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

Abstract

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.

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Fig. 1: Infection by Salmonella and Shigella are controlled by largely non-overlapping subsets of microRNAs.
Fig. 2: miRNAs regulate different stages of Salmonella and Shigella infection cycles.
Fig. 3: Shigella actin-based motility and intercellular spreading are inhibited by miR-3668, miR-4732-5p and miR-6073.
Fig. 4: Let-7i-3p inhibits Salmonella binding to host cells and bacterial intracellular replication.
Fig. 5: Inhibition of Salmonella replication by let-7i-3p is mediated by RGS2.
Fig. 6: Modulation of endolysosomal trafficking by let-7i-3p is critical for Salmonella infection.

Data availability

The data supporting the findings of this study are available from the corresponding authors upon reasonable request. The demultiplexed FASTQ files and gene-wise quantifications have been deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO Series accession numbers GSE53281 (mock-treated and Salmonella-infected samples) and GSE69660 (cells transfected with control miRNA or let-7i-3p mimic). Source data for Figs. 3e,i,k, 4f, 5f,g,i and 6h and Extended Data Figs. 3b,d, 5f,h,j and 6b are included in this article and its Supplementary Information files.

Code availability

Custom image analysis workflows implemented in Columbus or CellProfiler image analysis software are available upon reasonable request.

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Acknowledgements

C.A. is a recipient of a fellowship from the Bayerischen Gleichstellungsförderung (BGF) through the SCIENTIA Program; I.R.L. and R.J.S. are recipients of PhD fellowships (PD/BD/146464/2019 and PD/BD/129294/2017) of the Doctoral Programme in Experimental Biology and Biomedicine of the Center for Neuroscience and Cell Biology, University of Coimbra. The authors acknowledge the animal facility staff at the University of Leon, Spain, in particular A. Carvajal from the Department of Animal Health. This work was supported by grants from the Bavarian Ministry of Sciences, Research and the Arts in the framework of the Bavarian Molecular Biosystems Research Network (BioSysNet), DFG project BR 4837/1-1 and the Portuguese Foundation for Science and Technology (FCT Investigator Programme IF/01105/2015).

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Contributions

C.A., A.R.C., C.M., M.M. and A.E. designed the experiments; A.R.C., M.M. and A.E. performed the high-throughput screening and validation experiments; C.A., I.R.L., C.M., U.S., R.J.S. and C.L. performed the biochemical and cell biology assays for characterization of the role of miRNAs in infection; M.S. performed the analysis of the RNA-sequencing datasets; S.Z.-L. and J.J.G. performed the in vivo Salmonella infections; M.G. provided reagents and access to equipment; M.M. and A.E. wrote the manuscript, with input from all authors.

Corresponding authors

Correspondence to Miguel Mano or Ana Eulalio.

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Extended data

Extended Data Fig. 1 Screenings for miRNAs controlling infection by Salmonella and Shigella.

a. Microscopy images and corresponding image segmentation outlines of HeLa cells treated with control miRNA and infected with Salmonella or Shigella. Cells marked in green in the image segmentation panels correspond to cells with high intracellular bacterial load; cells touching the borders of the images were excluded from the analysis. Scale bar, 100 μm. b and c. Pairwise correlation between the three independent runs of the screenings to identify miRNAs that regulate Salmonella (b) and Shigella (c) infection. Results are based on quantification of the percentage of infected cells following treatment with the library of miRNA mimics and are shown as fold change compared to control miRNA (log2 scale). Spearman rank correlation coefficients are shown in the right bottom corner of graphs. d and e. Comparison of the results obtained in the Salmonella (d) and Shigella (e) screenings for the 110 selected miRNAs, with those obtained for the corresponding time point in the time-course experiments (20 hpi, Salmonella; 6 hpi, Shigella). Results shown are the mean of the three independent runs of each screen or time-course experiments. Colours in panels d and e correspond to the different miRNA categories identified in Figure 2a. Spearman rank correlation coefficients are shown in the right bottom corner of graphs.

Extended Data Fig. 2 Shigella intracellular motility and intercellular spreading are inhibited by miR-3668, miR-4732-5p and miR-6073.

