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Host nectin-1 is required for efficient Chlamydia trachomatis serovar E development.

Hall JV, Sun J, Slade J, Kintner J, Bambino M, Whittimore J, Schoborg RV - Front Cell Infect Microbiol (2014)

Bottom Line: However, inhibition of signal transduction molecules associated with HSV attachment and entry does not rescue EB production during C. trachomatis/HSV-2 co-infection.Additionally, immunofluorescence assays confirm that HSV infection decreases nectin-1 expression.Overall, the current study indicates that the actions of host molecule, nectin-1, are required for successful C. trachomatis development.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, Quillen College of Medicine, East Tennessee State University Johnson City, TN, USA ; Center for Inflammation, Infectious Disease and Immunity, Quillen College of Medicine, East Tennessee State University Johnson City, TN, USA.

ABSTRACT
Interaction of Herpes Simplex Virus (HSV) glycoprotein D (gD) with the host cell surface during Chlamydia trachomatis/HSV co-infection stimulates chlamydiae to become persistent. During viral entry, gD interacts with one of 4 host co-receptors: HVEM (herpes virus entry mediator), nectin-1, nectin-2 and 3-O-sulfated heparan sulfate. HVEM and nectin-1 are high-affinity entry receptors for both HSV-1 and HSV-2. Nectin-2 mediates HSV-2 entry but is inactive for HSV-1, while 3-O-sulfated heparan sulfate facilitates HSV-1, but not HSV-2, entry. Western blot and RT-PCR analyses demonstrate that HeLa and HEC-1B cells express nectin-1 and nectin-2, but not HVEM. Because both HSV-1 and HSV-2 trigger persistence, these data suggest that nectin-1 is the most likely co-receptor involved. Co-infections with nectin-1 specific HSV-1 mutants stimulate chlamydial persistence, as evidenced by aberrant body (AB) formation and decreased production of elementary bodies (EBs). These data indicate that nectin-1 is involved in viral-induced chlamydial persistence. However, inhibition of signal transduction molecules associated with HSV attachment and entry does not rescue EB production during C. trachomatis/HSV-2 co-infection. HSV attachment also does not activate Cdc42 in HeLa cells, as would be expected with viral stimulated activation of nectin-1 signaling. Additionally, immunofluorescence assays confirm that HSV infection decreases nectin-1 expression. Together, these observations suggest that gD binding-induced loss of nectin-1 signaling negatively influences chlamydial growth. Chlamydial infection studies in nectin-1 knockdown (NKD) HeLa cell lines support this hypothesis. In NKD cells, chlamydial inclusions are smaller in size, contain ABs, and produce significantly fewer infectious EBs compared to C. trachomatis infection in control HeLa cells. Overall, the current study indicates that the actions of host molecule, nectin-1, are required for successful C. trachomatis development.

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HSV co-infection-induced chlamydial persistence signal requires viral attachment/entry via nectin-1. (A) HeLa cells were either mock-, singly-, or co-infected with CtE and various wild type (HSV-2 and HSV-1 gDwt) or mutant viruses (gDG43P, gDQ27P, and gDA3C/Y38C). Cells were collected 20 h pvi and processed for chlamydial EB titration analyses. Results are expressed as the mean ±s.e.m. of three biological replicates. A single asterisk (*) indicates significant difference (p ≤ 0.05) compared to CtE. (B) CHO-C8, CHO-HVEM, CHO-nectin-1 and CHO-nectin-2, were infected with HSV-1 gDwt, gDG43P, gDQ27P, and gDA3C/Y38C for 6 h. Viral entry was analyzed by β-galactosidase assay. (C) HeLa cells were either mock, singly or co-infected with C. trachomatis and HSV-2, HSV-1 gDwt, gDG43P, gDQ27P, or gDA3C/Y38C for 6 h. Viral entry was analyzed by β-galactosidase assay. (D) HeLa cells were either mock-, CtE-infected or co-infected with CtE and HSV-2, HSV-1 gDwt, gDG43P, gDQ27P, or gDA3C/Y38C. Cells were harvested for TEM analyses. White arrows on electron micrographs indicate EBs and black arrows indicate abnormally enlarged RBs characteristic of persistence (i.e., abberent bodies or AB).
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Figure 1: HSV co-infection-induced chlamydial persistence signal requires viral attachment/entry via nectin-1. (A) HeLa cells were either mock-, singly-, or co-infected with CtE and various wild type (HSV-2 and HSV-1 gDwt) or mutant viruses (gDG43P, gDQ27P, and gDA3C/Y38C). Cells were collected 20 h pvi and processed for chlamydial EB titration analyses. Results are expressed as the mean ±s.e.m. of three biological replicates. A single asterisk (*) indicates significant difference (p ≤ 0.05) compared to CtE. (B) CHO-C8, CHO-HVEM, CHO-nectin-1 and CHO-nectin-2, were infected with HSV-1 gDwt, gDG43P, gDQ27P, and gDA3C/Y38C for 6 h. Viral entry was analyzed by β-galactosidase assay. (C) HeLa cells were either mock, singly or co-infected with C. trachomatis and HSV-2, HSV-1 gDwt, gDG43P, gDQ27P, or gDA3C/Y38C for 6 h. Viral entry was analyzed by β-galactosidase assay. (D) HeLa cells were either mock-, CtE-infected or co-infected with CtE and HSV-2, HSV-1 gDwt, gDG43P, gDQ27P, or gDA3C/Y38C. Cells were harvested for TEM analyses. White arrows on electron micrographs indicate EBs and black arrows indicate abnormally enlarged RBs characteristic of persistence (i.e., abberent bodies or AB).

