The secRNome of Listeria monocytogenes Harbors Small Noncoding RNAs That Are Potent Inducers of Beta Interferon

Abstract

Cellular sensing of bacterial RNA is increasingly recognized as a determinant of host-pathogen interactions. The intracellular pathogen Listeria monocytogenes induces high levels of type I interferons (alpha/beta interferons [IFN-α/β]) to create a growth-permissive microenvironment during infection. We previously demonstrated that RNAs secreted by L. monocytogenes (comprising the secRNome) are potent inducers of IFN-β. We determined the composition and diversity of the members of the secRNome and found that they are uniquely enriched for noncoding small RNAs (sRNAs). Testing of individual sRNAs for their ability to induce IFN revealed several sRNAs with this property. We examined ril32, an intracellularly expressed sRNA that is highly conserved for the species L. monocytogenes and that was the most potent inducer of IFN-β expression of all the sRNAs tested in this study, in more detail. The rli32-induced IFN-β response is RIG-I (retinoic acid inducible gene I) dependent, and cells primed with rli32 inhibit influenza virus replication. We determined the rli32 motif required for IFN induction. rli32 overproduction promotes intracellular bacterial growth, and a mutant lacking rli32 is restricted for intracellular growth in macrophages. rli32-overproducing bacteria are resistant to H2O2 and exhibit both increased catalase activity and changes in the cell envelope. Comparative transcriptome sequencing (RNA-Seq) analysis indicated that ril32 regulates expression of the lhrC locus, previously shown to be involved in cell envelope stress. Inhibition of IFN-β signaling by ruxolitinib reduced rli32-dependent intracellular bacterial growth, indicating a link between induction of the interferon system and bacterial physiology. rli32 is, to the best of our knowledge, the first secreted individual bacterial sRNA known to trigger the induction of the type I IFN response. IMPORTANCE Interferons are potent and broadly acting cytokines that stimulate cellular responses to nucleic acids of unusual structures or locations. While protective when induced following viral infections, the induction of interferons is detrimental to the host during L. monocytogenes infection. Here, we identify specific sRNAs, secreted by the bacterium, with the capacity to induce type I IFN. Further analysis of the most potent sRNA, rli32, links the ability to induce RIG-I-dependent induction of the type I IFN response to the intracellular growth properties of the bacterium. Our findings emphasize the significance of released RNA for Listeria infection and shed light on a compartmental strategy used by an intracellular pathogen to modulate host responses to its advantage.

Staphylococcus aureus secretes immunomodulatory RNA and DNA via membrane vesicles

“…After reaching mid-log phase (OD600 of 1.0), MVs were isolated from the supernatant of the culture as delineated in the "Membrane Vesicle Isolation" section. However, after the initial filtration step, supernatants were pelleted using a Beckman Avanti J -25 centrifuge (38,400 x g, 3h, 4˚C; JLA-16.250 rotor), followed by ultracentrifugation and filtration using a 0.22-µm pore size, hydrophilic polyvinylidene fluoride filter (Merck Millipore Germany). MV preparations were tested for sterility and the protein concentration was measured using a Bradford assay.…”

Staphylococcus aureus secretes immunomodulatory RNA and DNA via membrane vesicles

“…Cheng and Shorey showed that M. tuberculosis releases RNA into the cytosol of infected macrophages in a SecA2 and EsX-1 secretion system-dependent manner, however they only speculated that potential RNA-binding proteins could also shuttle bound RNA molecules via the SecA2 system into the extracellular milieu 37 . Frantz and colleagues recently showed that L. monocytogenes secretes both “naked” and MV-contained sRNAs that are potent IFN-ß inducers in macrophages and in HEK293 cells 38 . This group focused on characterizing specific “naked” sRNAs present in the non-MV containing supernatant fraction that also induced high levels of IFN-ß but did not examine the secretion mechanism for the RNA.…”

Interplay between Regulatory RNAs and Signal Transduction Systems during Bacterial Infection

“…Application of RNA-seq technologies to analyze bacterial secRNome provides promising perspectives to expand the role of sRNAs in host–pathogen interactions [ 227 ]. Secreted RNAs and RNAs in extracellular vesicles (membrane vesicles and OMVs) with potential regulatory function in host could be characterized in both Gram-negative and Gram-positive bacteria, including recent examples in P. aeruginosa and L. monocytogenes [ 79 , 228 ]. Interestingly, Rli32, one of these sRNAs that have been identified as potential inducers of type I interferons IFN response during L. monocytogenes infection is part of the VirR/VirS and Agr quorum sensing regulons [ 79 , 229 ].…”

“…Future characterization of secRNome of major pathogens will shed a new light on the role of sRNAs in host–pathogen interactions. The recent discovery of Rli32 sRNA inducing interferon response during L. monocytogenes infection illustrates well this yet unexplored regulatory potential [ 79 ]. This new field of research could suggest new ways to transport RNA inside the host and new possibilities for the modulation of host immune system.…”