Chlamydia trachomatis fails to protect its growth niche against pro-apoptotic insults

Barbara S Sixt,Guido Kroemer,Raphael H Valdivia,Oliver Kepp,Carlos Núñez-Otero

doi:10.1038/s41418-018-0224-2

Abstract

Chlamydia trachomatis is an obligate intracellular bacterial agent responsible for ocular infections and sexually transmitted diseases. It has been postulated that Chlamydia inhibits apoptosis in host cells to maintain an intact replicative niche until sufficient infectious progeny can be generated. Here we report that, while cells infected with C. trachomatis are protected from apoptosis at early and mid-stages of infection, they remain susceptible to the induction of other cell death modalities. By monitoring the fate of infected cells by time-lapse video microscopy and by analyzing host plasma membrane integrity and the activity of caspases, we determined that C. trachomatis-infected cells exposed to pro-apoptotic stimuli predominately died by a mechanism resembling necrosis. This necrotic death of infected cells occurred with kinetics similar to the induction of apoptosis in uninfected cells, indicating that C. trachomatis fails to considerably prolong the lifespan of its host cell when exposed to pro-apoptotic insults. Inhibitors of bacterial protein synthesis partially blocked necrotic death of infected cells, suggesting that the switch from apoptosis to necrosis relies on an active contribution of the bacteria. Tumor necrosis factor alpha (TNF-α)-mediated induction of necrosis in cells infected with C. trachomatis was not dependent on canonical regulators of necroptosis, such as RIPK1, RIPK3, or MLKL, yet was blocked by inhibition or depletion of CASP8. These results suggest that alternative signaling pathways regulate necrotic death in the context of C. trachomatis infections. Finally, consistent with the inability of C. trachomatis to preserve host cell viability, necrosis resulting from pro-apoptotic conditions significantly impaired production of infectious progeny. Taken together, our findings suggest that Chlamydia’s anti-apoptotic activities are not sufficient to protect the pathogen’s replicative niche.

Highlights

9 Department of Women’s and Children’s Health, Karolinska Institute, Karolinska University Hospital, 17176 Stockholm, Sweden cleavage activity in cell lysates was measured at 7 hpt, normalized to the activity detected in STS-treated uninfected cells, and plotted against the percentage of infected cells determined microscopically from parallel cultures. c Phase contrast microscopy reveals signs of necrotic cell death in C. trachomatis-infected (10 IFU/ cell) HeLa cells that were treated at 24 hpi for 7 h with STS (1 μM)
In the presence of TNF/CHX, inclusion-bearing cells predominately died by necrosis, displaying a sudden rupture of the host cell membrane in absence of morphological signs of apoptosis, while neighboring inclusion-free cells died by apoptosis (Fig. 2a, b, movie S2)
We observed that under pro-apoptotic conditions, infected cells die by necrosis, a type of death that is characterized by a sudden rupture of the host cell plasma membrane (Figs 1c, 2a, b, and movies S2-S4) and release of host cell contents (Fig. 3a), but is not accompanied by the lactate dehydrogenase (LDH) activity in culture supernatants was measured at 9 hpt (mean ± SD, n = 6, n = 3, ANOVA) (d). e Western blot analysis displaying the presence or absence of CASP8 in wild-type and CASP8-deficient HeLa cells

Summary

Introduction

C. trachomatis is the causative agent of blinding trachoma, an ocular disease that is endemic in many developing countries [1]. C. trachomatis is the most frequent agent of bacterial sexually transmitted disease worldwide cleavage activity in cell lysates was measured at 7 hpt, normalized to the activity detected in STS-treated uninfected cells, and plotted against the percentage of infected cells determined microscopically from parallel cultures (mean ± SD, n = 3). C Phase contrast microscopy reveals signs of necrotic cell death in C. trachomatis-infected (10 IFU/ cell) HeLa cells that were treated at 24 hpi for 7 h with STS (1 μM). Asterisks and arrowheads indicate examples of bacterial inclusions in apparently intact infected cells and examples of necrotic infected cells, respectively (scale bar, 20 μm). Acute Chlamydia urogenital tract infections are often asymptomatic, but repeated and recurrent infections increase the risk for complications, such as pelvic inflammatory disease, ectopic pregnancy, and infertility [3]

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