RNA-seq Brings New Insights to the Intra-Macrophage Transcriptome of Typhimurium

English version České info

The burden of Salmonellosis remains unacceptably high throughout the world and control measures have had limited success. Because Salmonella bacteria can be transmitted from the wider environment to animals and humans, the bacteria encounter diverse environments that include food, water, plant surfaces and the extracellular and intracellular phases of infection of eukaryotic hosts. An intricate transcriptional network has evolved to respond to a variety of environmental signals and control the “right time/ right place” expression of virulence genes. To understand how transcription is rewired during intracellular infection, we determined the primary transcriptome of Salmonella enterica serovar Typhimurium within murine macrophages. We report the coding genes, sRNAs and transcriptional start sites that are expressed within macrophages at 8 hours after infection, and use these to infer gene function. We identified gene promoters that are specifically expressed within macrophages and could drive the intracellular delivery of antigens by S. Typhimurium vaccine strains. These data contribute to our understanding of the mechanisms used by Salmonella to regulate virulence gene expression whilst replicating inside mammalian cells.

Vyšlo v časopise: RNA-seq Brings New Insights to the Intra-Macrophage Transcriptome of Typhimurium. PLoS Pathog 11(11): e32767. doi:10.1371/journal.ppat.1005262
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1005262

Souhrn

Zdroje

1. Majowicz SE, Musto J, Scallan E, Angulo FJ, Kirk M, O'Brien SJ, et al. The global burden of nontyphoidal Salmonella gastroenteritis. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America. 2010;50(6):882–9. doi: 10.1086/650733 20158401.

2. Crump JA, Mintz ED. Global trends in typhoid and paratyphoid Fever. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America. 2010;50(2):241–6. doi: 10.1086/649541 20014951; PubMed Central PMCID: PMC2798017.

3. Ao TT, Feasey NA, Gordon MA, Keddy KH, Angulo FJ, Crump JA. Global burden of invasive nontyphoidal Salmonella disease, 2010(1). Emerging infectious diseases. 2015;21(6). doi: 10.3201/eid2106.140999 25860298; PubMed Central PMCID: PMC4451910.

4. Fabrega A, Vila J. Salmonella enterica serovar Typhimurium skills to succeed in the host: virulence and regulation. Clinical microbiology reviews. 2013;26(2):308–41. doi: 10.1128/CMR.00066-12 23554419; PubMed Central PMCID: PMC3623383.

5. Hebrard M, Kroger C, Srikumar S, Colgan A, Handler K, Hinton JC. sRNAs and the virulence of Salmonella enterica serovar Typhimurium. RNA biology. 2012;9(4):437–45. doi: 10.4161/rna.20480 22546935; PubMed Central PMCID: PMC3384567.

6. Hurley D, McCusker MP, Fanning S, Martins M. Salmonella-host interactions—modulation of the host innate immune system. Frontiers in immunology. 2014;5:481. doi: 10.3389/fimmu.2014.00481 25339955; PubMed Central PMCID: PMC4188169.

7. Jones BD, Ghori N, Falkow S. Salmonella typhimurium initiates murine infection by penetrating and destroying the specialized epithelial M cells of the Peyer's patches. The Journal of experimental medicine. 1994;180(1):15–23. 8006579; PubMed Central PMCID: PMC2191576.

8. Vazquez-Torres A, Jones-Carson J, Baumler AJ, Falkow S, Valdivia R, Brown W, et al. Extraintestinal dissemination of Salmonella by CD18-expressing phagocytes. Nature. 1999;401(6755):804–8. doi: 10.1038/44593 10548107.

9. Kuhle V, Hensel M. Cellular microbiology of intracellular Salmonella enterica: functions of the type III secretion system encoded by Salmonella pathogenicity island 2. Cellular and molecular life sciences: CMLS. 2004;61(22):2812–26. doi: 10.1007/s00018-004-4248-z 15558211.

10. Haraga A, Ohlson MB, Miller SI. Salmonellae interplay with host cells. Nature reviews Microbiology. 2008;6(1):53–66. doi: 10.1038/nrmicro1788 18026123.

11. Helaine S, Holden DW. Heterogeneity of intracellular replication of bacterial pathogens. Current opinion in microbiology. 2013;16(2):184–91. doi: 10.1016/j.mib.2012.12.004 23485258.

