Salmonella produce microrna-like RNA fragment Sal-1 in the infected cells to. facilitate intracellular survival
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- Κλεισθένης Μέλιοι
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1 Salmonella produce microrna-like RNA fragment Sal-1 in the infected cells to facilitate intracellular survival Hongwei Gu 1,2*, Chihao Zhao 1,2*, Tianfu Zhang 1,2*, Hongwei Liang 1,2, Xiao-Ming Wang 1, Yi Pan 1,2, Xi Chen 1,2, Quan Zhao 1,2, Donghai Li 1,2, Fenyong Liu 3, Chen-Yu Zhang 1,2, and Ke Zen 1,2 1 State Key Laboratory of Pharmaceutical Biotechnology, Nanjing Advanced Institute for Life Sciences, School of Life Sciences, Nanjing University, Nanjing, Jiangsu ; 2 Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, Nanjing, Jiangsu , China; 3 School of Public Health, University of California at Berkeley, Berkeley, CA 94720, USA. Running title: Salmonella produce mirna-like Sal-1 Keywords: Salmonella; non-coding RNA; AGO2; infection; mirna * To whom correspondence should be addressed: Correspondences: Ke Zen, PhD kzen@nju.edu.cn Chen-Yu Zhang, PhD, MD cyzhang@nju.edu.cn Fenyong Liu, PhD liu_fy@berkeley.edu
2 Supplementary Table S1. Sequences and reads of mirna-like RNA fragments in salmonella-infected intestinal epithelial cells detected by Solexa (Top 10). No. Name Sequence* Reads 1 Sal-1 TGTGGGCACTCGAAGATACGGATT Sal-2 ATGCGAGAGTAGGGAACTGCCAGGCAT Sal-3 TCCTCTGTAGTTCAGTCGGTAGAACGGC Sal-4 GAAGGTGGCGGAATTGGTAGACG Sal-5 GCCCGGATGGTGGAATCGGTA Sal-6 TTCAGTCGGTAGAACGGCG Sal-7 AGGGGCGTAGTTCAATTGGT Sal-8 GAGTAGGGAACTGCCAGGCAT Sal-9 ACCTGTGTGACTGCGTACCTT Sal-10 GCGGGCATCGTATAATGGCTAT mir-107 AGCAGCATTGTACAGGGCTATCA 1569
3 Supplementary Table S2. Characterization of Pri-Sal-1 (A) in bacterial species. Species Locus Identities Strand (plus/minus) /402(99%) P S. Enteritidis (str. P125109) /402(98%) M /402(98%) P /402(99%) P S. Dublin (str. CT_ ) /402(98%) M /402(99%) P /402(99%) P S. Cubana (str. CFSAN002050) /402(97%) P /402(97%) M /402(99%) M S. Heidelberg (str ) /402(97%) P /402(97%) P /402(99%) P S. Newport (str. SL254) /402(97%) M /402(98%) P /402(99%) M S. Javiana (str. CFSAN001992) /402(99%) P /402(98%) M /402(99%) P S. Gallinarum/pullorum /402(99%) P (str. RKS5078) /402(98%) M /402(99%) P S. Paratyphi A /402(98%) P (str. ATCC 9150) /402(97%) M /402(98%) P S. Paratyphi B (str. SPB7) /402(97%) M /402(96%) P /402(99%) P S.Paratyphi C (str. RKS4594) /402(98%) M /402(96%) P /402(97%) P S. Schwarzengrund /402(98%) M (str. CVM19633) /402(98%) P /402(98%) P S. Typhimurium (str. LT2) /402(98%) M /402(97%) P /402(98%) P S. Typhi (str. CT18) /402(98%) M /402(98%) P E. coli O157:H /402(98%) P (str. TW14359) /402(98%) P Shigella flexneri /402(98%) P /402(97%) P
4 Supplementary Table S3. Characterization of Pri-Sal-1 (B) in bacterial species. Species Locus Identities Strand (plus/minus) S. Enteritidis (str. P125109) /291(98%) P /291(98%) M /291(99%) M /291(99%) P S. Dublin (str. CT_ ) /291(98%) P /291(99%) M /291(98%) M /291(99%) P S. Cubana (str. CFSAN002050) /291(99%) P /291(99%) M /291(98%) P /291(97%) M S. Heidelberg (str ) /291(98%) M /291(98%) M /291(98%) P /291(96%) P S. Newport (str. SL254) /291(99%) P /291(98%) P /291(98%) M /291(98%) M S. Javiana (str. CFSAN001992) /291(98%) M /291(98%) P /291(98%) P /291(97%) M S. Gallinarum/pullorum /291(98%) P (str. RKS5078) /291(98%) M /291(98%) M /291(98%) M /291(98%) M /291(98%) M /291(98%) P S. Paratyphi A (str. ATCC 9150) /291(98%) P /291(98%) P /291(98%) P /291(98%) P /291(97%) M /291(97%) M S. Paratyphi B (str. SPB7) /291(98%) P /291(97%) M /291(97%) M /291(97%) P S.Paratyphi C (str. RKS4594) /291(98%) P /291(98%) M /291(98%) M S. Schwarzengrund /291(99%) M (str. CVM19633) /291(98%) P /291(97%) M /291(96%) P S. Typhimurium (str. LT2) /291(99%) P /291(98%) M /291(98%) P /291(97%) M S. Typhi (str. CT18) /291(97%) P /291(97%) M /291(97%) M /291(97%) M /291(97%) M /291(97%) P /291(97%) P E. coli O157:H7 s(tr. TW14359) /291(96%) P /291(96%) M /291(96%) P /291(96%) P /291(96%) P Shigella sonnei 53G /291(97%) M /291(96%) P
