SummaryEpidemiological and clinical studies have shown that double infection with herpes simplex virus type 2 (HSV-2) and Chlamydia trachomatis occurs in vivo . We hypothesized that co-infection would alter replication of these agents. To test this hypothesis, HeLa cells were infected with C. trachomatis serovar E, followed 24 h later by HSV-2 strain 333. Transmission electron microscopic (TEM) analyses indicated that, by 10 h after HSV addition, reticulate bodies (RBs) in coinfected cells were swollen, aberrantly shaped and electron-lucent. In infectious titre assays, HSV-2 coinfection abrogated production of infectious chlamydial progeny. Western blot analyses indicated that accumulation of chlamydial major outer membrane protein (MOMP) was decreased by HSV co-infection while accumulation of chlamydial heat-shock protein 60-1 (HSP60-1) was increased. Polymerase chain reaction (PCR) experiments indicated that chlamydial genome copy number was unaltered by HSV-2 superinfection. Semi-quantitative, reverse transcription PCR (RT-PCR) experiments demonstrated that levels of chlamydial groEL , ftsK , ftsW , dnaA and unprocessed 16S rRNA transcripts were not changed by HSV-2 super-infection. These data indicate that HSV-2 superinfection drives chlamydia into a viable but noncultivable state, which is the hallmark of persistence. Because chlamydial HSP60-1 has been associated with immunopathology in vivo , these results also suggest that disease severity might be increased in coinfected individuals.

SummaryEpidemiological studies have demonstrated that co-infections of herpes simplex virus type 2 (HSV-2) and Chlamydia trachomatis occur in vivo. Data from a tissue culture model of C. trachomatis/HSV-2 co-infection indicate that viral co-infection stimulates the formation of persistent chlamydiae. Transmission electron microscopic (TEM) analyses demonstrated that in both HeLa and HEC-1B cells, co-infection caused developing chlamydiae to exhibit swollen, aberrantly shaped reticulate bodies (RBs), characteristically observed in persistence. Additionally, HSV-2 co-infection suppressed production of infectious chlamydial elementary bodies (EBs) in both host cell types. Co-infection with HSV type 1 (HSV-1) produced similar morphologic alterations and abrogated infectious EB production. These data indicate that virusinduced chlamydial persistence was neither host cellnor virus strain-specific. Purification of crude HSV-2 stocks demonstrated that viral particles were required for coinfection-induced chlamydial persistence to occur. Finally, co-infection with either UV-inactivated, replication-incompetent virus or replication-competent HSV-2 in the presence of cyclohexamide reduced chlamydial infectivity without altering chlamydial genomic DNA accumulation. These data demonstrate that productive viral replication is not required for the induction of chlamydial persistence and suggest that HSV attachment and entry can provide the necessary stimulus to alter C. trachomatis development.

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 HSVinduced 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.

Several inducers of chlamydial persistence have been described, including interferon-c (IFN-c), IFN-a, IFN-b, and tumour necrosis factor-a (TNF-a) exposure, and iron, amino acid or glucose deprivation. A tissue-culture model of Chlamydia trachomatis/herpes simplex virus type-2 (HSV-2) co-infection indicates that viral co-infection stimulates the formation of persistent chlamydiae. This study was designed to ascertain whether co-infection-induced persistence is mediated by a previously characterized mechanism. Luminex assays indicate that IFN-c, IFN-a, and TNF-a are not released from co-infected cells. Semiquantitative RT-PCR studies demonstrate that IFN-b, IFN-c, indoleamine 2,3-dioxygenase, lymphotoxin-a and inducible nitric oxide synthase are not expressed during co-infection. These data indicate that viral-induced persistence is not stimulated by any persistence-associated cytokine. Supplementation of co-infected cells with excess amino acids, iron-saturated holotransferrin, glucose or a combination of amino acids and iron does not restore chlamydial infectivity, demonstrating that HSV-2-induced persistence is not mediated by depletion of these nutrients. Finally, inclusions within co-infected cells continue to enlarge and incorporate C 6 -NBD-ceramide, indicating that HSV-2 co-infection does not inhibit vesicular transport to the developing inclusion. Collectively these data demonstrate that co-infection-induced persistence is not mediated by any currently characterized persistence inducer or anti-chlamydial pathway. Previous studies indicate that HSV-2 attachment and/or entry into the host cell is sufficient for stimulating chlamydial persistence, suggesting that viral attachment and/or entry may trigger a novel host pathway which restricts chlamydial development.