The obligate intracellular bacterial pathogen Chlamydia trachomatis (Ctr) has been associated with cervical and ovarian cancer development. However, establishment of causality and the underlying mechanisms remain outstanding. Our analysis of Ctr-induced alterations to global host histone modifications revealed distinct patterns of histone marks during acute and persistent infections. In particular, pH2AX (Ser139) and H3K9me3, hallmarks of DNA double-strand breaks (DSBs) and senescence-associated heterochromatin foci (SAHF), respectively, showed sustained upregulation during Ctr infection. Ctr-induced reactive oxygen species were found to contribute to persistent DSBs, which in turn elicited SAHF formation in an ERK-dependent manner. Furthermore, Ctr interfered with DNA damage responses (DDR) by inhibiting recruitment of the DDR proteins pATM and 53BP1 to damaged sites. Despite impaired DDR, Ctr-infected cells continued to proliferate, supported by enhanced oncogenic signals involving ERK, CyclinE, and SAHF. Thus, by perturbing host chromatin, DSB repair, and cell-cycle regulation, Ctr generates an environment favorable for malignant transformation.

The transition zones of the squamous and columnar epithelia constitute hotspots for the emergence of cancer, often preceded by metaplasia, in which one epithelial type is replaced by another. It remains unclear how the epithelial spatial organization is maintained and how the transition zone niche is remodelled during metaplasia. Here we used single-cell RNA sequencing to characterize epithelial subpopulations and the underlying stromal compartment of endo- and ectocervix, encompassing the transition zone. Mouse lineage tracing, organoid culture and single-molecule RNA in situ hybridizations revealed that the two epithelia derive from separate cervix-resident lineage-specific stem cell populations regulated by opposing Wnt signals from the stroma. Using a mouse model of cervical metaplasia, we further show that the endocervical stroma undergoes remodelling and increases expression of the Wnt inhibitor Dickkopf-2 (DKK2), promoting the outgrowth of ectocervical stem cells. Our data indicate that homeostasis at the transition zone results from divergent stromal signals, driving the differential proliferation of resident epithelial lineages.

Chlamydiae are obligate intracellular bacterial pathogens that have a major effect on human health. Because of their intimate association with their host, chlamydiae depend on various host cell functions for their survival. Here, we present an RNA-interference-based screen in human epithelial cells that identified 59 host factors that either positively or negatively influenced the replication of Chlamydia trachomatis (Ctr). Two factors, K-Ras and Raf-1, which are members of the canonical Ras-Raf-MEK (mitogen-activated or extracellular signal-regulated protein kinase kinase)-ERK (extracellular signal-regulated kinase) pathway, were identified as central components of signaling networks associated with hits from the screen. Depletion of Ras or Raf in HeLa cells increased pathogen growth. Mechanistic analyses revealed that ERK was activated independently of K-Ras and Raf-1. Infection with Ctr led to the Akt-dependent, increased phosphorylation (and inactivation) of Raf-1 at serine-259. Furthermore, phosphorylated Raf-1 relocalized from the cytoplasm to the intracellular bacterial inclusion in an Akt- and 14-3-3beta-dependent manner. Together, these findings not only show that Chlamydia regulates components of an important host cell signaling pathway, but also provide mechanistic insights into how this is achieved.

Mammalian cells possess sophisticated genome surveillance and repair mechanisms, executed by the so-called DNA damage response (DDR), failure of which leads to accumulation of DNA damage and genomic instability. Mounting evidence suggests that bacterial infections can elicit DNA damage in host cells, and certain pathogens induce such damage as part of their multi-faceted infection programme. Bacteria-mediated DNA damage can occur either directly through the formation of toxins with genotoxic activities or indirectly as a result of the activation of cell-autonomous or immune defence mechanisms against the pathogen. Moreover, host-cell signalling routes involved in the DDR can be altered in response to an infection, and this, in the context of DNA damage elicited by the pathogen, has the potential to trigger mutations and cancer.

Coinfections with pathogenic microbes continually confront cervical mucosa, yet their implications in pathogenesis remain unclear. Lack of in-vitro models recapitulating cervical epithelium has been a bottleneck to study coinfections. Using patient-derived ectocervical organoids, we systematically modeled individual and coinfection dynamics of Human papillomavirus (HPV)16 E6E7 and Chlamydia, associated with carcinogenesis. The ectocervical stem cells were genetically manipulated to introduce E6E7 oncogenes to mimic HPV16 integration. Organoids from these stem cells develop the characteristics of precancerous lesions while retaining the self-renewal capacity and organize into mature stratified epithelium similar to healthy organoids. HPV16 E6E7 interferes with Chlamydia development and induces persistence. Unique transcriptional and post-translational responses induced by Chlamydia and HPV lead to distinct reprogramming of host cell processes. Strikingly, Chlamydia impedes HPV-induced mechanisms that maintain cellular and genome integrity, including mismatch repair in the stem cells. Together, our study employing organoids demonstrates the hazard of multiple infections and the unique cellular microenvironment they create, potentially contributing to neoplastic progression.