5 Supplementary Table S4. Characterization of Pri-Sal-1 (C) in bacterial species. Species Locus Identities Strand (plus/minus) S. Enteritidis (str. P125109) /195(97%) P /195(97%) M /195(99%) M /195(100%) P S. Dublin (str. CT_ ) /195(97%) P /195(100%) M /195(97%) M /195(98%) P S. Cubana (str. CFSAN002050) /195(98%) P /195(99%) M /195(97%) P /195(95%) M S. Heidelberg (str ) /195(97%) M /195(97%) M /195(97%) P /195(94%) P S. Newport (str. SL254) /195(98%) P /195(98%) P /195(98%) M /195(97%) M S. Javiana (str. CFSAN001992) /195(97%) M /195(97%) P /195(97%) P /195(95%) M S. Gallinarum/pullorum /195(97%) P (str. RKS5078) /195(97%) M /195(97%) M /195(97%) M /195(97%) M /195(97%) M /195(97%) P S. Paratyphi A (str. ATCC 9150) /195(97%) P /195(97%) P /195(97%) P /195(97%) P /195(96%) M /195(95%) M S. Paratyphi B (str. SPB7) /195(97%) P /195(96%) M /195(96%) M /195(95%) P S.Paratyphi C (str. RKS4594) /195(98%) P /195(98%) M /195(98%) M S. Schwarzengrund /195(98%) M (str. CVM19633) /195(97%) P /195(96%) M /195(95%) P S. Typhimurium (str. LT2) /195(99%) P /195(97%) M /195(97%) P /191(96%) M S. Typhi (str. CT18) /191(95%) P /191(95%) M /191(95%) M /191(95%) M /191(95%) M /191(95%) P /191(95%) P E. coli O157:H7 s(tr. TW14359) /195(95%) P /195(95%) M /195(95%) P /195(95%) P /195(95%) P Shigella sonnei 53G /195(95%) M /195(95%) P
6 Supplementary Table S5. Oligonucleotide sequence lists No. Name Sequence (5 to 3 ) 1 sal-gsp1 TACGTGTTCACTCTTGAGACT 2 sal-gsp2 GCACTGCTCTTTAACAATTTATCAGAC 3 ago2-a AAGGAUAUGCCUUCAAGCCUCdTdT 4 ago2-b GAGGCUUGAAGGCAUAUCCUUdTdT 5 dicer-a UGCUUGAAGCAGCUCUGGAdTdT 6 dicer-b UCCAGAGCUGCUUCAAGCAdTdT 7 LNA probe AATCCGTATCTTCGAGTGCCCACA 8 U6 probe CTGCGCAAGGATGACACGCAAAT 9 Pre-Sal-1 P1 TGTGGGCACTCGAAGATAC 10 Pre-Sal-1 P2 TACGTGTTCACTCTTGAGACT
7 Figure S1. Multiple locations of Sal-1 sequence in Salmonella genome. (a) The seven sites of Sal-1 sequence in the Salmonella genome, referring the Salmonella enterica serovar Enteritidis strain P genome sequence. All seven Sal-1 copies were located in the non-coding region. (b) The adjacent genes of Sal-1 sequence at seven different sites.
8 Figure S2.Sequence analysis and alignment of Sal-1 among the seven Salmonella non-coding regions (referring to S. enteritidis strain P125109). The Sal-1 sequence is marked with a red frame, and the putative Pre-Sal-1 hairpin, arm, and loop are highlighted.
9 Figure S3. Sal-1 biogenesis is hairpin structure-dependent. (a) Schematic of the Pre-Sal-1 and its mutated hairpin. Site mutants were generated in the context of the Pre-Sal-1 plasmid (Pre-1). (b) Northern blot analysis of Sal-1 biogenesis. Note that Pre-Sal-1 (Pre-1) is cleaved by Ago2 into mature Sal-1, but Pre-1 (MUT) cannot be processed into mature Sal-1.
10 Figure S4. Biogenesis of Sal-1 in human intestinal HT-29 cells is Dicer-independent but Ago2-dependent. (a) Western blot detection of Dicer expression in HT-29 cells using Dicer-specific sirna. (b) Ago2-specific sirna was transfected into HT-29 cells to knockdown Ago2 expression. (c) Expression level of mir-451 and mir-143 in HT-29 cells after knocking down Dicer and Ago2 using Dicer- or Ago2-specific sirna, respectively. The data are presented as the mean ± SEM (n=3). **, P<0.01.
11 Figure S5. Diagram of 3 - and 5 -RT-RACE amplification of primary Sal-1 from infected HT-29 cells.
12 Figure S6. Depict of deletion of Sal-1 sequence at 4 locations.
13 Figure S7. Bacterial survival rate in HT-29 cells infected with SE2472 or SE2472ΔSal-1(1,2,5,7) with or without Ago2 silence. **, P<0.01.
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