A CEACAM-binding adhesin is sufficient to counteract the detachment of human cervical epithelial cells upon bacterial infection

Previous work demonstrated that a CEACAM-binding adhesin is necessary to allow Neisseria gonorrhoeae to increase extracellular matrix binding of infected cells [14,16]. To test if CEACAM-binding alone is sufficient to trigger this process, we separated the CEACAM-binding Opa_CEA adhesin from other gonococcal virulence determinants by expressing this neisserial outer membrane protein in E. coli. In the used expression plasmid pTrc, Opa_CEA-protein expression is under control of the IPTG-inducible lac promoter [22]. Due to the leakiness of this promoter, Opa_CEA protein-expression was already observed under non-inducing conditions in Opa_CEA-expressing E. coli (E. coli Opa_CEA) compared to an E. coli strain harbouring the empty pTrc plasmid (S1A Fig). The Opa_CEA protein expressed in E. coli showed a similar size to the native Opa_CEA protein expressed in N. gonorrhoeae, but levels were only about 20% of that observed in gonococci (S1A Fig). Both E. coli strains showed a comparable growth pattern in liquid culture, indicating that expression of the Opa_CEA protein in E. coli at this level did not interfere with growth (S1B Fig). In contrast, strong overexpression, e.g. upon IPTG induction, can retard E. coli growth (S1C Fig). When expressed in E. coli, the neisserial Opa protein adhesin was functional with regard to CEACAM-binding, as E. coli Opa_CEA was able to associate with the GFP-tagged amino-terminal domain of CEA (CEA-N; Fig 1A). The control E. coli strain did not bind to soluble CEA-N, and no binding of either E. coli strain to the CEACAM8-amino-terminal domain (CEA8-N) was observed (Fig 1A). This binding pattern of the heterologously expressed Opa_CEA protein is in agreement with the CEACAM binding profile observed for N. gonorrhoeae expressing this Opa_CEA protein (Fig 1A). To investigate, whether E. coli Opa_CEA interacts with CEA in a cellular context, we infected the human cervical epithelial cell line ME-180 for 2 h with the E. coli control strain or with E. coli Opa_CEA. ME-180 cells endogenously express CEACAM1, CEACAM6, and CEA, which were located in the plasma membrane of uninfected cells (S2A and S2B Fig). Upon infection with the E. coli control strain, the distribution of CEACAMs was unchanged and these bacteria did not associate with ME-180 cells (Fig 1B and S2B Fig). Importantly, E. coli Opa_CEA strongly adhered to ME-180 cells and triggered re-location and local concentration of CEACAMs at sites of bacteria-host cell contact (Fig 1B and S2B Fig; arrowheads). Similarly, Opa_CEA protein-expressing gonococci adhered in large numbers to ME-180 cells and induced CEACAM clustering (Fig 1B and S2B Fig). Together, these results demonstrated that the neisserial adhesin is functionally expressed in E. coli, where it promotes interaction with CEA-expressing cells.

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Fig 1. CEACAM engagement by E. coli strains expressing Opa_CEA adhesins blocks the detachment of human cervical epithelial cells.

(A) Cell culture supernatants containing GFP-fused Ig_V-like domains of CEA (CEA-N) or CEACAM8 (CEA8-N) or control supernatants (pcDNA) were prepared from transfected 293 cells and analysed by Western blotting with a monoclonal anti-GFP antibody to verify similar amounts of the soluble fusion proteins (Supe; left panel). E. coli, E. coli expressing a CEACAM-binding Opa protein from N. gonorrhoeae (E. coli Opa_CEA), or non-piliated, Opa_CEA-expressing N. gonorrhoeae MS11 (Ngo Opa_CEA) were incubated with the indicated CEA-N- or CEA8-N-containing or control cell culture supernatants for 2 h at room temperature. Bacteria were washed with PBS and bacteria-bound GFP-fusion proteins were detected by Western blotting with anti-GFP antibody (Pull-down; right panel). Shown is a representative experiment out of three independent biological replicates. (B) ME-180 cells were seeded on glasscover slips, infected with the indicated bacteria for 2 h, fixed and co-stained with a monoclonal antibody against CEACAMs (clone D14HD11; red) together with polyclonal rabbit antibodies against E. coli or against N. gonorrhoeae MS11 (green). Presented are merged images showing the recruitment of CEACAMs to cell-associated CEACAM-binding bacteria (arrowheads). Pictures are representative for four independent biological replicates. (C) ME-180 cells were seeded on 25 μg/ml collagen, confluent layers were left uninfected or infected for 14h with piliated, non-opaque N. gonorrhoeae (Ngo P+), Ngo Opa_CEA, E. coli, or E. coli Opa_CEA. Following infection, cells were washed and remaining cells were stained with crystal violet. Staining intensity was determined after dye elution in a spectrophotometer at 550 nm. Bars represent mean ± SD of eight replicates. Two-tailed student’s t-test; *** p < 0.001, n.s.—not significant. Shown is one representative experiment out of four independent biological replicates. (D) ME-180 cells were infected and stained as in (C). Representative areas with remaining cells were photographed. (E) Confluent layers of ME-180 cultures were infected with the indicated bacteria or left uninfected. After 14 h, cells were fixed in situ and analysed by scanning electron microscopy. Uninfected ME-180 cells were tightly adherent to the cell culture surface and displayed numerous cell-cell contacts (arrowheads). In contrast, cells infected with E. coli rounded up and reduced cell-cell contacts, whereas cells infected with Opa_CEA-expressing E. coli or N. gonorrhoeae retained cell-cell contacts (arrowheads) despite the presence of bacteria. Pictures are representative for four independent biological replicates.

