A Novel Mechanism of Host-Pathogen Interaction through sRNA in Bacterial Outer Membrane Vesicles

Abstract

Bacterial outer membrane vesicle (OMV)-mediated delivery of proteins to host cells is an important mechanism of host-pathogen communication. Emerging evidence suggests that OMVs contain differentially packaged short RNAs (sRNAs) with the potential to target host mRNA function and/or stability. In this study, we used RNA-Seq to characterize differentially packaged sRNAs in Pseudomonas aeruginosa OMVs, and to show transfer of OMV sRNAs to human airway cells. We selected one sRNA for further study based on its stable secondary structure and predicted mRNA targets. Our candidate sRNA (sRNA52320), a fragment of a P. aeruginosa methionine tRNA, was abundant in OMVs and reduced LPS-induced as well as OMV-induced IL-8 secretion by cultured primary human airway epithelial cells. We also showed that sRNA52320 attenuated OMV-induced KC cytokine secretion and neutrophil infiltration in mouse lung. Collectively, these findings are consistent with the hypothesis that sRNA52320 in OMVs is a novel mechanism of host-pathogen interaction whereby P. aeruginosa reduces the host immune response.

Fig 1. OMVs contain differentially packaged sRNAs.

(A) Relative abundance of 481,480 unique OMV sRNA sequence reads versus P. aeruginosa whole cell sRNA sequence reads. Sequences to the left of the solid diagonal line were more abundant in OMVs than in whole cells and sequences to the right were less abundant in OMVs. The median sequence length of the sRNAs was 24 nucleotides, with a minimum of 15 and a maximum of 45 nucleotides. (B) Relative abundance of sRNAs associated with a locus in OMVs versus P. aeruginosa. The 1733 most abundant OMV sRNA sequences (with a minimum of 64 counts, indicated by the horizontal dashed line) mapped to 68 PA14 loci. Loci to the left of the solid diagonal line were more abundant in OMVs than in P. aeruginosa, while those to the right of the line were less abundant in OMVs than in P. aeruginosa. On average, loci that were abundant in OMVs were 9-fold enriched in OMVs compared to P. aeruginosa (mean log₂ Ratio = 3.17, 95% CI = 2.28–4.06, p = 1.1*10⁻⁹). Significance was determined using a one-sample t-test. The 10 most abundant OMV loci (solid black circles) are numbered and annotated in more detail in Table 1.

Fig 2. sRNA52320 is inside of OMVs and protected from RNase digestion.

(A) RNase A digests free RNA including RNA associated with the outside of OMVs, while RNA inside of intact OMVs is protected from degradation. (B) Agarose gel showing profiles of OMV-associated RNAs from untreated control OMVs (lane 1), RNase A treated OMVs (lane 2) and OMV RNA extracted from QIAzol lysed OMVs after digestion with RNase A (lane 3). RNA was visualized by staining with SYBR Safe. Samples were run on the same gel and were re-arranged for presentation. (C) qPCR for sRNA52320 using RNA isolated from control OMVs or RNase A-treated OMVs. RNase A treatment prior to RNA-Isolation (filled circles) increased the relative abundance of sRNA52320 compared to untreated OMVs (open circles). The difference in mean cycle threshold (Ct) of -2.5 ± 0.6 was statistically significant (95% CI = -4.1 to -0.9, N = 3, p = 0.013 indicated by an asterisk).

Fig 3. sRNAs are transferred from OMVs to host cells.

(A) Counts per million (CPM) reads that uniquely aligned to the PA14 reference sequence in unexposed HBE cells (left, HBE ctrl) and HBE cells that had been exposed to OMVs and washed vigorously after exposure (right, HBE+OMV). (B) Counts per million (CPM) reads for the seven PA14 loci most abundantly detected in OMV-exposed HBE cells.

Fig 4. sRNA52320 targets kinases in the LPS-stimulated MAPK signaling pathway and is predicted to attenuate the innate immune response.