12. Shen S, Fang FC. Integrated stress responses in Salmonella. International journal of food microbiology. 2012;152(3):75–81. doi: 10.1016/j.ijfoodmicro.2011.04.017 21570144; PubMed Central PMCID: PMC3164900.

13. Hebrard M, Kroger C, Sivasankaran SK, Handler K, Hinton JC. The challenge of relating gene expression to the virulence of Salmonella enterica serovar Typhimurium. Current opinion in biotechnology. 2011;22(2):200–10. doi: 10.1016/j.copbio.2011.02.007 21388802.

14. Eriksson S, Lucchini S, Thompson A, Rhen M, Hinton JC. Unravelling the biology of macrophage infection by gene expression profiling of intracellular Salmonella enterica. Molecular microbiology. 2003;47(1):103–18. 12492857.

15. Hautefort I, Thompson A, Eriksson-Ygberg S, Parker ML, Lucchini S, Danino V, et al. During infection of epithelial cells Salmonella enterica serovar Typhimurium undergoes a time-dependent transcriptional adaptation that results in simultaneous expression of three type 3 secretion systems. Cellular microbiology. 2008;10(4):958–84. doi: 10.1111/j.1462-5822.2007.01099.x 18031307; PubMed Central PMCID: PMC2343689.

16. Hammarlof DL, Canals R, Hinton JC. The FUN of identifying gene function in bacterial pathogens; insights from Salmonella functional genomics. Current opinion in microbiology. 2013;16(5):643–51. doi: 10.1016/j.mib.2013.07.009 24021902.

17. Kröger C, Colgan A, Srikumar S, Handler K, Sivasankaran SK, Hammarlof DL, et al. An infection-relevant transcriptomic compendium for Salmonella enterica Serovar Typhimurium. Cell host & microbe. 2013;14(6):683–95. doi: 10.1016/j.chom.2013.11.010 24331466.

18. Govoni G, Canonne-Hergaux F, Pfeifer CG, Marcus SL, Mills SD, Hackam DJ, et al. Functional expression of Nramp1 in vitro in the murine macrophage line RAW264.7. Infection and immunity. 1999;67(5):2225–32. 10225878; PubMed Central PMCID: PMC115961.

19. Haas BJ, Chin M, Nusbaum C, Birren BW, Livny J. How deep is deep enough for RNA-Seq profiling of bacterial transcriptomes? BMC genomics. 2012;13:734. doi: 10.1186/1471-2164-13-734 23270466; PubMed Central PMCID: PMC3543199.

20. Wagner GP, Kin K, Lynch VJ. Measurement of mRNA abundance using RNA-seq data: RPKM measure is inconsistent among samples. Theory in biosciences = Theorie in den Biowissenschaften. 2012;131(4):281–5. doi: 10.1007/s12064-012-0162-3 22872506.

21. Kröger C, Dillon SC, Cameron AD, Papenfort K, Sivasankaran SK, Hokamp K, et al. The transcriptional landscape and small RNAs of Salmonella enterica serovar Typhimurium. Proceedings of the National Academy of Sciences of the United States of America. 2012;109(20):E1277–86. doi: 10.1073/pnas.1201061109 22538806; PubMed Central PMCID: PMC3356629.

22. Sharma CM, Hoffmann S, Darfeuille F, Reignier J, Findeiss S, Sittka A, et al. The primary transcriptome of the major human pathogen Helicobacter pylori. Nature. 2010;464(7286):250–5. Epub 2010/02/19. doi: nature08756 [pii] doi: 10.1038/nature08756 20164839.

23. Conway T, Creecy JP, Maddox SM, Grissom JE, Conkle TL, Shadid TM, et al. Unprecedented high-resolution view of bacterial operon architecture revealed by RNA sequencing. mBio. 2014;5(4). doi: 10.1128/mBio.01442-14 25006232.

24. Chakraborty S, Gogoi M, Chakravortty D. Lactoylglutathione lyase, a Critical Enzyme in Methylglyoxal Detoxification, Contributes to Survival of Salmonella in the Nutrient Rich Environment. Virulence. 2014:0. doi: 10.4161/21505594.2014.983791 25517857.