https://doi.org/10.1371/journal.ppat.1005608.g001

CEA engagement by pathogenic N. gonorrhoeae has been shown to trigger increased matrix adhesion and to counteract the detachment of epithelial cells [14]. Therefore, we infected confluent monolayers of ME-180 cells, grown on collagen, for 14 h or left the ME-180 cells uninfected. Next, monolayers were washed to remove detached and loosely adherent cells and the remaining cells were stained with crystal violet. Elution and quantification of the dye in a spectrophotometer served as a measure of the remaining adherent cells. Interestingly, in samples infected with Opa_CEA protein-expressing N. gonorrhoeae as well as in samples infected with Opa_CEA protein-expressing E. coli no reduction in the amount of adherent cells compared to uninfected cells was observed (Fig 1C). Indeed, ME-180 cells infected with CEACAM-binding bacteria even showed a slightly higher recovery than uninfected cells suggesting that cell-matrix adhesion is reinforced upon infection with Opa_CEA protein expressing bacteria (Fig 1C). In contrast to CEACAM-binding bacteria, cells infected with the E. coli control strain or with piliated, non-CEACAM-binding gonococci (Ngo P+) showed pronounced detachment of cells (Fig 1C). Microscopic observation of the infected cell cultures corroborated the massive loss of epithelial cells from monolayers infected with control E. coli or Ngo P+, whereas cell numbers in samples infected for prolonged times with Opa_CEA protein-expressing bacteria were even higher than in uninfected samples (Fig 1D). Similar results were obtained, when primary human vaginal epithelial (hVECs) cells were infected for 14 h with either Opa_CEA protein-expressing bacteria or non-CEACAM-binding strains. Again, expression of a CEACAM-binding adhesin was able to block the infection-induced detachment of the primary epithelial cells (S3A and S3B Fig). Scanning electron microscopy revealed that ME-180 cells infected for 14 h with control E. coli reduced cell-cell-contacts and rounded up, whereas cells infected with Opa_CEA protein-expressing E. coli remained well-spread on collagen similar to uninfected cultures and similar to cells infected with Opa_CEA-protein expressing gonococci (Fig 1E). These results demonstrate that CEACAM engagement via a CEACAM-binding adhesin, even when expressed in a heterologous background, is sufficient to counteract the detachment of infected epithelial cells in vitro.

E. coli Opa_CEA engagement of CEA leads to upregulation of CD105 expression, increased cell adhesion, and integrin activation

Both human primary vaginal epithelial cells as well as ME180 cervical carcinoma cells endogenously express members of the CEACAM family, in including CEA (S2B and S3C Figs). To rigorously test the contribution of CEACAMs to this process, we employed 293 cells, a human cell line that lacks endogenous CEACAM expression. Upon transient transfection with a CEA-encoding plasmid, about 40% of the cell population showed surface expression of the receptor (S4A Fig). Control transfected or CEA-expressing cells were then infected for 5 h with the indicated bacterial strains and used in cell adhesion assays on collagen. We again observed increased matrix binding of CEA-expressing cells upon infection with Opa_CEA protein-expressing E. coli (S4B Fig). The adhesion of CEA-expressing cells infected with E. coli Opa_CEA was comparable to the increased cell adhesion seen upon infection with Opa_CEA protein-expressing gonococci. In contrast, incubation with the E. coli control strain did not alter matrix adhesion of the infected cells (S4B Fig). Importantly, 293 cells transfected with an empty control plasmid (pcDNA) did not display changes in adhesiveness, irrespective of the bacterial strain used for infection (S4B Fig). These results demonstrate that it is the adhesin-CEACAM interaction, which serves as the trigger for increased extracellular matrix adhesion of the infected cells.

Previously, CEACAM engagement by bacteria has been shown to induce CD105 expression in epithelial cells, which was a pre-requisite for enhanced matrix adhesion of infected cells [16]. Indeed, infection of CEA-expressing 293 cells with Opa_CEA protein-expressing E. coli or N. gonorrhoeae resulted in the presence of CD105 on the surface of the cells, whereas uninfected cells or cells infected with control E. coli did not have detectable CD105 on their surface (S4C Fig). In line with the idea that CEACAM-triggered expression of CD105 is critical for increased cell adhesion, expression of CD105 in 293 cells led to strongly elevated cell-matrix adhesion in the absence of bacterial infection or CEACAM stimulation (S4B Fig).

Though bacterial infection or CD105 expression resulted in alterations in cell-matrix adhesion, the amount of surface exposed integrin β1 was unaltered in all infected samples compared to the uninfected control (S5A Fig). However, infection with CEACAM-binding gonococci or Opa_CEA protein-expressing E. coli promoted a conformational change of integrin β1 as detected by the conformation-sensitive anti-integrin β1 antibody 9EG7 (S5B Fig). Increased labeling by antibody 9EG7 indicated that CEA-engagement by bacteria led to pronounced activation of integrins. In contrast, integrin activity remained low in uninfected cells or cells infected with non-CEACAM-binding bacteria (S5B Fig). The increased integrin activity clearly depended on the presence of CEA, as 293 cells transfected with the empty control vector did not alter the amount of surface integrin nor integrin activity upon infection with diverse bacteria (S5C and S5D Fig). As a further control, cells were stimulated with Mn²⁺, an exogenous activator of integrins. Upon Mn²⁺ addition, a similar, maximal integrin activity was observed in all samples demonstrating that the total activatable integrin levels on the cell surface were similar (S5B and S5D Fig). Together, these findings imply that E. coli Opa_CEA, via engagement of CEACAMs, can trigger CD105 expression, which in turn enhances integrin activity in vitro.

