A Loss-of-Function Screen Reveals Ras- and Raf-Independent MEK-ERK Signaling During Chlamydia trachomatis Infection

Chlamydiae are obligate intracellular bacterial pathogens that have amajor effect on humanhealth. 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 14-3-3 beta-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.