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

Abstract

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.

Stress‐induced host membrane remodeling protects from infection by non‐motile bacterial pathogens

“…Analysis of Shigella cell‐to‐cell spreading in control and arsenite or anisomycin‐treated cells, by quantifying the area of Shigella infection foci using fluorescence microscopy and automated image analysis (Sunkavalli et al , 2017), excluded an effect of stress in the actin‐based spreading of Shigella to neighboring cells (Appendix Fig S1J). In these experiments, infections were performed at different MOIs (MOI 10 for control, MOI 50 for anisomycin, and MOI 100 for arsenite), to achieve comparable levels of bacterial invasion.…”

“…Image analysis was performed using Columbus image analysis software (PerkinElmer) as described previously (Sunkavalli et al , 2017). …”

“…For quantification of Shigella infection and spreading, image acquisition was performed using Operetta automated high-content screening fluorescence microscope (PerkinElmer) at a 20× and 10× magnification, respectively; a total of 9 (infection) or 1 (spreading) images were acquired per coverslip/well, corresponding to approximately 2,500 cells analyzed. Image analysis was performed using Columbus image analysis software (PerkinElmer) as described previously (Sunkavalli et al, 2017).…”

MicroRNA-29b-2-5p inhibits cell proliferation by directly targeting Cbl-b in pancreatic ductal adenocarcinoma

“…On the contrary, miR-29b-2-5p is rarely studied. Although miR-29b-2-5p is considered a promoter of bacterial binding to host cells in prokaryotes [ 40 ], its identity and function in pancreatic cancer remain unclear. In the current study, miR-29b-2-5p expression independently predicted good survival in PDAC as evaluated by multivariate Cox regression analysis.…”

miRNAs reshape immunity and inflammatory responses in bacterial infection

“…Filopodia are thin actin-rich cell protrusions, and forming host-cell filopodia is critical for phagocytosis and bacterial internalization. miR-29b-2-5p, by inhibiting its direct target UNC5C, rapidly increased filopodia in hosts upon Shigella flexneri infection 102 . These studies highlight the complicated transcriptional and posttranscriptional response mechanisms of host cells to bacterial infection.…”