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. 2017 May 24;7(1):2392.
doi: 10.1038/s41598-017-02669-1.

Salmonella Produce microRNA-like RNA Fragment Sal-1 in the Infected Cells to Facilitate Intracellular Survival

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Free PMC article

Salmonella Produce microRNA-like RNA Fragment Sal-1 in the Infected Cells to Facilitate Intracellular Survival

Hongwei Gu et al. Sci Rep. .
Free PMC article

Abstract

Salmonella have developed a sophisticated machinery to evade immune clearance and promote survival in the infected cells. Previous studies were mostly focused on either bacteria itself or host cells, the interaction mechanism of host-pathogen awaits further exploration. In the present study, we show that Salmonella can exploit mammalian cell non-classical microRNA processing machinery to further process bacterial small non-coding RNAs into microRNA-like fragments. Sal-1, one such fragment with the highest copy number in the infected cells, is derived from Salmonella 5'-leader of the ribosomal RNA transcript and has a 'stem' structure-containing precursor. Processing of Sal-1 precursors to mature Sal-1 is dependent on host cell Argonaute 2 (AGO2) but not Dicer. Functionally, depleting cellular Sal-1 strongly renders the Salmonella bacteria less resistant to the host defenses both in vitro and in vivo. In conclusion, we demonstrate a novel strategy for Salmonella evading the host immune clearance, in which Salmonella produce microRNA-like functional RNA fragments to establish a microenvironment facilitating bacterial survival.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Production of small RNA fragments encoded by Salmonella in human intestinal epithelial cells infected by Salmonella strain SE2472. (a) Size distribution of Salmonella-encoded small RNA fragments. (b) Levels of five Salmonella-encoded small RNA fragments in HT-29 cells infected by SE2472. (c) Sal-1 level in RAW264.7 and HeLa cells infected with various Salmonella strains. (d) Sal-1 level in HT-29 cells at 12 h post-infection with SE2472 at MOI of 1 (low), 10 (medium) and 100 (high), respectively. (e) Sal-1 level in HT-29 cells at 6, 12 and 24 h post-infection with SE2472. The data are presented as the mean ± SEM (n = 4). ND, not detected.
Figure 2
Figure 2
Production of mature Sal-1 in the Salmonella-infected intestinal epithelial cells. (a) Northern blot detection of Sal-1 in SE2472-infected HT-29 cells. (b) Sal-1 level in LB-grown SE2472, bacteria culture supernatant (SP) and HT-29 cells with Mock or SE2472 infection. (c-d) Bacteria number (c) and Sal-1 level (d) in the SE2472-infected HT-29 cells treated with or without T3SS blocker INP0403. The data are presented as the mean ± SEM (n = 4). ND, not detected. **P < 0.01.
Figure 3
Figure 3
Biogenesis of Sal-1 in the Salmonella–infected HT-29 cells is AGO2-dependent. (a) Prediction of putative hairpin structure of Pre-Sal-1 for the biogenesis of mature Sal-1. (b) Construction of pcDNATM6.2-GW/EmGFP vector expressing Pre-Sal-1. Pre-Sal-1 was inserted immediately downstream of GFP and promoted by the CMV promoter. (c,e) Mature Sal-1 in HT-29 cells transfected with two Pre-Sal-1 expression plasmids (Pre-1 and Pre-2) detected by qRT-PCR (c) and northern blot analysis (e). (d) qRT-PCR detection of Sal-1 level in Pre-1- or Pre-2-transfected HT-29 cells with or without Dicer or Ago2 silence. (e) Northern blot analysis of Sal-1 in Pre-1- or Pre-2-transfected HT-29 cells with or without Ago2 silence. (f) qRT-PCR detection of Sal-1 level in SE2472-infected HT-29 cells with or without Dicer or Ago2 silence. (g) Northern blot of Sal-1 and Pre-Sal-1 in SE2472-infected HT-29 cells with or without Dicer or Ago2 silence. (h) RT-PCR amplification of Pre-Sal-1 and mature Sal-1 that are associated with AGO2 complex in HT-29 cells directly infected with SE2472 or transfected with Pre-Sal-1. Pre-Sal-1 was amplified, inserted into a T-vector and sequenced. The data are presented as the mean ± SEM (n = 3). **P < 0.01. ND, not detected.
Figure 4
Figure 4
Identification of ‘primary’ form of Sal-1 in SE2472-infected HT-29 cells, which can be processed into pre-Sal-1 and mature Sal-1 sequentially. (a) Analysis of the sequences of three primary Sal-1 sites consisting of non-coding RNA and a partial region of the 5′ terminus of the 16 sRNA. (b) AGO2-dependent of Sal-1 generation from 3 possible Pri-Sal-1 (A, B and C) in HT-29 cells. Pri-Sal-1 (A, B and C) were amplified from infected HT-29 cells with 3′- and 5′- RT-RACE cDNA amplification followed by insertion into a pGEM-11Zf(+) vector for in vitro transcription. These primary Sal-1 RNA transcription fragments were then transfected into HT-29 for pre-Sal-1 and Sal-1 production. (c-d) Levels of pre-Sal-1 (c) and Sal-1 (d) in the pri-Sal-1-transfected HT-29 cells with or without silencing Ago2. The data are presented as the mean ± SEM (n = 3). **P < 0.01. ND, not detected.
Figure 5
Figure 5
Sal-1 facilitates the intracellular bacterial survival following Salmonella infection. (a) Depletion of cellular Sal-1 by anti-Sal-1 oligonucleotide in HT-29 cells infected with SE2472 at MOI of 1, 10 and 100. (b) Bacterial survival rate in HT-29 cells infected with SE2472 at MOI of 1, 10 or 100. (c) Depletion of Sal-1 by anti-Sal-1 oligonucleotide in HT-29 cells infected with SE2472 for 6, 12 or 24 h. (d) Bacterial survival rate in HT-29 cells infected with SE2472 for 6, 12 or 24 h. Data are presented as the mean ± SEM (n = 3). ND, not detected. **P < 0.01.
Figure 6
Figure 6
Deletion of Sal-1 sequence in bacterial genome decreases the bacterial intracellular survival rate following Salmonella infection. (a) Bacterial growth rate. (b) Level of Sal-1 in HT-29 cells infected with WT SE2472 or mutant SE2472∆Sal-1(1,2,5,7). (c) The level of bacterial intracellular survival rate in HT-29 cells infected with SE2472 or mutant SE2472∆Sal-1(1,2,5,7). Note that delivery of Sal-1 into SE2472∆Sal-1(1,2,5,7) -infected cells via Sal-1-expressing lentivirus largely repairs the defect of SE2472∆Sal-1(1,2,5,7) in promoting bacterial survival. ND, not detected. **P < 0.01.
Figure 7
Figure 7
Role of Sal-1 in facilitating Salmonella infection in mice. The lentivirus expressing Sal-1 (LV-Sal-1) or Sal-1 sponge (LV-Sal-1 sponge) were constructed to overexpress or deplete Sal-1 in mouse colon epithelium. Prior to Salmonella infection, the lentiviruses were slowly administered into the lumen of mouse colon via a catheter. Female BALB/c mice (6–8 weeks) were then inoculated intragastrically with Salmonella. (a) Experimental design. (b) Sal-1 levels in mouse colon tissues. (c) Salmonella bacteria count in the mouse colon tissues. (d) The clinical scores of the mice after Salmonella infection. ND, not detected. **P < 0.01.

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