25. Sasikaran J, Ziemski M, Zadora PK, Fleig A, Berg IA. Bacterial itaconate degradation promotes pathogenicity. Nature chemical biology. 2014;10(5):371–7. doi: 10.1038/nchembio.1482 24657929.

26. Rollenhagen C, Bumann D. Salmonella enterica highly expressed genes are disease specific. Infection and immunity. 2006;74(3):1649–60. doi: 10.1128/IAI.74.3.1649–1660.2006 16495536; PubMed Central PMCID: PMC1418657.

27. Figueira R, Holden DW. Functions of the Salmonella pathogenicity island 2 (SPI-2) type III secretion system effectors. Microbiology. 2012;158(Pt 5):1147–61. doi: 10.1099/mic.0.058115–0 22422755.

28. Beuzon CR, Banks G, Deiwick J, Hensel M, Holden DW. pH-dependent secretion of SseB, a product of the SPI-2 type III secretion system of Salmonella typhimurium. Molecular microbiology. 1999;33(4):806–16. 10447889.

29. Deiwick J, Nikolaus T, Erdogan S, Hensel M. Environmental regulation of Salmonella pathogenicity island 2 gene expression. Molecular microbiology. 1999;31(6):1759–73. 10209748.

30. Lober S, Jackel D, Kaiser N, Hensel M. Regulation of Salmonella pathogenicity island 2 genes by independent environmental signals. International journal of medical microbiology: IJMM. 2006;296(7):435–47. doi: 10.1016/j.ijmm.2006.05.001 16904940.

31. Cirillo DM, Valdivia RH, Monack DM, Falkow S. Macrophage-dependent induction of the Salmonella pathogenicity island 2 type III secretion system and its role in intracellular survival. Molecular microbiology. 1998;30(1):175–88. 9786194.

32. Feng X, Oropeza R, Kenney LJ. Dual regulation by phospho-OmpR of ssrA/B gene expression in Salmonella pathogenicity island 2. Molecular microbiology. 2003;48(4):1131–43. 12753201.

33. Feng X, Walthers D, Oropeza R, Kenney LJ. The response regulator SsrB activates transcription and binds to a region overlapping OmpR binding sites at Salmonella pathogenicity island 2. Molecular microbiology. 2004;54(3):823–35. doi: 10.1111/j.1365-2958.2004.04317.x 15491370.

34. Tomljenovic-Berube AM, Mulder DT, Whiteside MD, Brinkman FS, Coombes BK. Identification of the regulatory logic controlling Salmonella pathoadaptation by the SsrA-SsrB two-component system. PLoS genetics. 2010;6(3):e1000875. doi: 10.1371/journal.pgen.1000875 20300643; PubMed Central PMCID: PMC2837388.

35. Richardson EJ, Limaye B, Inamdar H, Datta A, Manjari KS, Pullinger GD, et al. Genome sequences of Salmonella enterica serovar typhimurium, Choleraesuis, Dublin, and Gallinarum strains of well- defined virulence in food-producing animals. Journal of bacteriology. 2011;193(12):3162–3. doi: 10.1128/JB.00394-11 21478351; PubMed Central PMCID: PMC3133203.

36. Sabbagh SC, Lepage C, McClelland M, Daigle F. Selection of Salmonella enterica serovar Typhi genes involved during interaction with human macrophages by screening of a transposon mutant library. PloS one. 2012;7(5):e36643. doi: 10.1371/journal.pone.0036643 22574205; PubMed Central PMCID: PMC3344905.

37. Chakraborty S, Chaudhuri D, Balakrishnan A, Chakravortty D. Salmonella methylglyoxal detoxification by STM3117-encoded lactoylglutathione lyase affects virulence in coordination with Salmonella pathogenicity island 2 and phagosomal acidification. Microbiology. 2014;160(Pt 9):1999–2017. doi: 10.1099/mic.0.078998–0 24961952.

38. Shah DH, Lee MJ, Park JH, Lee JH, Eo SK, Kwon JT, et al. Identification of Salmonella Gallinarum virulence genes in a chicken infection model using PCR-based signature-tagged mutagenesis. Microbiology. 2005;151(Pt 12):3957–68. doi: 10.1099/mic.0.28126–0 16339940.