The CEA–E. coli Opa_CEA interaction facilitates mucosal colonization in vivo

Because Opa_CEA-expressing E. coli was able to enhance integrin activity and suppress cell detachment in vitro, we next analyzed bacterial colonization of the urogenital tract. For this purpose, we used either wildtype mice or transgenic mice, expressing human CEA on all mucosal surfaces (CEAtg mice) [14,23]. Accordingly, 8–10 week old female mice were vaginally infected with 1 x 10⁶ bacteria and colonizing bacteria were recovered by urogenital swabs 24 hours later. Only few bacterial colonies of the E. coli control strain could be isolated from wildtype mice and a slightly elevated (~3-fold) recovery of this non-CEACAM binding strain from CEA-tg mice was observed (Fig 2A). In contrast, more than 30-fold higher numbers of Opa_CEA protein-expressing E. coli were recovered from CEAtg mice than from wildtype mice (Fig 2A). Analysis of re-isolated Opa_CEA protein-expressing E. coli showed that expression of the Opa adhesin was unaltered by in vivo growth conditions (Fig 2B). Immunohistochemical staining of tissue sections from the urogenital tract revealed that Opa_CEA-expressing E. coli were closely associated with the CEA-positive tissue surface (Fig 2C). In the case of E. coli control only few bacteria could be detected on the mucosal surface (Fig 2C). Importantly, CD105 was expressed by mucosal epithelial cells of CEAtg mice in contact with Opa_CEA-protein expressing E. coli, whereas the E. coli control strain did not trigger CD105 expression (Fig 2D). Furthermore, E. coli did not trigger CD105 expression in wildtype mice, irrespective of the Opa protein status of the bacteria (S6 Fig). These data demonstrate that Opa_CEA-expressing E. coli are able to associate with CEA-positive epithelial cells and trigger CD105 expression in the urogenital tract in vivo. Together, these data suggest that a CEACAM-binding adhesin is not only necessary, but also sufficient to promote colonization of the mucosal surface via stimulating CD105 expression and enhanced integrin activity in superficial epithelial cells.

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Fig 2. The interaction of E. coli Opa_CEA with CEA facilitates mucosal colonization and leads to CD105 expression in epithelial cells.

(A) Wild-type (●) or CEAtg (▼) female mice were infected with the indicated bacterial strains and 24 h later, bacteria were re-isolated. Each data point in the graph reflects the number of bacteria re-isolated from an individual animal (n = 10; except for CEAtg animals infected with E. coli Opa_CEA, where n = 15). Data were compiled from four independent experiments. The median for each experimental group of animals is indicated by a line; groups were compared by Mann-Whitney U-test and highly significant differences (p<0.001) are indicated by ***. (B) Individual re-isolated bacterial colonies from the genital tract of CEAtg mice infected with either E. coli or E. coli Opa_CEA were plated on LB-ampicillin agar plates (post infection). Five isolates for each strain were analysed by Western blotting with an antibody against Opa protein. As a control, lysates of the E. coli or E. coli Opa_CEA used for infection (input) were also analysed. (C) Genital tracts from CEAtg mice infected for 24 hours with E. coli or E. coli Opa_CEA were excised, and cryosections were co-stained with antibodies against E. coli (green) and against CEA (red). Cell nuclei were stained with Hoechst dye (blue). Arrowheads indicate host-associated E. coli. Pictures are representative for three independent biological replicates. (D) Cryosections as in (C) were co-stained with antibodies against E. coli (green) and a rat monoclonal antibody against murine CD105 (red). Cell nuclei were visualized by Hoechst (blue). CD105 expression on the mucosal surface of CEAtg mice infected with E. coli Opa_CEA is highlighted by small arrows. Pictures are representative for three independent biological replicates.

https://doi.org/10.1371/journal.ppat.1005608.g002

Uropathogenic E. coli AfaE-III engage CEACAMs in a species-specific manner

Besides the neisserial Opa_CEA proteins, additional CEACAM-binding bacterial proteins have been described. For example, uropathogenic E. coli harbouring the Afa/Dr locus of afimbrial adhesins have also been shown to engage CEACAMs including CEACAM1 and CEA [24]. Therefore, we used the uropathogenic E. coli (UPEC) strain A30, which expresses the AfaE-III adhesin (E. coli AfaE-III) encoded within the afa gene cluster on a large virulence plasmid [25]. To monitor AfaE-III-dependent events, we cured strain A30 from the virulence plasmid generating the AfaE-III-negative strain E. coli ΔAfaE-III, which lacks the afa gene cluster (S7A Fig). The wildtype UPEC strain and the ΔAfaE-III strain grew with similar growth kinetics (S7B Fig). Clearly, E. coli AfaE-III was able to associate with human CEACAM1 and CEA in the form of soluble GFP-tagged receptor domains and this property was lost in E. coli ΔAfaE-III (Fig 3A). Similar to other CEACAM-binding bacteria such as gonococci, E. coli AfaE-III selectively bound to human CEACAM1, but not to CEACAM1 orthologues from other mammalian species, including mouse, dog and cattle (Fig 3B). Furthermore, E. coli AfaE-III strongly associated with CEA-expressing ME-180 cells and clustered CEA on the surface of the infected cells, whereas E. coli ΔAfaE-III hardly attached to the cell surface (Fig 3C). These results suggest that E. coli pathovars associated with urogenital infections target human CEACAM family members present on epithelial cells of the urogenital tract via their AfaE-III adhesin.

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Fig 3. Uropathogenic E. coli expressing the AfaE-III adhesin selectively bind to the amino terminal domain of human CEACAM family members.