Mentions: Finally, we used β-galactosidase (β-gal) assays to assess nectin-1, nectin-2 and/or HVEM mediated HSV entry in HeLa cells. HeLa monolayers were infected with wild-type HSV-1 or HSV-1 gD mutants that exhibit specific binding efficiencies to HSV co-receptors, as previously shown by Yoon and Spear (2004). The parental and mutant HSV-1 strains express β-gal upon host cell entry (Yoon and Spear, 2004). The parental strain, HSV-1 KOS/FRT-gDwt (gDwt), expresses wild type gD protein and enters host cells via HVEM, nectin-1 and 3-O-S-HS with high/moderate efficiency and via nectin-2 with very low efficiency. HSV-1 KOS/FRT-gDG43P(gDG43P) can only enter using nectin-1, whereas HSV-1 KOS/FRT-gDQ27P (gDQ27P) uses both nectin-1 and nectin-2 equally well. Neither gDG43P nor gDQ27P use HVEM or 3-O-S-HS, even when infections are performed at 200 MOI. Both HVEM and 3-O-S-HS facilitate entry of HSV-1 KOS/FRT-gDA3C/Y38C(gDA3C/Y38C), but nectin 1 and 2 do not (Yoon and Spear, 2004). As shown in Figure 1C, β-gal activity was present in HeLa cells infected with gDwt, gDQ27P, and gDG43P. However, gDA3C/Y38C did not enter HeLa cells, as evidenced by the lack of β-gal activity, confirming that HeLa cells express little or no HVEM co-receptor (Figure S1E).


Host nectin-1 is required for efficient Chlamydia trachomatis serovar E development.

Hall JV, Sun J, Slade J, Kintner J, Bambino M, Whittimore J, Schoborg RV - Front Cell Infect Microbiol (2014)