39. Jha AK, Huang SC, Sergushichev A, Lampropoulou V, Ivanova Y, Loginicheva E, et al. Network Integration of Parallel Metabolic and Transcriptional Data Reveals Metabolic Modules that Regulate Macrophage Polarization. Immunity. 2015;42(3):419–30. doi: 10.1016/j.immuni.2015.02.005 25786174.

40. Blanc-Potard AB, Solomon F, Kayser J, Groisman EA. The SPI-3 pathogenicity island of Salmonella enterica. Journal of bacteriology. 1999;181(3):998–1004. 9922266; PubMed Central PMCID: PMC93469.

41. Lee EJ, Groisman EA. Control of a Salmonella virulence locus by an ATP-sensing leader messenger RNA. Nature. 2012;486(7402):271–5. doi: 10.1038/nature11090 22699622; PubMed Central PMCID: PMC3711680.

42. Lee EJ, Pontes MH, Groisman EA. A bacterial virulence protein promotes pathogenicity by inhibiting the bacterium's own F1Fo ATP synthase. Cell. 2013;154(1):146–56. doi: 10.1016/j.cell.2013.06.004 23827679; PubMed Central PMCID: PMCPMC3736803.

43. Wood MW, Jones MA, Watson PR, Hedges S, Wallis TS, Galyov EE. Identification of a pathogenicity island required for Salmonella enteropathogenicity. Molecular microbiology. 1998;29(3):883–91. 9723926.

44. Knodler LA, Celli J, Hardt WD, Vallance BA, Yip C, Finlay BB. Salmonella effectors within a single pathogenicity island are differentially expressed and translocated by separate type III secretion systems. Molecular microbiology. 2002;43(5):1089–103. 11918798.

45. Knodler LA, Vallance BA, Hensel M, Jackel D, Finlay BB, Steele-Mortimer O. Salmonella type III effectors PipB and PipB2 are targeted to detergent-resistant microdomains on internal host cell membranes. Molecular microbiology. 2003;49(3):685–704. 12864852.

46. Knodler LA, Steele-Mortimer O. The Salmonella effector PipB2 affects late endosome/lysosome distribution to mediate Sif extension. Mol Biol Cell. 2005;16(9):4108–23. doi: 10.1091/mbc.E05-04-0367 15987736; PubMed Central PMCID: PMC1196323.

47. Blondel CJ, Jimenez JC, Contreras I, Santiviago CA. Comparative genomic analysis uncovers 3 novel loci encoding type six secretion systems differentially distributed in Salmonella serotypes. BMC genomics. 2009;10:354. doi: 10.1186/1471-2164-10-354 19653904; PubMed Central PMCID: PMC2907695.

48. Pezoa D, Yang HJ, Blondel CJ, Santiviago CA, Andrews-Polymenis HL, Contreras I. The type VI secretion system encoded in SPI-6 plays a role in gastrointestinal colonization and systemic spread of Salmonella enterica serovar Typhimurium in the chicken. PloS one. 2013;8(5):e63917. doi: 10.1371/journal.pone.0063917 23691117; PubMed Central PMCID: PMC3653874.

49. Mulder DT, Cooper CA, Coombes BK. Type VI secretion system-associated gene clusters contribute to pathogenesis of Salmonella enterica serovar Typhimurium. Infection and immunity. 2012;80(6):1996–2007. doi: 10.1128/IAI.06205-11 22493086; PubMed Central PMCID: PMC3370595.

50. Brunet YR, Khodr A, Logger L, Aussel L, Mignot T, Rimsky S, et al. H-NS Silencing of the Salmonella Pathogenicity Island 6-Encoded Type VI Secretion System Limits Salmonella enterica Serovar Typhimurium Interbacterial Killing. Infection and immunity. 2015;83(7):2738–50. doi: 10.1128/IAI.00198-15 25916986.

51. Galan JE, Curtiss R 3rd. Cloning and molecular characterization of genes whose products allow Salmonella typhimurium to penetrate tissue culture cells. Proceedings of the National Academy of Sciences of the United States of America. 1989;86(16):6383–7. 2548211; PubMed Central PMCID: PMC297844.

52. Watson PR, Galyov EE, Paulin SM, Jones PW, Wallis TS. Mutation of invH, but not stn, reduces Salmonella-induced enteritis in cattle. Infection and immunity. 1998;66(4):1432–8. 9529064; PubMed Central PMCID: PMC108071.