(A) The indicated soluble CEACAM-GFP fusion proteins (human CEACAM1-NT, CEA-NT, and CEACAM8-NT) were produced in 293 cells and the resulting cell culture supernatants were incubated with E. coli AfaE-III or with the AfaE-III-deficient strain (E. coli ΔAfaE-III). After washing, bacteria-associated CEACAM-GFP fusion proteins were detected by Western blotting with a monoclonal anti-GFP antibody (Pull-down; upper two panels). To verify the presence of equal amounts of the CEACAM-GFP fusion proteins, cell culture supernatants were analyzed by Western blotting with anti-GFP antibody (Supe; lowest panel). Shown is a representative experiment out of three independent biological replicates. (B) Cell culture supernatants containing the soluble N-terminal domains of murine CEACAM1 (mCEA1), bovine CEACAM1a (bCEA1a), bovine CEACAM1b (bCEA1b), canine CEACAM1 (cCEA1), human CEACAM1 (hCEA1) as GFP-fusion proteins, or GFP alone (GFP) were incubated with E. coli AfaE-III. After washing, bacteria-associated CEACAM1 was detected as in (A) (Pull-down; upper panel). The presence of CEACAM-GFP fusion proteins was analyzed as in (A) (Supe; lower panel). Shown is a representative experiment out of three independent biological replicates. (C) ME-180 cells were infected or not with the indicated bacteria for 2 h, fixed and co-stained with antibodies against endogenous CEACAMs (clone D14HD11; red) and rabbit anti-E. coli (green). Arrowheads indicate bacteria bound to clustered CEA. Pictures are representative for four independent biological replicates.

https://doi.org/10.1371/journal.ppat.1005608.g003

CEACAM-binding uropathogenic E. coli AfaE-III promotes integrin activity and epithelial cell adhesion

To investigate, if a CEACAM-binding pathogenic E. coli strain is able to modulate the matrix-adhesion of infected epithelial cells, we again employed 293 cells transfected either with a GFP-encoding control vector or a CEA-encoding expression vector. As observed for CEACAM-binding gonococci, E. coli AfaE-III promoted cell-matrix adhesion in 293 cells expressing CEA, but not in control transfected cells (Fig 4A). Neither the E. coli control strain nor E. coli ΔAfaE-III led to enhanced extracellular matrix adhesion of infected cells (Fig 4A). In agreement with the enhanced extracellular matrix adhesion, CEA-expressing 293 cells did not detach upon infection with E. coli AfaE-III, whereas detachment of cells infected with non-CEACAM-binding E. coli (E. coli control strain or E. coli ΔAfaE-III), could be readily detected under the microscope (Fig 4B and 4C). Treatment of the infected cells with Mn²⁺, a general inducer of integrin activity, increased the matrix adhesion of cells infected with the E. coli control strain or E. coli ΔAfaE-III, but did not further enhance collagen binding of cells infected with CEACAM-binding E. coli AfaE-III (Fig 4D). Moreover, infection of CEA-expressing 293 cells with E. coli AfaE-III, but not E. coli ΔAfaE-III, triggered CD105 expression by 293 cells (Fig 4E). These results indicate that E. coli AfaE-III is able to exploit CEACAMs to modulate host cell adhesion and to counteract bacteria-induced cell detachment via CD105 expression and integrin activation. In line with this idea, infection with E. coli AfaE-III, but not with but not E. coli ΔAfaE-III, resulted in enhanced integrin β1 activity in CEA-expressing cells (Fig 4F and 4G). These results demonstrate that CEACAM-binding pathogenic strains of E. coli can modulate host cell adhesion and integrin activity on human epithelial cells.

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Fig 4. CEA engagement by E. coli AfaE-III triggers enhanced cell-matrix adhesion via integrin activation.

(A) 293 cells were transfected with a control vector (GFP), or plasmids encoding CEA or CD105. Cells were left uninfected or infected for 8 h with the indicated bacteria. After infection, cells were used in adhesion assays on collagen. Bars represent means ± S.D. of 8 replicates. Two-tailed student’s t-test; *** p < 0.001, n.s.–not significant. Shown is one representative experiment out of three independent biological replicates (B) 293 cells were transfected with CEA and seeded on 25 μg/ml collagen. Confluent layers were left uninfected or infected for 14 h with E. coli, E. coli AfaE-III, or E. coli ΔAfaE-III. Following infection, cells were washed and remaining cells were stained with crystal violet. Representative areas with remaining cells were photographed. Pictures are representative for three independent biological replicates. (C) 293 cells were infected and stained as in (B). Staining intensity was determined after dye elution in a spectrophotometer at 550 nm. Bars represent mean ± S.D. of 8 replicates. Two-tailed student’s t-test; *** p < 0.001, n.s.—not significant. Shown is one representative experiment out of three independent biological replicates. (D) 293 cells were transfected with plasmids encoding CEA or CD105. Cells were left uninfected or infected for 8 h with the indicated bacteria. After infection, cells were used in adhesion assays on collagen in the absence or presence of 1 mM Mn²⁺. Bars represent means ± S.D. of 8 replicates. Two-tailed student’s t-test; *** p < 0.001, n.s.—not significant. Shown is one representative experiment out of three independent biological replicates. (E) CEA-transfected 293 cells were infected for 14 h with the indicated bacteria and analyzed by flow cytometry for CD105 expression. Gray area indicates staining of uninfected cells with an isotype matched control antibody. Shown is one representative experiment out of three independent biological replicates. (F, G) 293 cells were transfected with plasmids encoding either CEA or CD105. As indicated, transfected cells were infected with bacteria for 14 h or left uninfected. After infection, cells were replated onto collagen-coated culture dishes for 90 min and stimulated or not for 5 min with 1 mM Mn²⁺ before fixation. Fixed samples were either stained with a rat monoclonal integrin β1 antibody (clone AIIB2), which recognizes the integrin β1 extracellular domain irrespective of its conformation (total integrin) (F) or samples were stained with an activation-epitope specific rat monoclonal integrin β1 antibody (clone 9EG7), which recognizes the extended, ligand-bound conformation of integrin β1 (active integrin)(G). Bars represent the mean ± S.D. of 5 replicates. Two-tailed student’s t-test; *** p < 0.001, n.s.—not significant. Shown is one representative experiment out of three independent biological replicates.

https://doi.org/10.1371/journal.ppat.1005608.g004

E. coli AfaE-III colonization of the urogenital tract is facilitated by CEACAM-binding and results in CD105 expression in vivo

