Functional screenings reveal different requirements for host microRNAs in Salmonella and Shigella infection.

MicroRNAs (miRNAs) are increasingly recognized for their role in infection by bacterial pathogens, although the effect of each individual miRNA remains largely unknown. Here, we used a comparative genome-wide microscopy-based functional screening approach to identify miRNAs controlling infection by two bacterial pathogens-Salmonella enterica serovar Typhimurium and Shigella flexneri. Despite the similarities between these pathogens, we found infections to be controlled by largely non-overlapping subsets of miRNAs, seemingly reflecting different requirements prompted by their distinct intracellular lifestyles. By characterizing a small subset of miRNAs chosen among the strongest inhibitors of Shigella infection, we discovered that miR-3668, miR-4732-5p and miR-6073 exert a selective effect on Shigella infection by impairing bacterial actin-based motility by downregulating N-WASP. Additionally, by identifying let-7i-3p miRNA as a strong inhibitor of Salmonella replication and performing in-depth analysis of its mechanisms of action, we showed that this miRNA specifically inhibits Salmonella infection via modulation of endolysosomal trafficking and the vacuolar environment by targeting the host RGS2 protein. These findings illustrate two paradigms underlying miRNA-mediated regulation of bacterial infection, acting as part of the host response to infection, or as part of bacterial strategies to modulate the host environment and favour pathogenesis.

Analysis of host microRNA function uncovers a role for miR-29b-2-5p in Shigella capture by filopodia.

MicroRNAs play an important role in the interplay between bacterial pathogens and host cells, participating as host defense mechanisms, as well as exploited by bacteria to subvert host cellular functions. Here, we show that microRNAs modulate infection by Shigella flexneri, a major causative agent of bacillary dysentery in humans. Specifically, we characterize the dual regulatory role of miR-29b-2-5p during infection, showing that this microRNA strongly favors Shigella infection by promoting both bacterial binding to host cells and intracellular replication. Using a combination of transcriptome analysis and targeted high-content RNAi screening, we identify UNC5C as a direct target of miR-29b-2-5p and show its pivotal role in the modulation of Shigella binding to host cells. MiR-29b-2-5p, through repression of UNC5C, strongly enhances filopodia formation thus increasing Shigella capture and promoting bacterial invasion. The increase of filopodia formation mediated by miR-29b-2-5p is dependent on RhoF and Cdc42 Rho-GTPases. Interestingly, the levels of miR-29b-2-5p, but not of other mature microRNAs from the same precursor, are decreased upon Shigella replication at late times post-infection, through degradation of the mature microRNA by the exonuclease PNPT1. While the relatively high basal levels of miR-29b-2-5p at the start of infection ensure efficient Shigella capture by host cell filopodia, dampening of miR-29b-2-5p levels later during infection may constitute a bacterial strategy to favor a balanced intracellular replication to avoid premature cell death and favor dissemination to neighboring cells, or alternatively, part of the host response to counteract Shigella infection. Overall, these findings reveal a previously unappreciated role of microRNAs, and in particular miR-29b-2-5p, in the interaction of Shigella with host cells.

Functional high-throughput screening identifies the miR-15 microRNA family as cellular restriction factors for Salmonella infection.

Increasing evidence suggests an important role for miRNAs in the molecular interplay between bacterial pathogens and host cells. Here we perform a fluorescence microscopy-based screen using a library of miRNA mimics and demonstrate that miRNAs modulate Salmonella infection. Several members of the miR-15 miRNA family were among the 17 miRNAs that more efficiently inhibit Salmonella infection. We discovered that these miRNAs are downregulated during Salmonella infection, through the inhibition of the transcription factor E2F1. Analysis of miR-15 family targets revealed that derepression of cyclin D1 and the consequent promotion of G1/S transition are crucial for Salmonella intracellular proliferation. In addition, Salmonella induces G2/M cell cycle arrest in infected cells, further promoting its replication. Overall, these findings uncover a mechanism whereby Salmonella renders host cells more susceptible to infection by controlling cell cycle progression through the active modulation of host cell miRNAs.