Die RNA-Sekretion via SecA2 am Beispiel der sRNA rli32 in Listeria monocytogenes
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Beschreibung
The recognition of pathogen-associated molecules by the innate immune system is an essential component of an efficient immune response to eliminate pathogens. The recognition of bacterial RNA and its immune stimulatory properties has been focused in recent years of research. Bacterial RNAs stimulate the production of inflammatory cytokines and induce a strong type I interferon (IFN) response. The facultative intracellular foodborn pathogen Listeria monocytogenes (Lm) stimulates a strong type I IFN response, which supports its intracellular growth. Living intracellular Lm are a prerequisite for the stimulation of a type I IFN response. Secreted RNA from Lm was described as a potential trigger for IFN-β induction, but little is known about the secretion mechanism of such RNAs. Sequencing data of this RNA fraction identified the accumulation of small non-coding RNAs (sRNA). Some of these individuall sRNAs have the potential to stimulate the IFN-β expression.
SecA2 is an additional motor protein of the general Sec secretion system in several Gram-positive bacteria and is related to virulence of some pathogens. Besides a small selection of SecA2-dependent proteins, a SecA2-dependent secretion of RNAs has been suspected. In this work, it was shown that the deletion mutant Lm-ΔsecA2 grown in defined nutrient medium shows reduced amounts of freely secreted RNA in the supernatant (called sec-RNA), which was strictly separated from the membrane vesicles (MVs) fraction. The sec-RNA of Lm-ΔsecA2 shows a weaker potential for IFN-β induction than sec-RNA from Lm. A possible involvement of SecA2-dependent enolase (Eno) in this issue was shown by the deletion of RNA-binding Eno, which has an adverse effect on the secretion of individual potential IFN-β-inducing sRNAs. In this work, isolated recombinant SecA2 was found in association with short RNAs that have the potential to induce IFN-β. SecA1 is required in Lm for the secretion of a large number of proteins, which associate with RNAs of high diversity and these have a lower capacity for IFN-β expression in macrophages. The recent sequencing data of SecA2-associated RNA identified the enrichment of some sRNA as potential trigger of IFN-β induction. Among these sRNAs the small non-coding sRNA rli32 was enriched, which is one of the most potential IFN-β inducing sRNAs and also part of the secreted RNA. Rli32 is highly expressed intracellularly and highly conserved in Lm, and shows RIG-I (retinoic acid inducible gene I)-dependent stimulation of the IFN-β response. For the recognition of rli32 via the cytosolic receptor RIG-I, a motif (motif2) of the rli32 sequence is essential, which was shown in this work by mutations in motif2. Furthermore, it could be shown that the overexpression of rli32 leads to a strong increase of IFN-β induction in macrophages and this effect improves the intracellular growth of Lm. In contrast, the deletion of rli32 leads to limited intracellular growth. The growth-promoting effect of rli32 on intracellular Lm can be prevented by the Janus kinase (JAK) inhibitor Ruxolitinib (Rux). In addition, this work showed that the overexpression of rli32 promotes the resistance of Lm to hydrogen peroxide (H2O2) and makes Lm more susceptible to the cephalosporin cefuroxime. A comparative RNA sequencing identified a regulation of the sRNAs LhrC1-4 by rli32, which were shown to be induced at iron toxicity and cell wall stress. The comparative proteomic data of this work revealed altered protein compositions in the cell envelope of Lm by altered rli32 expression levels and hypothesized a bacterial physiological function of rli32 in nitrogen metabolism. In addition, the overexpression of rli32 showed an increased secretion of eno, which indirectly indicates an increased activity of the SecA2-dependent secretion pathway. SecA2 is in close proximity to RNA-protein complexes such as actively translating ribosomes, whose association with SecA2 was observed in this work.
The present work provides new insights into the secretion of type I IFN-inducing sRNAs by SecA2 and reveals first evidence for the involvement of SecA2-dependent Eno. Furthermore, the results on sRNA rli32 show for the first time a relationship between the type I IFN-stimulating capacities of individual secreted sRNAs and the intracellular physiology of Lm.
Autor*in
Lisa Teubner
Themen in »Die RNA-Sekretion via SecA2 am Beispiel der sRNA rli32 in Listeria monocytogenes«
Bakterium Genetik Signalpeptid