Sess the relevance of our findings in the hostpathogen interaction for the duration of infection we investigated whether bacRNA is able to reach RIGI localized within the cytosol on the host cell. The bacterium L. monocytogenes is actually a wellcharacterized model organism for studying intracellular bacteriahost interaction. In particular, 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). In the following experiments, we selected cell lines that are derived from tissues or cell sorts 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 in to the cytosol of cells making use of a lately developed sensitive, nonradioactive but nontoxic method to label RNA inRIGI Detects RNA of Listeria in NonImmune CellsFigure 1. Bacterial RNA is recognized by human monocytes in a TLRindependent and 59phosphorylationdependent pathway. Human PBMC had been preincubated with chloroquine and transfected with indicated nucleic acids. IFNa production was analyzed 24 hours following stimulation.3-Bromo-6-fluoro-2-methylbenzoic acid Data Sheet 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), were compared. doi:10.1371/journal.pone.0062872.gliving cells [52] (Fig. 2): 5ethynyluridine (EU) was shown to be 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 had been then infected with labeled bacteria. 1 or 4 hours post infection, host cells containing Listeria inside the cytosol had been fixed, permeabilized and incubated with fluorescence dye coupled to a reactive azide group (Alexa594azide). In this setting, the azideselectively couples towards the ethynyl group of EU incorporated into the bacRNA. Entire Listeria are labeled green with FITC; RNA is visible as red fluorescence (Alexa594), nuclei are stained by DAPI (blue). As evident from fluorescence photos, 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 with a mutant lacking LLO (hly) (Fig. 2A suitable panel) and as a result impaired in its escape from the endosome. Bacterial RNA accumulated inside the cytosol of host cells upon prolonged infection time with wt L.737790-46-4 Purity monocytogenes (Fig.PMID:35227773 2A, left and middle panel). In concordance with findings from THP1 cells, similar benefits have been obtained with epithelial (A549, Fig. 2B ) and hepatocarcinoma cell lines (HepG2, Fig. 2C). Throughout Listeria infection only bacRNA delivered towards the cytosol in the host cell could possibly be detected. As direct labeling of RNA from gram negative E. coli, which lack a gram optimistic cell wall, was achievable (Fig. S2A), the gram good cell wall appeared to be the explanation for the absence of RNA staining in the bacteria. To confirm equal incorporation of EU inside the RNA of L. monocytogenes strains, bacterial RNA of wt and hly L. monocytogenes incubated in EUcontaining medium.