Because CEACAM-binding by E. coli AfaE-III results in enhanced integrin activity in vitro, we wondered whether these pathogens have an advantage during colonization of the urogenital tract. Therefore, wildtype or CEAtg mice were vaginally infected for 24 h with 10⁶ E. coli AfaE-III or with E. coli ΔAfaE-III. 24h after infection, colonization was analyzed by dilution plating. Whereas only low numbers of bacteria were recovered from the urogenital tract of wildtype mice, recovery of E. coli AfaE-III from CEAtg mice increased more than 80-fold (Fig 5A). In contrast, numbers of non-CEACAM-binding E. coli ΔAfaE-III were only slightly (~3-fold) elevated in CEAtg mice compared to wildtype mice (Fig 5A). Immunohistochemistry revealed that numerous E. coli AfaE-III were found associated with the epithelial surface of CEAtg mice, which stained positive for human CEA (Fig 5B). Furthermore, superficial epithelial cells of CEAtg mice infected with E. coli AfaE-III showed local expression of CD105 (Fig 5C). While occasionally non-CEACAM-binding E. coli were also detected on the mucosal surface of CEAtg mice, no increase in CD105 expression was evident (Fig 5C). Together, these results indicate that the enhanced colonization of CEAtg mice by E. coli AfaE-III might be due to modulation of epithelial cell-extracellular matrix adhesion via CD105 expression and integrin activation, which would provide the mechanistic explanation for the observed phenotype.

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Fig 5. E. coli AfaE-III shows improved mucosal colonization and induces CD105 expression.

(A) Wild-type or CEAtg female mice were infected with E. coli AfaE-III or E. coli ΔAfaE-III. 24 h later, bacteria were re-isolated. Each data point in the graph reflects the number of bacteria re-isolated from an individual animal (n = 10). Data were compiled from two independent experiments. The median for each experimental group of animals is indicated by a line; numbers of recovered bacteria were compared by Mann-Whitney U-test and highly significant differences (p<0.001) are indicated by ***. (B, C) Animals were infected as in (A) and genital tracts were excised, fixed and frozen. (B) Cryosections of genital tracts were co-stained with rabbit antibodies against E. coli (green) and a mouse monoclonal antibody against CEA. Cell nuclei were visualized by Hoechst (blue). Numerous E. coli AfaE-III can be detected in close association with the CEA-positive epithelium (arrowhead), whereas non-CEACAM binding E. coli are rarely observed. (C) Cryosections were co-stained with rabbit antibodies against E. coli (green) and a rat monoclonal antibody against murine CD105 (red). Cell nuclei were visualized by Hoechst (blue). Strong local expression of CD105 can be observed on the mucosal surface of CEAtg mice upon association with E. coli AfaE-III (arrowhead). Pictures in B) and C) are representative for three independent biological replicates.

https://doi.org/10.1371/journal.ppat.1005608.g005

CEACAM-binding by E. coli AfaE-III suppresses epithelial exfoliation in the genital tract

In the case of gonococcal infection, bacteria-triggered CD105 expression on the epithelial surface translates into suppression of host cell exfoliation in vivo [14]. To investigate the level of host cell exfoliation in response to bacterial infection, we used scanning electron microscopy (SEM) of the urogenital tract. In uninfected wildtype and CEAtg animals, the surface of the upper vaginal epithelium showed few detaching superficial cells, indicating low tissue turnover under these conditions (Fig 6A). However, infection of wildtype mice with E. coli AfaE-III or E. coli ΔAfaE-III triggered a dramatic increase in exfoliation of superficial epithelial cells (Fig 6B and 6C). In strong contrast, infection with the CEACAM-binding UPEC strain did not result in an increased exfoliation of epithelial cells in CEAtg mice (Fig 6B and 6C). Despite the presence of numerous E. coli AfaE-III as well as E. coli ΔAfaE-III on the vaginal epithelium, only the non-CEACAM-binding strain E. coli ΔAfaE-III led to a strong increase in exfoliation (Fig 6B and 6C). The low number of exfoliating cells in CEAtg mice upon infection with E. coli AfaE-III was comparable to the uninfected situation indicating that CEACAM-binding by the AfaE-III adhesin is able to completely suppress the exfoliation response (Fig 6B and 6C). Together, these results suggest that CEACAM-binding pathogenic E. coli, such as E. coli AfaE-III, can interfere with the detachment of infected epithelial cells in a manner reminiscent of Opa_CEA-expressing Neisseria gonorrhoeae. Suppression of exfoliation clearly is a means to increase the likelihood of a successful and lasting colonization. Therefore, CEACAM-triggered interference with epithelial exfoliation seems to be more common amongst human pathogens than previously appreciated and appears to be an evolutionarily favourable strategy to colonize human mucosal surfaces.

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Fig 6. E. coli AfaE-III suppresses epithelial exfoliation in CEAtg mice.

(A) Whole mount urogenital tracts of uninfected wild-type or CEAtg female mice were fixed and processed for scanning electron microscopy (SEM). Pictures show the luminal surface of the upper vaginal and cervical regions and are representative for five animals each treatment group. (B) Wild-type or CEAtg female mice were infected with E. coli AfaE-III or E. coli ΔAfaE-III for 24 h and the genital tracts processed as in (A). SEM pictures (at two different magnifications, as indicated by the scale bars) show the luminal surface of the upper vaginal and cervical regions. Whereas massive epithelial exfoliation is evident in infected wildtype mice and in CEAtg mice infected with E. coli ΔAfaE-III, a strongly reduced detachment of epithelial cells is observed in CEAtg mice infected with CEACAM-binding E. coli AfaE-III. Adherent bacteria can be observed at higher magnifications (arrows). Pictures are representative for five animals each treatment group. (C) Quantification of exfoliating epithelial cells from samples in (B). Bars represent mean ± S.D. of exfoliating cells from at least n = 26 areas (~0.075 mm²) derived from at least five animals each treatment group. Results were compared by Mann-Whitney U-test and highly significant differences (p<0.001) are indicated by ***.

