Given the growing antibiotic resistance crisis, novel ways to target bacterial infections are becoming increasingly important. One potential strategy is to manipulate bacterial genes at the transcriptional level by silencing their resistance genes. However, siRNAs have not been applied to regulate bacterial genes due to the lack of RNAi regulatory machinery, i.e., RNA-induced silencing complexes (RISCs), in bacteria. In addition, efficient methods for delivering siRNAs to bacteria in vivo are not currently available.
In a new study, researchers demonstrate that exosomes can serve as delivery vehicles to introduce AGO2-loaded siRNA into the cytoplasm of bacteria which downregulate gene expression of the mRNA that shares sequence complementarity to the siRNA.
This work is published in Cell Reports Medicine in the paper, “siRNA-AGO2 complex inhibits bacterial gene translation: a promising therapeutic strategy for superbug infection.”
Despite the fact that the siRNA machinery is absent in prokaryotes, this study demonstrates that exosomal siRNAs can be efficiently delivered into bacteria and silence target bacterial genes. The exosomal Argonaute 2 (AGO2) protein forms a complex with siRNAs, which is essential for siRNA-mediated gene silencing in bacteria. Even though the delivered siRNA was designed to possess full sequence complementarity to the bacterial mRNA, downregulation of the target protein was achieved at the translational level rather than on the level of mRNA stability (as would be expected in mammalian systems).
Exosomal siMecA (an siRNA designed to target the mecA gene) can downregulate the mecA gene which encodes the penicillin-binding protein 2a (PBP2a), a protein at the heart of the drug-resistance phenotype in MRSA. Both in vitro and in vivo (using MRSA-infected mice) data demonstrated that by reducing PBP2a levels by exosome-delivered siRNA-AGO2 complex, MRSA can be converted into methicillin-sensitive bacteria.
To induce exosome production in vivo, the authors demonstrated that by intravenous injection of a plasmid encoding all relevant genes for the production of a particular siRNA into mice, the mouse liver was producing siRNA-AGO2 loaded exosomes (siMecA-Exos) efficiently targeting MRSA. These findings have potential clinical relevance since it might be possible to use this approach to also target multi-drug resistant bacterial infections in the human system.
Exosome-mediated small RNA-AGO2 transport and bacterial gene silencing is a potential pathway by which mammalian cells regulate interacting bacteria in vivo. This is demonstrated in the treatment of MRSA infection by mouse liver-produced siMecA-Exos. In addition, exosome-mediated RNAi may function with imperfectly matched sequences, so exosomal miRNAs, which have multiple bacterial targets with imperfect base pairing, may regulate bacterial genes in the same way as exosomal siRNAs. It is intriguing to consider the possibility that mammalian hosts employ exosomes to transport molecules with biological functions to the mammalian microbiome for interspecies communication and regulation under physiological conditions.
graphical abstract
siMecA-AGO2 complex inhibits the translation of the mecA gene which encodes the penicillin-binding protein 2a (PBP2a), a protein at the heart of the drug-resistance phenotype in MRSA. By reducing PBP2a levels by exosome-delivered siMecA-AGO2 complex, MRSA can be converted into methicillin-sensitive bacteria. Thus, coadministration of methicillin (i.p.) and siMecA-Exos (i.v.) effectively protected mice from lethal infection (70% survival rate). [Cell Reports Medicine]