RNAi Screen Reveals Host Cell Kinases Specifically Involved in Listeria monocytogenes Spread from Cell to Cell

Ryan Chong,Hervé Agaisse,Raynal Squires,Rachel Swiss,Stefan Bereswill

doi:10.1371/journal.pone.0023399

Abstract

Intracellular bacterial pathogens, such as Listeria monocytogenes and Rickettsia conorii display actin-based motility in the cytosol of infected cells and spread from cell to cell through the formation of membrane protrusions at the cell cortex. Whereas the mechanisms supporting cytosolic actin-based motility are fairly well understood, it is unclear whether specific host factors may be required for supporting the formation and resolution of membrane protrusions. To address this gap in knowledge, we have developed high-throughput fluorescence microscopy and computer-assisted image analysis procedures to quantify pathogen spread in human epithelial cells. We used the approach to screen a siRNA library covering the human kinome and identified 7 candidate kinases whose depletion led to severe spreading defects in cells infected with L. monocytogenes. We conducted systematic validation procedures with redundant silencing reagents and confirmed the involvement of the serine/threonine kinases, CSNK1A1 and CSNK2B. We conducted secondary assays showing that, in contrast with the situation observed in CSNK2B-depleted cells, L. monocytogenes formed wild-type cytosolic tails and displayed wild-type actin-based motility in the cytosol of CSNK1A1-depleted cells. Furthermore, we developed a protrusion formation assay and showed that the spreading defect observed in CSNK1A1-depleted cells correlated with the formation of protrusion that did not resolve into double-membrane vacuoles. Moreover, we developed sending and receiving cell-specific RNAi procedures and showed that CSNK1A was required in the sending cells, but was dispensable in the receiving cells, for protrusion resolution. Finally, we showed that the observed defects were specific to Listeria monocytogenes, as Rickettsia conorii displayed wild-type cell-to-cell spread in CSNK1A1- and CSNK2B-depleted cells. We conclude that, in addition to the specific host factors supporting cytosolic actin-based motility, such as CSNK2B, Listeria monocytogenes requires specific host factors, such as CSNK1A1 in order to form productive membrane protrusions and spread from cell to cell.

Highlights

Various intracellular bacterial pathogens display actin-based motility within human cells, including Listeria monocytogenes and Rickettsia conorii [1,2]
With the objective of identifying host factors involved in pathogen spread, we have developed a high-throughput screening strategy for quantification of L. monocytogenes spread in human epithelial cells using automated fluorescence microscopy and computer-assisted image analysis (Figure 1)
We showed that the accumulation of nonspreading bacteria in primarily infected cells resulted in a dramatic increase of average intensity of the GFP signal (AI-GFP) (Figure 1, MOCK, 10403SDactA, Image analysis, Z-score = 8.8)

Summary

Introduction

Various intracellular bacterial pathogens display actin-based motility within human cells, including Listeria monocytogenes and Rickettsia conorii [1,2]. L. monocytogenes is a Gram-positive bacterium that invades epithelial cells of the intestinal mucosa and causes rare but potentially lethal food-borne infections. R. conorii is an obligate Gram-negative bacterium responsible for Mediterranean spotted fever (MSF), a disease transmitted to humans by the brown dog tick Rhipicephalus sanguineus. The ability to spread from cell to cell is an important determinant of virulence since infection in animal models with strains of L. monocytogenes and R. rickettsii impaired in actin-based motility leads to attenuated pathogenesis [3,4,5]. The bacterial and host factors supporting actin-based motility have been extensively investigated. Seminal genetic studies identified ActA as the bacterial factor required for L. monocytogenes actin tail formation [3,4]. It was initially thought that, similar to L. monocytogenes, Rickettsia spp

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