RNA interference screen identifies AbI kinase and PDGFR signaling in Chlamydia trachomatis entry

• Authors: Cherilyn A. Elwell, University of California San Francisco; Alhaji Ceesay, University of California San Francisco; Jung Hwa Kim, University of California San Francisco; Daniel Kalman, Emory University; Joanne N. Engel, University of California San Francisco
• Journal Title: PLoS Pathogens
• Volume: Volume 4, Number 3
• Publisher: Public Library of Science | 2008-03-01, Pages e1000021-e1000021
• Type of Work: Article | Final Publisher PDF
• Publisher DOI: http://doi.org/10.1371/journal.ppat.1000021
• Subjects: Health Sciences, Pathology; Biology, Microbiology; Health Sciences, Immunology
• Keywords: Science & Technology; Life Sciences & Biomedicine; Microbiology; Parasitology; Virology; MICROBIOLOGY; PARASITOLOGY; VIROLOGY; PROTEIN-TYROSINE KINASE; GROWTH-FACTOR RECEPTORS; SRC FAMILY KINASES; C-ABL; HELA-CELLS; HEPARAN-SULFATE; ACTIN CYTOSKELETON; MEMBRANE-PROTEIN; DORSAL RUFFLES; HOST-CELLS

Abstract

The strain designated Chlamydia trachomatis serovar L2 that was used for experiments in this paper is Chlamydia muridarum, a species closely related to C. trachomatis (and formerly termed the Mouse Pneumonitis strain of C. trachomatis). This conclusion was verified by deep sequencing and by PCR using species-specific primers. All data presented in the results section that refer to C. trachomatis should be interpreted as referring to C. muridarum. Since C. muridarum TARP lacks the consensus tyrosine repeats present in C. trachomatis TARP, we cannot make any conclusions about the role of TARP phosphorylation and C. muridarum entry. However, the conclusion that C. trachomatis L2 TARP is a target of Abl kinase is still valid as these experiments were performed with C. trachomatis L2 TARP. 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.