Host translation initiation and ribosome networks suppress Wolbachia levels.

(A) The host translation initiation and ribosome networks identified by COMPLEAT and KEGG analysis are enriched for genes that increase Wolbachia levels when knocked down by RNAi in the primary screen. Wolbachia level changes are indicated by color: increases (magenta) and no effect (grey). Asterisks mark network components that are not expressed in the JW18 cell line. Changes in cell proliferation in the whole genome screen assay are indicated by icon shape: no change (circle) or decrease (square). (B) Representative genes from each network were validated by RNAi. Effects on Wolbachia levels were assessed quantitatively by a DNA qPCR assay measuring the number of Wolbachia genomes (wspB copy number) relative to Drosophila nuclei (RpL11 copy number). Network validation is represented relative to untreated JW18 cells. Note that even in those cases where knockdown of a host gene results in slowed cell proliferation the resulting increase in Wolbachia cell proliferation exceeds that expected. In control culturing conditions, JW18 cells maintain a stable Wolbachia infection level within the population when cells are split 1:1 every 4 days implying that the cells as well as the Wolbachia double in this timeframe. Thus, we would expect that if knockdown of a host gene results in slowed cell proliferation then the resulting increase in Wolbachia would be a two-fold increase at most. Instead, our results show far greater increases in Wolbachia levels. As such, we suggest that the Wolbachia level increases observed cannot be explained by slowed host cell proliferation rate. (C) Quantification of Wolbachia-infected (magenta) and uninfected (black) cells within the JW18 cell population in control and ribosome (RpS3) RNAi knockdown conditions. Wolbachia infection was detected using the Wolbachia-detecting 23s rRNA FISH probe and cells were identified by DAPI staining of host nuclei. (D) Classification of the level of Wolbachia infection within infected cells of the JW18 cell population under control and ribosome (RpS3) knockdown conditions (seen in C). For each cell population >500 cells were quantified for Wolbachia infection level by the following criteria: low (1–10 Wolbachia), medium (11–30 Wolbachia), and high infection (>30 Wolbachia). See S8 and S10 Figs for further network analysis results.