(A) Volcano plot of -log₁₀ p-values and mean log₂ ratios for proteins from LPS-stimulated HBE cells transfected with sRNA52320 compared to LPS-stimulated HBE cells transfected with negative control RNA (siNC). A total of 3902 proteins were identified in all three HBE cell donor samples. Proteins that were differentially expressed (320 total), as determined by a one-sample t-test, are shown as red circles (darker shade indicates higher abundance). sRNA52320 decreased the abundance of the majority of the 320 proteins. (B) The 320 proteins whose abundance was altered by sRNA52320 were analyzed with Ingenuity Pathway Analysis (IPA). The depicted IPA canonical pathway “LPS-stimulated MAPK signaling” was one of the top 10 canonical pathways identified (p = 0.005, activation z-score = -2.236). The green circles identify proteins whose abundance was reduced by sRNA52320 compared to siNC. Five kinases in the LPS-stimulated MAPK signaling pathway were predicted to be direct targets of sRNA52320, including MAP2K2, MAP2K3, MAP2K4, MAP3K7, and PIK3R2 (indicted by purple text). *denotes significantly decreased protein abundance by sRNA52320 (p < 0.05). Blue shading indicates predicted inhibition and orange shading stands for predicted activation.

Fig 5. Transfection with sRNA52320 reduces LPS-stimulation of IL-8 mRNA abundance and IL-8 cytokine secretion in HBE cells.

(A) HBE cells transfected with sRNA52320 (filled circles) had a lower induction of IL-8 mRNA (ΔCt = control Ct minus LPS-stimulated Ct) in response to LPS than control cells transfected with siNC (open circles). There was a statistically significant difference in the ΔCt means of -0.6 ± 0.2 (95% CI = -1.2 to -0.1, N = 5, p = 0.034 indicated by an asterisk). (B) sRNA52320 reduced IL-8 secretion by HBE cells in response to LPS. The difference in mean IL-8 secretion between cells transfected with siNC (open circles) and sRNA52320 (filled circles) of -149 ± 47 pg/ml was statistically significant (95% CI = -269 to -29, N = 6, p = 0.02 indicated by an asterisk).

Fig 6. sRNA52320 reduces OMV-stimulated IL-8 secretion by HBE cells.

OMV-induced IL-8 secretion was significantly attenuated in HBE cells exposed to ΔsRNA+sRNA OMVs (closed circles) compared to HBE cells exposed to ΔsRNA+vector (open circles). The difference in means of -2645 ± 685 pg/ml was statistically significant (95% CI = -4548 to -743, N = 5, p = 0.018 indicated by an asterisk).

Fig 7. sRNA52320 reduces OMV-induced secretion of the functional IL-8 homolog, KC, and neutrophil infiltration in mouse lung.

(A) Heatmap of Z-scores for 31 cytokines in mouse BALF scaled by row (N = 4 mice per group). In each row, relative cytokine abundance ranges from red (low), to orange (medium) to yellow (high). Rows are ordered by mean cytokine abundance with a range of 22,985 pg/ml for G-CSF (top row) to 1.2 pg/ml for IL-4 (bottom row). MIP-1a was excluded from the analysis because it was out of detection range in three of the samples. (B) KC concentration in BALF of mice exposed to vehicle (blue), ΔsRNA+vector OMVs (purple) or ΔsRNA+sRNA OMVs (green). The difference between the means of ΔsRNA+vector OMVs and ΔsRNA+sRNA OMVs of -3933 ± 756 pg/ml was statistically significant (95% CI = -5783 to -2083, N = 4, p = 0.011 indicated by an asterisk). (C) Number of neutrophils per ml BALF of mice exposed to vehicle (blue), ΔsRNA OMVs (purple) or wt OMVs (green). The difference between the means of ΔsRNA OMVs and wt OMVs of -6023 ± 1708 was statistically significant (95% CI = -9962 to -2085, N = 5, p = 0.008 indicated by **).

Fig 8. Model of OMV sRNA mechanism of action.

(1) P. aeruginosa (P.a.) resides in the airway mucus layer and produces OMVs. (2) OMVs traverse the mucus layer and reach the airway epithelial cells. (3) Pathogen-associated molecular patterns (PAMPs) on the outside of OMVs induce the host innate immune response by activating the Toll-like receptor and MAP-kinase (TLR/MAPK) signaling pathway. (4) Activation of transcription factors leads to up-regulation of IL-8 mRNA and IL-8 protein secretion. (5) IL-8 is a potent chemoattractant for neutrophils, which infiltrate the lungs and phagocytose P. aeruginosa. (6) OMVs also fuse with and deliver sRNA52320 into cells, which targets the mRNA of MAP-kinases upstream of IL-8, leading to reduced host IL-8 secretion and neutrophil recruitment. sRNA52320-mediated attenuation of the innate immune response to LPS is a novel mechanism of pathogen-host interaction that may facilitate chronic infection by P. aeruginosa.