar increase in the transmigration efficiency over non-piliated MS11Opa+ bacteria (Fig 3C). The similar levels of adherence between MS11Opa+ and MS11ΔOpa and similar increases in the transmigration efficiency of non-piliated MS11ΔOpa over non-piliated MS11Opa+ GC suggest that pili phase variation has no significant impact on GC adherence to and transmigration across polarized epithelial cells. Since Opa deletion does not change the level of GC adherence, this indicates that the different aggregative ability of the Opa+ and ΔOpa strains does not significantly affect bacterial quantification.

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