a. Fluorescence microscopy images extracted from the time-lapse microscopy analysis of HeLa cells infected with Shigella wild-type, upon treatment with control miRNA, miR-3668, miR-4732-5p or miR-6073 mimics. Full time-lapse sequence is included as Supplementary Videos 1-4. Scale bar, 100 μm. b and c. Representative images (b) and area of Shigella infection foci (c) in HCT-8 cells transfected with control miRNA, miR-3668, miR-4732-5p or miR-6073 mimics and infected with Shigella, analyzed at 3 hpi. Shigella ΔicsA mutant is shown for comparison. Scale bar, 25 µm. d. Area of Listeria infection foci in HeLa cells transfected with the indicated miRNAs, analyzed at 8 hpi. e. Sequences of mature human miR-3668, miR-4732-5p and miR-6073. The 7-mer seed sequences of the miRNAs are highlighted in red. f. N-WASP levels, determined by western blot, in HeLa cells transfected with control miRNA, miR-3668, miR-4732-5p or miR-6073 mimics, or with control siRNA or N-WASP siRNA. g and h. Representative images (g) and percentage of bacteria forming actin tails (h) in HeLa cells transfected with control siRNA or N-WASP siRNA, analyzed at 1.5 hpi. Staining of F-actin using fluorescently labeled phalloidin allows the visualization of actin tails. Scale bar, 10 µm. i and j. Area of Shigella infection foci in HeLa (i) and HCT-8 (j) cells transfected with control miRNA or N-WASP siRNA, analyzed at 3 hpi. Shigella ΔicsA mutant is shown for comparison. k. Area of Listeria infection foci in HeLa cells transfected with control miRNA or N-WASP siRNA, analyzed at 3 hpi. Shigella infection was performed at MOI 25 (b,g,h), 10 (a,i) or 5 (c,j); Listeria infection was performed at MOI 20. Results are shown as mean ± s.e.m. of n=4 (h) or n=5 (c,f,i-k) biologically independent experiments; microscopy images are representative of n=3 (a), n=4 (g) or n=5 (b) biologically independent experiments; centre values indicate means (c,d,h-j); ***P<0.001 (statistical analyses are detailed in Supplementary Table 4).

Extended Data Fig. 3 Targeting of N-WASP by miR-3668, miR-4732-5p and miR-6073 inhibits Shigella intercellular spreading.

a. Area of Shigella infection foci (in parental HeLa cells, HeLa cells stably expressing a siRNA resistant version of N-WASP fused with EGFP (EGFP-N-WASPr) or wild-type N-WASP fused with EGFP (EGFP-N-WASP), transfected with a control siRNA or a siRNA targeting N-WASP, analyzed at 3 hpi. b. N-WASP expression, determined by western blot, in HeLa cells (parental, EGFP-N-WASPr, EGFP-N-WASP) transfected with a control siRNA or a siRNA targeting N-WASP. c. Area of Shigella infection foci in HeLa cells transfected with control siRNA or 4 independent siRNAs targeting N-WASP, analyzed at 3 hpi. d. N-WASP expression, determined by western blot, in HeLa cells transfected with control siRNA or 4 independent siRNAs targeting N-WASP. e. Sequences of the six active binding sites for miR-3668 (2 sites), miR-4732-5p (1 site) and miR-6073 (3 sites) identified in the 3’UTR of N-WASP. Complementarity of the binding sites with the miRNAs, as predicted by TargetScan, is shown. f. N-WASP levels, determined by western blot, in HeLa cells mock-treated or infected with Shigella, at different times post-infection (0.5, 3, 6 and 8 hpi). g. Expression of mature miR-4732-5p or miR-6073, determined by RT-qPCR, in HeLa cells transfected with control miRNA inhibitor or inhibitors of miR-4732-5p and miR-6073, separately or simultaneously. h. N-WASP levels, determined by western blot, in HeLa cells mock-treated or infected with Shigella, and transfected with control miRNA inhibitor or inhibitors of miR-4732-5p and miR-6073 simultaneously, analyzed at 8 hpi. Shigella infection was performed at MOI 100 (f,h) or 10 (a,c). Results are shown as mean ± s.e.m. of n=3 (g,h) or n=5 (a,c,f) biologically independent experiments; western blots (b,d) are representative of n=3 biologically independent experiments; centre values indicate means (a,d); **P<0.01, ***P<0.001 (statistical analyses are detailed in Supplementary Table 4). Source Data

Extended Data Fig. 4 Disruption of actin stress fibers reverts the inhibitory effect of let-7i-3p on Salmonella binding to host cells.

a. Sequences of the mature miRNAs derived from the 5’ and 3’ arm of the precursors of the let-7 family. Common seed sequence of the let-7 miRNA family (-5p miRNAs) is highlighted in blue and that of let-7i-3p is highlighted in green. b, c, e and f. Representative images (b,e) and quantification by cfu (c,f) of Salmonella bound to HeLa cells transfected with control miRNA or let-7i-3p mimics, and incubated with Salmonella wild-type or Δ4 mutant strain for 10 min. Cells were pre-treated with cytochalasin D for 1 h (b,c). Salmonella was previously coated with PL (e,f). d, g and h. Representative images (d,g) and quantification by cfu (h) of Shigella bound to HeLa cells transfected with control miRNA or let-7i-3p mimics, and incubated with Shigella wild-type or ΔipaB mutant strains for 25 min. Shigella was previously coated with PL (g,h). i-l. Representative images (i,k) and quantification by cfu (j,l) of Listeria bound to (i,j) or intracellular Listeria (k,l) of HeLa cells transfected with control miRNA or let-7i-3p mimics. Binding was performed for 25 min, and intracellular bacteria analyzed at 1 and 20 hpi. m-p. Expression of mature let-7i-3p (m,n) and RGS2 (o,p), evaluated by RT-qPCR, in HeLa cells infected with Shigella (m,o) or Listeria (n,p). Shigella and Listeria infections were analyzed at 6 or 20 hpi, respectively. Salmonella infection was performed at MOI 50. Shigella infection was performed at MOI 50 (d,g,h) or MOI 100 (m,o). Listeria infection was performed at MOI 25 (k,l,n,p) or 50 (i,j). Scale bars, 50 μm. Results are shown as mean ± s.e.m. of n=5 (c,f,h,l-p) or n=6 (j) biologically independent experiments; microscopy images are representative of n=3 (i,k) or n=5 (b,d,e,g) biologically independent experiments; ***P<0.001 (statistical analyses are detailed in Supplementary Table 4).

