Sess the relevance of our findings within the hostpathogen interaction throughout infection we investigated no matter if bacRNA is capable to reach RIGI localized within the cytosol of your host cell. The bacterium L. monocytogenes is usually a wellcharacterized model organism for studying intracellular bacteriahost interaction. In certain, it can be able to escape the endosome and migrate into the cytosol of macrophages, human (not murine) epithelial cells [49,50] or hepatocytes [51], a method mediated by the bacterial poreforming toxin “listeriolysin” (LLO, encoded by the hly gene). Inside the following experiments, we chosen cell lines that are derived from tissues or cell kinds involved in L. monocytogenes pathogenesis in vivo. THP1 is an acute monocytic leukemia cell line related to human monocytederived macrophages. We visualized transfer of bacRNA into the cytosol of cells using a not too long ago created sensitive, nonradioactive but nontoxic strategy to label RNA inRIGI Detects RNA of Listeria in NonImmune CellsFigure 1. Bacterial RNA is recognized by human monocytes within a TLRindependent and 59phosphorylationdependent pathway. Human PBMC had been preincubated with chloroquine and transfected with indicated nucleic acids. IFNa production was analyzed 24 hours just after stimulation. Error bars represent s.d. A: The IFNainducing activity of bacterial RNA (untreated or DNase treated) and DNA of L. monocytogenes, Staphylococcus aureus and Escherichia coli was analyzed. B: The IFNainducing activity of RNAs from L. monocytogenes, L. ivanovii, E. coli, S. aureus and Acinetobacter baumannii, treated with DNase or calf intestine alkaline phosphatase (CIAP), had been compared. doi:10.1371/journal.pone.0062872.gliving cells [52] (Fig. 2): 5ethynyluridine (EU) was shown to become incorporated into RNA transcripts generated by RNA polymerases I, II and III in mammalian cells but not into DNA [52]. Listeria had been grown in medium containing EU and FITC. The host cells were then infected with labeled bacteria. One or 4 hours post infection, host cells containing Listeria within the cytosol have been fixed, permeabilized and incubated with fluorescence dye coupled to a reactive azide group (Alexa594azide). Within this setting, the azideselectively couples towards the ethynyl group of EU incorporated in to the bacRNA. Complete Listeria are labeled green with FITC; RNA is visible as red fluorescence (Alexa594), nuclei are stained by DAPI (blue). As evident from fluorescence images, the cytosol of THP1 cells was clearly labeled for EUcontaining RNA when infected with wild variety (wt) L. monocytogenes (Fig. 2A left and middle panel) but not when infected having a mutant lacking LLO (hly) (Fig.Azetidin-2-one structure 2A suitable panel) and thus impaired in its escape in the endosome.Formula of [Ir[dF(CF3)2ppy]2(bpy)]PF6 Bacterial RNA accumulated inside the cytosol of host cells upon prolonged infection time with wt L.PMID:22943596 monocytogenes (Fig. 2A, left and middle panel). In concordance with findings from THP1 cells, similar final results have been obtained with epithelial (A549, Fig. 2B ) and hepatocarcinoma cell lines (HepG2, Fig. 2C). During Listeria infection only bacRNA delivered towards the cytosol with the host cell may very well be detected. As direct labeling of RNA from gram negative E. coli, which lack a gram positive cell wall, was attainable (Fig. S2A), the gram positive cell wall appeared to become the purpose for the absence of RNA staining in the bacteria. To confirm equal incorporation of EU within the RNA of L. monocytogenes strains, bacterial RNA of wt and hly L. monocytogenes incubated in EUcontaining medium.