53. Tsolis RM, Adams LG, Ficht TA, Baumler AJ. Contribution of Salmonella typhimurium virulence factors to diarrheal disease in calves. Infection and immunity. 1999;67(9):4879–85. 10456944; PubMed Central PMCID: PMC96822.

54. Bustamante VH, Martinez LC, Santana FJ, Knodler LA, Steele-Mortimer O, Puente JL. HilD-mediated transcriptional cross-talk between SPI-1 and SPI-2. Proceedings of the National Academy of Sciences of the United States of America. 2008;105(38):14591–6. doi: 10.1073/pnas.0801205105 18799744; PubMed Central PMCID: PMC2567235.

55. Ellermeier JR, Slauch JM. Adaptation to the host environment: regulation of the SPI1 type III secretion system in Salmonella enterica serovar Typhimurium. Current opinion in microbiology. 2007;10(1):24–9. doi: 10.1016/j.mib.2006.12.002 17208038.

56. Schechter LM, Damrauer SM, Lee CA. Two AraC/XylS family members can independently counteract the effect of repressing sequences upstream of the hilA promoter. Molecular microbiology. 1999;32(3):629–42. 10320584.

57. Ellermeier CD, Slauch JM. RtsA and RtsB Coordinately Regulate Expression of the Invasion and Flagellar Genes in Salmonella enterica Serovar Typhimurium. Journal of bacteriology. 2003;185(17):5096–108. doi: 10.1128/jb.185.17.5096–5108.2003 12923082

58. Petrone BL, Stringer AM, Wade JT. Identification of HilD-Regulated Genes in Salmonella enterica Serovar Typhimurium. Journal of bacteriology. 2014;196(5):1094–101. doi: 10.1128/JB.01449-13 24375101.

59. Gerlach RG, Jackel D, Stecher B, Wagner C, Lupas A, Hardt WD, et al. Salmonella Pathogenicity Island 4 encodes a giant non-fimbrial adhesin and the cognate type 1 secretion system. Cellular microbiology. 2007;9(7):1834–50. doi: 10.1111/j.1462-5822.2007.00919.x 17388786.

60. Wagner C, Barlag B, Gerlach RG, Deiwick J, Hensel M. The Salmonella enterica giant adhesin SiiE binds to polarized epithelial cells in a lectin-like manner. Cellular microbiology. 2014;16(6):962–75. doi: 10.1111/cmi.12253 24345213.

61. Gerlach RG, Claudio N, Rohde M, Jackel D, Wagner C, Hensel M. Cooperation of Salmonella pathogenicity islands 1 and 4 is required to breach epithelial barriers. Cellular microbiology. 2008;10(11):2364–76. doi: 10.1111/j.1462-5822.2008.01218.x 18671822.

62. La MV, Raoult D, Renesto P. Regulation of whole bacterial pathogen transcription within infected hosts. FEMS microbiology reviews. 2008;32(3):440–60. doi: 10.1111/j.1574-6976.2008.00103.x 18266740.

63. Allam US, Krishna MG, Sen M, Thomas R, Lahiri A, Gnanadhas DP, et al. Acidic pH induced STM1485 gene is essential for intracellular replication of Salmonella. Virulence. 2012;3(2):122–35. doi: 10.4161/viru.19029 22460643; PubMed Central PMCID: PMC3396692.

64. Rathman M, Sjaastad MD, Falkow S. Acidification of phagosomes containing Salmonella Typhimurium in murine macrophages. Infection and immunity. 1996;64(7):2765–73. 8698506; PubMed Central PMCID: PMC174137.

65. Abu Kwaik Y, Bumann D. Host Delivery of Favorite Meals for Intracellular Pathogens. PLoS pathogens. 2015;11(6):e1004866. doi: 10.1371/journal.ppat.1004866 26110434; PubMed Central PMCID: PMC4482385.

66. Steeb B, Claudi B, Burton NA, Tienz P, Schmidt A, Farhan H, et al. Parallel exploitation of diverse host nutrients enhances Salmonella virulence. PLoS pathogens. 2013;9(4):e1003301. doi: 10.1371/journal.ppat.1003301 23633950; PubMed Central PMCID: PMC3636032.