HSV co-infection-induced chlamydial persistence signal requires viral attachment/entry via nectin-1. (A) HeLa cells were either mock-, singly-, or co-infected with CtE and various wild type (HSV-2 and HSV-1 gDwt) or mutant viruses (gDG43P, gDQ27P, and gDA3C/Y38C). Cells were collected 20 h pvi and processed for chlamydial EB titration analyses. Results are expressed as the mean ±s.e.m. of three biological replicates. A single asterisk (*) indicates significant difference (p ≤ 0.05) compared to CtE. (B) CHO-C8, CHO-HVEM, CHO-nectin-1 and CHO-nectin-2, were infected with HSV-1 gDwt, gDG43P, gDQ27P, and gDA3C/Y38C for 6 h. Viral entry was analyzed by β-galactosidase assay. (C) HeLa cells were either mock, singly or co-infected with C. trachomatis and HSV-2, HSV-1 gDwt, gDG43P, gDQ27P, or gDA3C/Y38C for 6 h. Viral entry was analyzed by β-galactosidase assay. (D) HeLa cells were either mock-, CtE-infected or co-infected with CtE and HSV-2, HSV-1 gDwt, gDG43P, gDQ27P, or gDA3C/Y38C. Cells were harvested for TEM analyses. White arrows on electron micrographs indicate EBs and black arrows indicate abnormally enlarged RBs characteristic of persistence (i.e., abberent bodies or AB).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 1: HSV co-infection-induced chlamydial persistence signal requires viral attachment/entry via nectin-1. (A) HeLa cells were either mock-, singly-, or co-infected with CtE and various wild type (HSV-2 and HSV-1 gDwt) or mutant viruses (gDG43P, gDQ27P, and gDA3C/Y38C). Cells were collected 20 h pvi and processed for chlamydial EB titration analyses. Results are expressed as the mean ±s.e.m. of three biological replicates. A single asterisk (*) indicates significant difference (p ≤ 0.05) compared to CtE. (B) CHO-C8, CHO-HVEM, CHO-nectin-1 and CHO-nectin-2, were infected with HSV-1 gDwt, gDG43P, gDQ27P, and gDA3C/Y38C for 6 h. Viral entry was analyzed by β-galactosidase assay. (C) HeLa cells were either mock, singly or co-infected with C. trachomatis and HSV-2, HSV-1 gDwt, gDG43P, gDQ27P, or gDA3C/Y38C for 6 h. Viral entry was analyzed by β-galactosidase assay. (D) HeLa cells were either mock-, CtE-infected or co-infected with CtE and HSV-2, HSV-1 gDwt, gDG43P, gDQ27P, or gDA3C/Y38C. Cells were harvested for TEM analyses. White arrows on electron micrographs indicate EBs and black arrows indicate abnormally enlarged RBs characteristic of persistence (i.e., abberent bodies or AB).
Mentions: Finally, we used β-galactosidase (β-gal) assays to assess nectin-1, nectin-2 and/or HVEM mediated HSV entry in HeLa cells. HeLa monolayers were infected with wild-type HSV-1 or HSV-1 gD mutants that exhibit specific binding efficiencies to HSV co-receptors, as previously shown by Yoon and Spear (2004). The parental and mutant HSV-1 strains express β-gal upon host cell entry (Yoon and Spear, 2004). The parental strain, HSV-1 KOS/FRT-gDwt (gDwt), expresses wild type gD protein and enters host cells via HVEM, nectin-1 and 3-O-S-HS with high/moderate efficiency and via nectin-2 with very low efficiency. HSV-1 KOS/FRT-gDG43P(gDG43P) can only enter using nectin-1, whereas HSV-1 KOS/FRT-gDQ27P (gDQ27P) uses both nectin-1 and nectin-2 equally well. Neither gDG43P nor gDQ27P use HVEM or 3-O-S-HS, even when infections are performed at 200 MOI. Both HVEM and 3-O-S-HS facilitate entry of HSV-1 KOS/FRT-gDA3C/Y38C(gDA3C/Y38C), but nectin 1 and 2 do not (Yoon and Spear, 2004). As shown in Figure 1C, β-gal activity was present in HeLa cells infected with gDwt, gDQ27P, and gDG43P. However, gDA3C/Y38C did not enter HeLa cells, as evidenced by the lack of β-gal activity, confirming that HeLa cells express little or no HVEM co-receptor (Figure S1E).

Bottom Line: However, inhibition of signal transduction molecules associated with HSV attachment and entry does not rescue EB production during C. trachomatis/HSV-2 co-infection.Additionally, immunofluorescence assays confirm that HSV infection decreases nectin-1 expression.Overall, the current study indicates that the actions of host molecule, nectin-1, are required for successful C. trachomatis development.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, Quillen College of Medicine, East Tennessee State University Johnson City, TN, USA ; Center for Inflammation, Infectious Disease and Immunity, Quillen College of Medicine, East Tennessee State University Johnson City, TN, USA.

ABSTRACT
Interaction of Herpes Simplex Virus (HSV) glycoprotein D (gD) with the host cell surface during Chlamydia trachomatis/HSV co-infection stimulates chlamydiae to become persistent. During viral entry, gD interacts with one of 4 host co-receptors: HVEM (herpes virus entry mediator), nectin-1, nectin-2 and 3-O-sulfated heparan sulfate. HVEM and nectin-1 are high-affinity entry receptors for both HSV-1 and HSV-2. Nectin-2 mediates HSV-2 entry but is inactive for HSV-1, while 3-O-sulfated heparan sulfate facilitates HSV-1, but not HSV-2, entry. Western blot and RT-PCR analyses demonstrate that HeLa and HEC-1B cells express nectin-1 and nectin-2, but not HVEM. Because both HSV-1 and HSV-2 trigger persistence, these data suggest that nectin-1 is the most likely co-receptor involved. Co-infections with nectin-1 specific HSV-1 mutants stimulate chlamydial persistence, as evidenced by aberrant body (AB) formation and decreased production of elementary bodies (EBs). These data indicate that nectin-1 is involved in viral-induced chlamydial persistence. However, inhibition of signal transduction molecules associated with HSV attachment and entry does not rescue EB production during C. trachomatis/HSV-2 co-infection. HSV attachment also does not activate Cdc42 in HeLa cells, as would be expected with viral stimulated activation of nectin-1 signaling. Additionally, immunofluorescence assays confirm that HSV infection decreases nectin-1 expression. Together, these observations suggest that gD binding-induced loss of nectin-1 signaling negatively influences chlamydial growth. Chlamydial infection studies in nectin-1 knockdown (NKD) HeLa cell lines support this hypothesis. In NKD cells, chlamydial inclusions are smaller in size, contain ABs, and produce significantly fewer infectious EBs compared to C. trachomatis infection in control HeLa cells. Overall, the current study indicates that the actions of host molecule, nectin-1, are required for successful C. trachomatis development.

Show MeSH
Related in: MedlinePlus