Extended Data Fig. 5 Let-7i-3p targets the coding sequence of RGS2.

a. Schematic representation of RGS2 3′UTR, CDS, and CDS fragments and mutant constructs used for miRNA binding site reporter assays. The RGS2 3’UTR was cloned downstream of a Renilla luciferase reporter (psiCHECK2-RGS2 3’UTR) and the RGS2 CDS and CDS fragments were cloned as a C-terminal fusion of green fluorescent protein (EGFP-RGS2 CDS). b-d. Results of the reporter assays using the RGS2 3’UTR (b), RGS2 CDS (c), or RGS2 CDS fragments and mutant (d). Results in cells treated with the let-7i-3p mimic are shown compared to the control miRNA. e. Sequence of the binding site for let-7i-3p in fragment F1 of the N-WASP CDS. Complementarity of the miRNA with the binding site of human RGS2 sequence is shown. f. RGS2 expression, determined by western blot, in HeLa cells transfected with control siRNA or RGS2 siRNA. g. Quantification of intracellular Salmonella by cfu in parental HeLa cells, HeLa cells stably expressing a siRNA resistant version of RGS2 fused with EGFP (EGFP-RGS2r) or wild-type RGS2 fused with EGFP (EGFP-RGS2), transfected with a control siRNA or a siRNA targeting RGS2, analyzed at 20 hpi. h. RGS2 expression, determined by western blot, in HeLa cells (parental, EGFP-RGS2r or EGFP-RGS2), transfected with a control siRNA or a siRNA targeting RGS2. i. Quantification of intracellular Salmonella by cfu in HeLa cells transfected with control siRNA or 4 independent siRNAs targeting RGS2, analyzed at 20 hpi. j. RGS2 expression, determined by western blot, in HeLa cells transfected with control siRNA or 4 independent siRNAs targeting RGS2. Salmonella infection was performed at MOI 25. Results are shown as mean ± s.e.m. of n=3 (b), n=5 (g,i), n=7 (d) or n=10 (c) biologically independent experiments; western blots (f,h,j) are representative of n=3 biologically independent experiments; **P<0.01, ***P<0.001 (statistical analyses are detailed in Supplementary Table 4). Source Data

Extended Data Fig. 6 Inhibition of let-7i-3p favours Salmonella infection.

a. Expression of mature let-7i-3p, determined by RT-qPCR, in HeLa cells transfected with control miRNA or let-7i-3p inhibitors. b. RGS2 expression, determined by western blot, in HeLa cells transfected with control miRNA or let-7i-3p inhibitors; non-transfected and mock-transfected cells are shown for comparison. c. Representative images of HeLa cells transfected with control miRNA or let-7i-3p inhibitors and infected with Salmonella. Infection was analyzed at 1, 4 and 20 hpi. Salmonella infection was performed at MOI 25. Scale bar, 50 μm. d-f. Representative images of HeLa cells transfected with control miRNA or let-7i-3p inhibitors and stained with Lysotracker (d), for LAMP-1 (e), or with DQ Red BSA (f). Scale bar, 50 μm. Results are shown as mean ± s.e.m. of n=4 (a) biologically independent experiments; western blots and microscopy images are representative of n=3 (b,d-f) or n=5 (c) biologically independent experiments; ***P<0.001 (statistical analyses are detailed in Supplementary Table 4). Source Data

Supplementary information

Reporting Summary

Supplementary Video 1

Time-lapse microscopy of Shigella infection in HeLa cells transfected with control miRNA. Data are representative of n = 3 biologically independent experiments.

Supplementary Video 2

Time-lapse microscopy of Shigella infection in HeLa cells transfected with miR-3668. Data are representative of n = 3 biologically independent experiments.

Supplementary Video 3

Time-lapse microscopy of Shigella infection in HeLa cells transfected with miR-4732-5p. Data are representative of n = 3 biologically independent experiments.

Supplementary Video 4

Time-lapse microscopy of Shigella infection in HeLa cells transfected with miR-6073. Data are representative of n = 3 biologically independent experiments.

Supplementary Tables

Supplementary Tables 1–4.

Source data

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Aguilar, C., Cruz, A.R., Rodrigues Lopes, I. et al. Functional screenings reveal different requirements for host microRNAs in Salmonella and Shigella infection. Nat Microbiol 5, 192–205 (2020). https://doi.org/10.1038/s41564-019-0614-3

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