RNA Interference Screen Identifies Abl Kinase and PDGFR Signaling in Chlamydia trachomatis Entry

Abstract

To elucidate the mechanisms involved in early events in Chlamydia trachomatis infection, we conducted a large scale unbiased RNA interference screen in Drosophila melanogaster S2 cells. This allowed identification of candidate host factors in a simple non-redundant, genetically tractable system. From a library of 7,216 double stranded RNAs (dsRNA), we identified ∼226 host genes, including two tyrosine kinases, Abelson (Abl) kinase and PDGF- and VEGF-receptor related (Pvr), a homolog of the Platelet-derived growth factor receptor (PDGFR). We further examined the role of these two kinases in C. trachomatis binding and internalization into mammalian cells. Both kinases are phosphorylated upon infection and recruited to the site of bacterial attachment, but their roles in the infectious process are distinct. We provide evidence that PDGFRβ may function as a receptor, as inhibition of PDGFRβ by RNA interference or by PDGFRβ neutralizing antibodies significantly reduces bacterial binding, whereas depletion of Abl kinase has no effect on binding. Bacterial internalization can occur through activation of PDGFRβ or through independent activation of Abl kinase, culminating in phosphorylation of the Rac guanine nucleotide exchange factor (GEF), Vav2, and two actin nucleators, WAVE2 and Cortactin. Finally, we show that TARP, a bacterial type III secreted actin nucleator implicated in entry, is a target of Abl kinase. Together, our results demonstrate that PDGFRβ and Abl kinases function redundantly to promote efficient uptake of this obligate intracellular parasite.

Author Summary

Chlamydia trachomatis infections are a worldwide problem; they are the leading cause of preventable blindness in developing nations and the most common cause of sexually transmitted disease in the Western world. Binding and entry into host cells are critical steps to the pathogenesis of this obligate intracellular parasite; however little is known regarding the mechanism of these processes. In this work, we describe a large scale RNA interference screen to identify host factors essential for early steps in C. trachomatis infection. We discover that the Platelet Derived Growth Factor Receptor β (PDGFRβ) can function as a receptor for C. trachomatis, and that activation of both PDGFRβ and Abl kinase signaling pathways by C. trachomatis leads to phosphorylation of a Rac guanine nucleotide exchange factor, Vav2, and several actin nucleators, including WAVE2, Cortactin, and TARP, a Chlamydia type III secreted effector. Our work suggests a model of redundant activation of PDGFRβ and Abl kinase upon C. trachomatis binding that culminates in cytoskeletal rearrangements that modulate efficient uptake of this obligate intracellular parasite.