Interaction of herpes simplex virus type 2 (HSV-2) glycoprotein D with the host cell surface is sufficient to induce Chlamydia trachomatis persistence

Abstract

When presented with certain unfavourable environmental conditions, Chlamydia trachomatis reticulate bodies (RBs) enter into a viable, yet non-cultivable state called persistence. Previously, we established an in vitro C. trachomatis and herpes simplex virus type 2 (HSV-2) co-infection model. These data indicate that (i) viral co-infection stimulates chlamydial persistence, (ii) productive HSV replication is not required for persistence induction, and (iii) HSV-induced persistence is not mediated by any currently characterized anti-chlamydial pathway or persistence inducer. In this study we demonstrated that chlamydial infectivity, though initially suppressed, recovered within 44 h of co-infection with UV-inactivated HSV-2, demonstrating that HSV-induced persistence is reversible. Co-incubation of chemically fixed, HSV-2-infected inducer cells with viable, C. trachomatis-infected responder cells both suppressed production of infectious chlamydial progeny and stimulated formation of swollen, aberrantly shaped RBs. In addition, pre-incubation of viral particles with viral glycoprotein D (gD)-specific neutralizing antibody prevented co-infection-induced persistence. Finally, exposure of C. trachomatis-infected cells to a soluble, recombinant HSV-2 gD : Fc fusion protein decreased production of infectious EBs to a degree similar to that observed in co-infected cultures. Thus, we conclude that interaction of HSV gD with the host cell surface is sufficient to trigger a novel host anti-chlamydial response that restricts chlamydial development.

Fig. 1.

Chlamydial infectivity recovers during long-term co-infection with HSV-2_UV. HeLa cell monolayers were mock-, singly or co-infected with C. trachomatis and HSV-2_UV. Replicate samples were harvested at days 1, 3 and 6 post-HSV-2_UV infection and processed for DNA isolation (a) and chlamydial titration (b). (a) PCR was used to determine relative HSV (HSV G2), chlamydial (Ct 16S rRNA Gene) and host (GAPDH) genome accumulation in co-infected cells. (b) EB titres are expressed as IFU (ml sample)⁻¹ ±sem, n=3. Asterisks indicate titres that were significantly different (by t test) from those of C. trachomatis singly infected cells (P<0.05) collected at the same time.

Fig. 2.

Co-incubation of HSV-2-infected fixed inducer cells with C. trachomatis-infected responder cells. (a) Mock- (MI) or HSV-2-infected (HI) inducer cells were fixed and immunostained with α-Sal, α-gB, α-gC, α-gD or α-gH mAbs and Alexa Fluor 488-conjugated secondary antibody. Photomicrographs are at ×320 magnification; bar, 120 μm. (b) MI or HI inducer cells were co-incubated with mock- or chlamydiae-infected responder cells for 20 h (day 1), 44 h (day 2), 68 h (day 3) and 140 h (day 6), and then harvested for EB titration. Singly infected and co-infected HeLa cultures were included as positive controls. Asterisks indicate titres that are significantly different (by t test) from those of either C. trachomatis singly infected cultures (for Ct/HSV controls) or C. trachomatis+MI cultures (for HI+Ct samples) (P<0.05). (c) Co-incubated cultures were harvested at 20 h.p.c.i. (day 1), fixed with glutaraldehyde/paraformaldehyde and processed for TEM. Both photomicrographs are at ×7000 magnification; bars, 2 μm.

Fig. 3.

Co-infection of C. trachomatis with antibody-pre-incubated HSV-1 tk12. (a) Cultures of HeLa cells were mock-, singly or co-infected with C. trachomatis and HSV-1 tk12 or HSV-1 tk12+α-Sal, α-gB, α-gC, α-gD, α-gH, cMEM or cMEM/α-HPV and assayed for β-gal activity. (b, c) Replicate HeLa monolayers were similarly infected and processed for chlamydial titration. EB titres are expressed as IFU (ml sample)⁻¹; error bars, sem, n=3. Asterisks indicate titres that are significantly different (by t test) from those of C. trachomatis singly infected cells (P<0.05).

Fig. 4.

Interaction of HSV-2 gD : Fc fusion proteins with C. trachomatis-infected cultures decreases chlamydial infectivity. (a) HeLa cell cultures were immunostained with 1× PBS (Mock), Blk : Fc or gD : Fc with or without Texas red-conjugated anti-Rabbit IgG Fc antibodies (αIgG-TR). Cells were photographed at ×320 magnification; bar, 120 μm. (b) Model for activation of signalling from HSV co-receptors by antibody–gD cross-linking. The gD fusion protein is cross-linked by addition of α-IgG FcAb, which may increase activation of receptor-associated signal transduction pathways by promoting receptor aggregation in the plasma membrane. (c) Replicate C. trachomatis singly infected samples were incubated with α-IgG, gD : Fc, Blk : Fc or mixtures of α-IgG+gD : Fc (αIgG/gD : Fc) or α-IgG+Blk : Fc (αIgG/Blk : Fc) and harvested for EB titration. Replicate HeLa cultures were co-infected with both C. trachomatis and HSV-2 (Ct/HSV). (d) C. trachomatis singly infected samples were incubated with α-IgG+gD : Fc (αIgG/gD : Fc) or α-IgG+gD : Fc+α-gD NuAb (αIgG/gD : Fc+αgD) and harvested for EB titration. Asterisks indicate titres that are significantly different (by t test) from those of parallel C. trachomatis singly infected cultures (P<0.05).