67. Mastroeni P, Grant A, Restif O, Maskell D. A dynamic view of the spread and intracellular distribution of Salmonella enterica. Nature reviews Microbiology. 2009;7(1):73–80. doi: 10.1038/nrmicro2034 19079353.

68. Nairz M, Haschka D, Demetz E, Weiss G. Iron at the interface of immunity and infection. Front Pharmacol. 2014;5:152. doi: 10.3389/fphar.2014.00152 25076907; PubMed Central PMCID: PMC4100575.

69. Zaharik ML, Cullen VL, Fung AM, Libby SJ, Kujat Choy SL, Coburn B, et al. The Salmonella enterica serovar typhimurium divalent cation transport systems MntH and SitABCD are essential for virulence in an Nramp1G169 murine typhoid model. Infection and immunity. 2004;72(9):5522–5. doi: 10.1128/IAI.72.9.5522–5525.2004 15322058; PubMed Central PMCID: PMC517450.

70. Osman D, Cavet JS. Metal sensing in Salmonella: implications for pathogenesis. Advances in microbial physiology. 2011;58:175–232. doi: 10.1016/B978-0-12-381043-4.00005–2 21722794.

71. Faucher SP, Porwollik S, Dozois CM, McClelland M, Daigle F. Transcriptome of Salmonella enterica serovar Typhi within macrophages revealed through the selective capture of transcribed sequences. Proceedings of the National Academy of Sciences of the United States of America. 2006;103(6):1906–11. doi: 10.1073/pnas.0509183103 16443683; PubMed Central PMCID: PMC1413645.

72. Chilcott GS, Hughes KT. Coupling of flagellar gene expression to flagellar assembly in Salmonella enterica serovar typhimurium and Escherichia coli. Microbiology and molecular biology reviews: MMBR. 2000;64(4):694–708. 11104815; PubMed Central PMCID: PMC99010.

73. Saini S, Slauch JM, Aldridge PD, Rao CV. Role of cross talk in regulating the dynamic expression of the flagellar Salmonella pathogenicity island 1 and type 1 fimbrial genes. Journal of bacteriology. 2010;192(21):5767–77. doi: 10.1128/JB.00624-10 20833811; PubMed Central PMCID: PMC2953706.

74. Saini S, Brown JD, Aldridge PD, Rao CV. FliZ Is a posttranslational activator of FlhD4C2-dependent flagellar gene expression. Journal of bacteriology. 2008;190(14):4979–88. doi: 10.1128/JB.01996-07 18469103; PubMed Central PMCID: PMC2447003.

75. Knodler LA, Vallance BA, Celli J, Winfree S, Hansen B, Montero M, et al. Dissemination of invasive Salmonella via bacterial-induced extrusion of mucosal epithelia. Proceedings of the National Academy of Sciences of the United States of America. 2010;107(41):17733–8. doi: 10.1073/pnas.1006098107 20876119; PubMed Central PMCID: PMC2955089.

76. Hinton JC. The Escherichia coli genome sequence: the end of an era or the start of the FUN? Molecular microbiology. 1997;26(3):417–22. 9402013.

77. Strugnell RA, Scott TA, Wang N, Yang C, Peres N, Bedoui S, et al. Salmonella vaccines: lessons from the mouse model or bad teaching? Current opinion in microbiology. 2014;17:99–105. doi: 10.1016/j.mib.2013.12.004 24440968.

78. Toussaint B, Chauchet X, Wang Y, Polack B, Le Gouellec A. Live-attenuated bacteria as a cancer vaccine vector. Expert review of vaccines. 2013;12(10):1139–54. doi: 10.1586/14760584.2013.836914 24124876.

79. Xu X, Husseiny MI, Goldwich A, Hensel M. Efficacy of intracellular activated promoters for generation of Salmonella-based vaccines. Infection and immunity. 2010;78(11):4828–38. doi: 10.1128/IAI.00298-10 20732994; PubMed Central PMCID: PMC2976355.

80. McKelvie ND, Stratford R, Wu T, Bellaby T, Aldred E, Hughes NJ, et al. Expression of heterologous antigens in Salmonella Typhimurium vaccine vectors using the in vivo-inducible, SPI-2 promoter, ssaG. Vaccine. 2004;22(25–26):3243–55. doi: 10.1016/j.vaccine.2004.05.014 15308346.