https://doi.org/10.1371/journal.ppat.1005608.g006

Bacteria and infection

The E. coli and E. coli Opa_CEA strains were derived from DH5α and have been described previously [22]. The heterologous expression of Opa proteins in E. coli allows these strains to interact with human CEACAMs in a manner analogous to Opa protein expressing gonococci and meningococci [18]. The opaque phenotype was regularly controlled by Western blotting of bacterial lysates with the monoclonal anti-Opa protein antibody (clone 4B12/C11). The uropathogenic E. coli harbouring the afa-3 gene cluster (strain A30; isolated from a cystitis patient [25,58] is a non-hemolytic, serotype O75 strain expressing the CEACAM-binding AfaE-III adhesin (E. coli AfaE-III). All E. coli strains were grown on LB agar plates with or without adequate antibiotics and cultured at 37°C. To select for afa-deficient variants of E. coli AfaE-III, bacteria were sequentially cultured overnight at 37°C, then for 24 h at 40°C, at 42°C, and at 45°C. Serial dilutions of the final culture were plated on LB agar and individual colonies were tested by colony-PCR for the presence of the afa locus (primer pair afa-f: 5’-ggcagagggccggcaacaggc-3’; afa-r: 5’-cccgtaacgcgccagcatctc-3‘) and the K5 capsule determinant (K5-f: 5‘-cagtatcagcaatcgttctgta-3‘ / kpsII-r: 5’-catccagacgataagcatgagca-3’) as described [59].

In addition, we have attempted to generate a complemented strain, but have not succeeded in re-expressing the afa-gene cluster of E. coli strain A30, despite subcloning into low-copy or inducible vectors. Sequencing of subcloned gene clusters in multiple independent clones revealed that all the tested subclones carried point mutations resulting in premature stop codons or frame shifts within the coding regions of the afa-gene cluster, indicating that the maintenance of heterologous fimbrial gene clusters in multiple copies in E. coli laboratory strains may select for subcloned PCR products with accumulated mutations preventing expression. For determining growth curves of E. coli strains, LB broth cultures were initiated and grown at 37°C for 4 h. These cultures were used as 1:25 inoculation into 100 ml LB medium at 37°C. Absorbance at 600nm was recorded every 60 min over a course of 10–25 h using a Libra S4 spectrophotometer (Biochrom, Cambridge, UK). For infection, bacteria were suspended in DMEM, the optical density of the suspension at 600 nm (OD₆₀₀) was used to estimate the number of bacteria according to a standard curve, and the bacteria were added to the cells at the indicated multiplicity of infection (MOI).

Neisseria gonorrhoeae strains used in this study were described previously and were derived from strain MS11 [16]. The gonococci were either non-piliated and expressed a CEACAM-binding Opa protein (Ngo Opa_CEA; strain N309), were non-piliated and expressed a heparansulphate proteoglycan-binding Opa protein (Ngo Opa_HSPG; strain N303) [22], or the bacteria did not express an Opa protein (non-opaque phenotype), but expressed pili to bind to human cells (Ngo P+, strain N280, a non-opaque derivative of MS11-F3 [60]). The opacity and the piliation status of the used bacteria were regularly monitored by colony morphology as well as Western Blotting with monoclonal anti-Opa antibodies. Gonococci were grown on GC agar plates (Difco BRL, Paisley, UK) supplemented with vitamins, chloramphenicol (10 μg/ml) and erythromycin (7 μg/ml) at 37°C, 5% CO₂ and subcultured daily.

Cell culture and plasmid transfection

The human cervix carcinoma cell line ME-180 (ATCC, Rockville, MD) and the embryonic kidney cell line 293T (293 cells; ACC-635, DSMZ, Braunschweig, Germany) were cultured in DMEM containing 10% calf serum (293 cells) or DMEM containing 10% FCS (ME-180 cells) at 37°C in 5% CO₂ and subcultured every second to third day. 293 cells were transfected by calciumphosphate co-precipitation using a total of 5 μg of plasmid DNA for each 10 cm culture dish and employed in experiments 2 days after transfection. In some cases, 293 cells were serum-starved in DMEM containing 0.5% CS. The human vaginal epithelial cell (hVEC) line MS74 was obtained from A.J. Schaeffer (Feinberg School of Medicine, Northwestern University, Chicago, IL), cultured on gelatine-coated dishes in DMEM containing 10% fetal calf serum (FCS), and subcultured every third day.

Recombinant DNA

The expression plasmids used in this study included the commercially available vectors pcDNA3.1 Hygro (pcDNA; Invitrogen, Karlsruhe, Germany), pEGFP-N1, pLPS-3’-EGFP and pDsRed2-C1 (Clontech, Palo Alto, CA). Plasmid pcDNA3.1 CEA (pcDNA-CEA) was described previously [61]. The expression plasmid pcDNA3.1 CEACAM1-4L-HA (pcDNA-CEACAM1) was constructed by PCR amplification of the human CEACAM1-4L cDNA (generous gift of W. Zimmermann, LMU München, Germany) with primers CEACAM1-HA-sense (5’-GGGAAGCTTGCCATGGGGCACCTCTCAGCCCCACTTCAC-3’) and CEACAM1-HA-anti (5’-GGGGACGTCATAGGGATACTGCTTTTTTACTTCTGAATAAATTATTTCTG-3’) and was cloned into the HindIII-AatII—digested plasmid pBluescript CEACAM3-HA [62] before further subcloning via HindIII-NotI into pcDNA3.1 Hygro (Invitrogen). The vector pLPS-3'-RFP2 was constructed by PCR amplifying the RFP2-coding sequence from vector pDsRed2-C1 with primers RFP2-AgeI-sense 5’-ATAACCGGTCGCCTCCTCCGAGAACGTCATCACC-3’ and RFP2-NotI-anti 5’-ATAGCGGCCGCTTACAGGAACAGGTGGTGGCGGCC-3’ and subcloning into the AgeI/NotI sites of pLPS-3’-EGFP resulting in the exchange of the GFP by the DsRed2 coding sequence. The human CD105 cDNA was transferred by Cre-mediated recombination from pDNR-dual CD105 [16] into pLPS-3'-RFP2 resulting in RFP2 fused to the carboxy-terminus of full-length CD105.

GFP-fusion proteins of the human CEACAM1, CEA, and CEACAM8 amino-terminal IgV-like domains as well as the corresponding constructs encoding the amino-terminal IgV-like domains of CEACAM1 orthologues from dog (cCEACAM1), the two CEACAM1 alleles from cattle (bCEACAM1a; bCEACAMb), and from mouse (mCEACAM1) have been constructed and employed previously [20,63].

Production of soluble CEACAM N-terminal domains and binding to bacteria

293 cells were transfected with 5 μg plasmid DNA encoding secreted GFP-fusion proteins of the N-terminal domains of human or mammalian CEACAM family members. After 24 h, the medium of the transfected 293 cell cultures was replaced by serum-reduced OptiMEM (Life Technologies, Darmstadt, Germany). Two days later the culture supernatants (supe) containing the secreted fusion proteins were collected, centrifuged for 15 min at 5000 rpm and either stored at -20°C or used immediately for bacterial pull-down experiments. The GFP-derived fluorescence was analysed using a Varioskan Flash reader (Thermo Scientific) to adjust equal amounts of the secreted fusion proteins. For pull-down experiments, indicated bacteria were suspended in PBS and binding to the indicated receptor protein contained in cell culture supernatants was determined essentially as described [63].

Immunofluorescence staining, cell lysis, Western blotting, and antibodies used

Immunofluorescence staining, cell lysis and Western blotting were performed as described previously [62] using mAbs against CEACAMs (clone D14HD11) against CEACAM1 (clone GM-8G5), against CEACAM6 (clone 9A6) (all from Aldevron, Freiburg, Germany), against CEA (clone COL-1; Zymed, San Francisco, CA), against green fluorescent protein (GFP; clone JL-8; BD Biosciences), against E. coli LPS (AbD Serotec, Oxford, UK), or against murine CD105 (clone MJ7/18; Southern-Biotech, Birmingham, USA). Mouse monoclonal antibodies against human CD105 (clone P4A4; provided by Developmental Studies Hybridoma Bank (DSHB), University of Iowa) or against Opa proteins (clone 4B12/C11; generous gift of Marc Achtman, University of Warwick, UK) as well as rat monoclonal anti-integrin β1 (clone 9EG7; provided by D. Vestweber (MPI for Molecular Medicine, Münster, Germany)) and rat monoclonal anti-integrin β1 (clone AIIB2; DSHB) were purified from hybridoma culture supernatants. Further, rabbit polyclonal antisera raised against paraformaldehyde-fixed N. gonorrhoeae MS11 (IG-511) was custom produced by Immunoglobe (Himmelstadt, Germany). All secondary antibodies were from Jackson Immuno-Research (West Grove, PA).

Electron microscopy of isolated cells

For scanning electron microscopy, ME-180 cells were seeded at 2.5 x 10⁵ cells/well in 24-well plates on acid-washed glass coverslips coated with 25 μg/ml collagen and grown to confluency. Medium was replaced with DMEM, 0.5% FCS for 8 h. Then cells were infected for 14 h at a MOI of 20 or left uninfected. Samples were fixed in situ for at least 1 h at 4°C. The samples were washed and dehydrated in a graded series of aceton on ice. After critical point drying samples were sputter-coated with 5 nm gold-palladium in a BAL-TEC SCD 030 and examined at 15 kV accelerating voltage in a Philipps 505 scanning electron microscope using the secondary electron detector. Images were digitally recorded with a DISS 5 system (remX GmbH, Bruchsal, Germany) and processed in Adobe Photoshop 6.

Animal husbandry and breeding

C57BL/6J mice transgenic for human CEA (CEAtg mice) have been described before [14,23]. Wildtype C57BL/6J mice (originally obtained from Elevage Janvier, Le Genest Saint Isle, France) and CEAtg mice as well as TLR4+/+(HeN) and TLR-/- (HeJ) female mice were maintained under specified pathogen-free conditions under a 12-h light cycle in the animal facility of University of Konstanz in accordance with the institutional guidelines. The CEAtg mice were kept heterozygous for the transgene by crossing male CEAtg mice with female WT mice. Offspring of these crosses (age 3–4 weeks old) was genotyped by PCR.

Ethics statement

Experiments involving animals were performed in accordance with the German Law for the Protection of Animal Welfare (Tierschutzgesetz). The animal care and use protocol, including the protocol of experimental vaginal infection of female mice, was approved by the appropriate state ethics committee and state authorities regulating animal experiments (Regierungspräsidium Freiburg, Germany) under the permit file numbers G-10/108 and G-15/43.

In vivo infection of CEAtg and wildtype mice with E. coli strains

Experimental vaginal infection of female mice with E. coli strains was performed as previously described for N. gonorrhoeae [14]. Briefly, CEAtg and wildtype mice were subcutaneously injected with 17-β-estradiol 4 days prior to infection. The drinking water was supplemented with ampicillin (1 mg/ml) to reduce the overgrowth of commensal bacteria during hormone treatment. Ampicillin containing water was changed to normal drinking water 1 day before infection. Mice were inoculated intravaginally with 10⁶ CFU of the different E. coli strains suspended in 20 μl of PBS. 24h later, the mucosa-associated bacteria were re-isolated by cotton swaps. Serial dilutions of re-isolated bacteria were plated on LB agar (E. coli AfaE-III) or LB agar containing ampicillin (E. coli Opa_CEA and E. coli). In order to analyse the Opa protein profile of re-isolated bacteria, single colonies were expanded on agar plates, lysed and analysed by Western blotting using Opa specific antibodies.