81. Brown NF, Vallance BA, Coombes BK, Valdez Y, Coburn BA, Finlay BB. Salmonella pathogenicity island 2 is expressed prior to penetrating the intestine. PLoS pathogens. 2005;1(3):e32. doi: 10.1371/journal.ppat.0010032 16304611; PubMed Central PMCID: PMC1287911.

82. Osborne SE, Coombes BK. Transcriptional priming of Salmonella Pathogenicity Island-2 precedes cellular invasion. PloS one. 2011;6(6):e21648. doi: 10.1371/journal.pone.0021648 21738750; PubMed Central PMCID: PMC3125303.

83. Rollenhagen C, Sorensen M, Rizos K, Hurvitz R, Bumann D. Antigen selection based on expression levels during infection facilitates vaccine development for an intracellular pathogen. Proceedings of the National Academy of Sciences of the United States of America. 2004;101(23):8739–44. doi: 10.1073/pnas.0401283101 15173591; PubMed Central PMCID: PMC423265.

84. Vogel J. A rough guide to the non-coding RNA world of Salmonella. Molecular microbiology. 2009;71(1):1–11. doi: 10.1111/j.1365-2958.2008.06505.x 19007416.

85. Papenfort K, Vogel J. Small RNA functions in carbon metabolism and virulence of enteric pathogens. Frontiers in cellular and infection microbiology. 2014;4:91. doi: 10.3389/fcimb.2014.00091 25077072; PubMed Central PMCID: PMC4098024.

86. Papenfort K, Vogel J. Regulatory RNA in bacterial pathogens. Cell host & microbe. 2010;8(1):116–27. doi: 10.1016/j.chom.2010.06.008 20638647.

87. Storz G, Vogel J, Wassarman KM. Regulation by small RNAs in bacteria: expanding frontiers. Molecular cell. 2011;43(6):880–91. doi: 10.1016/j.molcel.2011.08.022 21925377; PubMed Central PMCID: PMC3176440.

88. Caldelari I, Chao Y, Romby P, Vogel J. RNA-mediated regulation in pathogenic bacteria. Cold Spring Harbor perspectives in medicine. 2013;3(9):a010298. doi: 10.1101/cshperspect.a010298 24003243.

89. Vogel J, Luisi BF. Hfq and its constellation of RNA. Nature reviews Microbiology. 2011;9(8):578–89. doi: 10.1038/nrmicro2615 21760622.

90. Papenfort K, Pfeiffer V, Mika F, Lucchini S, Hinton JCD, Vogel J. σE-dependent small RNAs of Salmonella respond to membrane stress by accelerating global omp mRNA decay. Molecular microbiology. 2006;62(6):1674–88. doi: 10.1111/j.1365-2958.2006.05524.x 17427289

91. Humphreys S, Stevenson A, Bacon A, Weinhardt AB, Roberts M. The alternative sigma factor, sigmaE, is critically important for the virulence of Salmonella Typhimurium. Infection and immunity. 1999;67(4):1560–8. 10084987; PubMed Central PMCID: PMC96497.

92. Guo MS, Updegrove TB, Gogol EB, Shabalina SA, Gross CA, Storz G. MicL, a new sigmaE-dependent sRNA, combats envelope stress by repressing synthesis of Lpp, the major outer membrane lipoprotein. Genes & development. 2014;28(14):1620–34. doi: 10.1101/gad.243485.114 25030700; PubMed Central PMCID: PMC4102768.

93. Padalon-Brauch G, Hershberg R, Elgrably-Weiss M, Baruch K, Rosenshine I, Margalit H, et al. Small RNAs encoded within genetic islands of Salmonella Typhimurium show host-induced expression and role in virulence. Nucleic Acids Res. 2008;36(6):1913–27. doi: 10.1093/nar/gkn050 18267966; PubMed Central PMCID: PMC2330248.

94. Calderon IL, Morales EH, Collao B, Calderon PF, Chahuan CA, Acuna LG, et al. Role of Salmonella Typhimurium small RNAs RyhB-1 and RyhB-2 in the oxidative stress response. Res Microbiol. 2014;165(1):30–40. doi: 10.1016/j.resmic.2013.10.008 24239962.