Immunohistochemistry of tissue samples

The genital tract of infected animals was excised and the longitudinally opened vaginal and uteral tissue was mounted and analysed by scanning electron microscopy essentially as described previously [14]. For immunohistochemistry, tissue samples were immediately fixed with 4% paraformaldehyde for at least 24 h and transferred to 10% sucrose, 0.05% cacodylic acid for 1 h at 4°C. Next, samples were transferred to 20% sucrose for 1 h and then into 30% sucrose at 4°C over night. Organs were mounted in embedding medium (Cryo-M-Bed; Bright Instrument, Huntingdon, UK) and frozen at -20°C. 10 μm thick sections were cut at -20°C using a cryostat (Vacutom HM500, Microm, Germany). Sections were incubated with a mouse monoclonal antibody against CEA (clone COL-1; dilution 1:200) or a rat monoclonal antibody against murine CD105 (clone MJ7/18; dilution 1:1200) together with a polyclonal rabbit antibody against N. gonorrhoeae MS11 (dilution 1:100) or a polyclonal rabbit antibody against E.coli (dilution 1:200). Detection of the primary antibodies was accomplished by incubation with a combination of Cy5-conjugated goat-anti-rabbit antibody (1:250) and rhodamine-conjugated goat-anti-rat antibody (1:250; in the case of CD105 detection) or Cy3-conjugated goat-anti-mouse antibody (1:250; in the case of CEA detection). Cell nuclei were visualized by the addition of Hoechst 33342 (1:30,000; Life Technologies, Darmstadt, Germany) in the final staining step. Samples were analysed with a TCS SP5 confocal laser scanning microscope (Leica, Mannheim, Germany). Images were digitally processed with Photoshop CS (Adobe Systems, Mountain View, CA) and merged to yield pseudo-coloured images.

Analysis of receptor expression by flow cytometry

293 cells were transfected with pcDNA3.1 Hygro (pcDNA), pcDNA-CEA, or pLPS-3’-RFP2-CD105. Cells were infected or not with E. coli, E. coli Opa_CEA, E. coli AfaE-III, or gonococci for 14 h. After infection, cells were stained with monoclonal antibodies against CEA (clone COL-1) or against human CD105 (clone P4A4) for 1 h at 4°C. Following washing, samples were stained with a Cy2-conjugated goat-anti-mouse antibody for 30 min, 4°C. ME180 cells and hVEC cells were analysed for the presence of endogenous CEACAMs using mouse monoclonal antibodies specific for CEACAM1 (clone GM-8G5), CEACAM6 (clone 9A6), or CEA (clone COL-1). Stained samples were analysed for Cy2-derived fluorescence by flow cytometry on an LSRII (BD Biosciences) using FACS Diva software.

Cell adhesion, cell detachment and integrin activity assays

Cell adhesion and cell detachment assays were performed essentially as described [14,16]. Briefly, the wells of 96-well plates were coated with PBS containing collagen type 1 from calf skin (ICN Biomedicals, Irvine, CA) for 24 hours at 4°C. 293 cells were transfected with pEGFP-N1 (GFP), CEA or pLPS3’-CD105 (CD105). After serum starvation, cells were infected or not with the indicated bacterial strains at a MOI of 30 for 8 hours. Following infection, the cells were detached and kept in suspension medium (DMEM, 0.2% BSA) with or without 1 mM Mn²⁺ (1 h at 37C°), and then replated at 4 x 10⁴ cells/well onto collagen-coated wells. Cells were allowed to adhere for 90 min in the presence or absence of 1 mM Mn²⁺ at 37°C, before non-adherent cells were removed by gentle washing with PBS. For the Mn²⁺-treated samples, the washing buffer also contained 1 mM Mn²⁺. Adherent cells were fixed and stained for 60 min with 0.1% crystal violet in 0.1 M borate, pH 9. After washing and drying, the crystal violet was eluted in 10 mM acetic acid and the staining intensity was measured at 550 nm with a Varioskan Flash (Thermo Fisher Scientific Oy Microplate Instrumentation (Vantaa, Finland).

For measuring integrin activity, cells were serum-starved over-night, before they were infected or not with the indicated bacteria at an MOI of 30 for 8 h. Following infection, cells were detached by limited trypsin/EDTA digestion that was stopped by addition of soybean trypsin inhibitor (0.5 mg/ml in DMEM). Detached cells were kept in suspension medium (DMEM, 0.2% BSA) for 1 h at 37°C, and then replated at 5 x 10⁴ cells/well into wells coated with collagen type 1. After 75 min, wells were treated or not with 1 mM MnCl₂, incubated for 5 min and transferred to ice. Cells were fixed with 4% paraformaldehyde in PBS for at least 30 min, washed with PBS and permeabilized with Triton X-100 (0.1% in PBS) for 15 min. Cells were washed with PBS and blocked with 2% BSA in PBS (blocking buffer) for 20 min, before incubation for 1 h with rat monoclonal antibodies against active integrin (clone 9EG7; dilution 1:600) or against total integrin (clone AIIB2; dilution 1:750) in blocking buffer as described [14]. After washing and incubation with ProteinA/G-HRP (1:250), 100 μl/well of substrate solution (substrate solution was prepared by mixing 10 ml of 2.4 mg/ml tetramethylbenzidine in 10% acetone, 90% ethanol with 0.5 ml of 30 mM potassium citrate, pH 4.1) were added. The enzymatic colour reaction was stopped using 2 M H₂SO₄ (100 μl/well) and the absorbance was determined at 450 nm in a Varioskan Flash (Thermo Scientific) microplate reader.

Statistical analysis

For cell adhesion, cell detachment, and integrin activity assays, values were analysed for normal distribution and mean values were compared by two-tailed unpaired t-test. For in vivo infection assays, including enumeration of exfoliating cells, differences between samples were assessed using the Mann-Whitney U-test. Differences between samples with p<0.001 are indicated by ***.