95. Leclerc JM, Dozois CM, Daigle F. Role of the Salmonella enterica serovar Typhi Fur regulator and small RNAs RfrA and RfrB in iron homeostasis and interaction with host cells. Microbiology. 2013;159(Pt 3):591–602. doi: 10.1099/mic.0.064329–0 23306672.

96. Ortega AD, Gonzalo-Asensio J, Garcia-del Portillo F. Dynamics of Salmonella small RNA expression in non-growing bacteria located inside eukaryotic cells. RNA biology. 2012;9(4):469–88. doi: 10.4161/rna.19317 22336761.

97. Hoiseth SK, Stocker BA. Aromatic-dependent Salmonella Typhimurium are non-virulent and effective as live vaccines. Nature. 1981;291(5812):238–9. 7015147.

98. Tedin K, Blasi U. The RNA chain elongation rate of the lambda late mRNA is unaffected by high levels of ppGpp in the absence of amino acid starvation. The Journal of biological chemistry. 1996;271(30):17675–86. 8663373.

99. Hinton JC, Hautefort I, Eriksson S, Thompson A, Rhen M. Benefits and pitfalls of using microarrays to monitor bacterial gene expression during infection. Current opinion in microbiology. 2004;7(3):277–82. doi: 10.1016/j.mib.2004.04.009 15196496.

100. Hoffmann S, Otto C, Kurtz S, Sharma CM, Khaitovich P, Vogel J, et al. Fast mapping of short sequences with mismatches, insertions and deletions using index structures. PLoS computational biology. 2009;5(9):e1000502. doi: 10.1371/journal.pcbi.1000502 19750212; PubMed Central PMCID: PMC2730575.

101. Nicol JW, Helt GA, Blanchard SG Jr., Raja A, Loraine AE. The Integrated Genome Browser: free software for distribution and exploration of genome-scale datasets. Bioinformatics. 2009;25(20):2730–1. doi: 10.1093/bioinformatics/btp472 19654113; PubMed Central PMCID: PMC2759552.

102. Skinner ME, Uzilov AV, Stein LD, Mungall CJ, Holmes IH. JBrowse: a next-generation genome browser. Genome research. 2009;19(9):1630–8. doi: 10.1101/gr.094607.109 19570905; PubMed Central PMCID: PMC2752129.

103. Wagner GP, Kin K, Lynch VJ. A model based criterion for gene expression calls using RNA-seq data. Theory in biosciences = Theorie in den Biowissenschaften. 2013;132(3):159–64. doi: 10.1007/s12064-013-0178-3 23615947.

104. Li B, Ruotti V, Stewart RM, Thomson JA, Dewey CN. RNA-Seq gene expression estimation with read mapping uncertainty. Bioinformatics. 2010;26(4):493–500. doi: 10.1093/bioinformatics/btp692 20022975; PubMed Central PMCID: PMC2820677.

105. Argaman L, Hershberg R, Vogel J, Bejerano G, Wagner EG, Margalit H, et al. Novel small RNA-encoding genes in the intergenic regions of Escherichia coli. Current biology: CB. 2001;11(12):941–50. 11448770.

106. Pearson WR, Lipman DJ. Improved tools for biological sequence comparison. Proceedings of the National Academy of Sciences of the United States of America. 1988;85(8):2444–8. 3162770; PubMed Central PMCID: PMC280013.

107. Walthers D, Carroll RK, Navarre WW, Libby SJ, Fang FC, Kenney LJ. The response regulator SsrB activates expression of diverse Salmonella pathogenicity island 2 promoters and counters silencing by the nucleoid-associated protein H-NS. Molecular microbiology. 2007;65(2):477–93. doi: 10.1111/j.1365-2958.2007.05800.x 17630976.

108. Kidwai AS, Mushamiri I, Niemann GS, Brown RN, Adkins JN, Heffron F. Diverse secreted effectors are required for Salmonella persistence in a mouse infection model. PloS one. 2013;8(8):e70753. doi: 10.1371/journal.pone.0070753 23950998; PubMed Central PMCID: PMC3741292.

109. Chao Y, Papenfort K, Reinhardt R, Sharma CM, Vogel J. An atlas of Hfq-bound transcripts reveals 3' UTRs as a genomic reservoir of regulatory small RNAs. EMBO J. 2012;31(20):4005–19. doi: 10.1038/emboj.2012.229 22922465; PubMed Central PMCID: PMC3474919.