Comprehensive Assignment of Roles for Typhimurium Genes in Intestinal Colonization of Food-Producing Animals
Chickens, pigs, and cattle are key reservoirs of Salmonella enterica, a foodborne pathogen of worldwide importance. Though a decade has elapsed since publication of the first Salmonella genome, thousands of genes remain of hypothetical or unknown function, and the basis of colonization of reservoir hosts is ill-defined. Moreover, previous surveys of the role of Salmonella genes in vivo have focused on systemic virulence in murine typhoid models, and the genetic basis of intestinal persistence and thus zoonotic transmission have received little study. We therefore screened pools of random insertion mutants of S. enterica serovar Typhimurium in chickens, pigs, and cattle by transposon-directed insertion-site sequencing (TraDIS). The identity and relative fitness in each host of 7,702 mutants was simultaneously assigned by massively parallel sequencing of transposon-flanking regions. Phenotypes were assigned to 2,715 different genes, providing a phenotype–genotype map of unprecedented resolution. The data are self-consistent in that multiple independent mutations in a given gene or pathway were observed to exert a similar fitness cost. Phenotypes were further validated by screening defined null mutants in chickens. Our data indicate that a core set of genes is required for infection of all three host species, and smaller sets of genes may mediate persistence in specific hosts. By assigning roles to thousands of Salmonella genes in key reservoir hosts, our data facilitate systems approaches to understand pathogenesis and the rational design of novel cross-protective vaccines and inhibitors. Moreover, by simultaneously assigning the genotype and phenotype of over 90% of mutants screened in complex pools, our data establish TraDIS as a powerful tool to apply rich functional annotation to microbial genomes with minimal animal use.
Vyšlo v časopise:
Comprehensive Assignment of Roles for Typhimurium Genes in Intestinal Colonization of Food-Producing Animals. PLoS Genet 9(4): e32767. doi:10.1371/journal.pgen.1003456
Kategorie:
Research Article
prolekare.web.journal.doi_sk:
https://doi.org/10.1371/journal.pgen.1003456
Souhrn
Chickens, pigs, and cattle are key reservoirs of Salmonella enterica, a foodborne pathogen of worldwide importance. Though a decade has elapsed since publication of the first Salmonella genome, thousands of genes remain of hypothetical or unknown function, and the basis of colonization of reservoir hosts is ill-defined. Moreover, previous surveys of the role of Salmonella genes in vivo have focused on systemic virulence in murine typhoid models, and the genetic basis of intestinal persistence and thus zoonotic transmission have received little study. We therefore screened pools of random insertion mutants of S. enterica serovar Typhimurium in chickens, pigs, and cattle by transposon-directed insertion-site sequencing (TraDIS). The identity and relative fitness in each host of 7,702 mutants was simultaneously assigned by massively parallel sequencing of transposon-flanking regions. Phenotypes were assigned to 2,715 different genes, providing a phenotype–genotype map of unprecedented resolution. The data are self-consistent in that multiple independent mutations in a given gene or pathway were observed to exert a similar fitness cost. Phenotypes were further validated by screening defined null mutants in chickens. Our data indicate that a core set of genes is required for infection of all three host species, and smaller sets of genes may mediate persistence in specific hosts. By assigning roles to thousands of Salmonella genes in key reservoir hosts, our data facilitate systems approaches to understand pathogenesis and the rational design of novel cross-protective vaccines and inhibitors. Moreover, by simultaneously assigning the genotype and phenotype of over 90% of mutants screened in complex pools, our data establish TraDIS as a powerful tool to apply rich functional annotation to microbial genomes with minimal animal use.
Zdroje
1. CrumpJA, LubySP, MintzED (2004) The global burden of typhoid fever. Bull World Health Organ 82: 346–353.
2. MajowiczSE, MustoJ, ScallanE, AnguloFJ, KirkM, et al. (2010) The global burden of nontyphoidal Salmonella gastroenteritis. Clin Infect Dis 50: 882–889.
3. StevensMP, HumphreyTJ, MaskellDJ (2009) Molecular insights into farm animal and zoonotic Salmonella infections. Philos Trans R Soc Lond B Biol Sci 364: 2709–2723.
4. TurnerAK, LovellMA, HulmeSD, Zhang-BarberL, BarrowPA (1998) Identification of Salmonella typhimurium genes required for colonization of the chicken alimentary tract and for virulence in newly hatched chicks. Infect Immun 66: 2099–2106.
5. HenselM, SheaJE, GleesonC, JonesMD, DaltonE, et al. (1995) Simultaneous identification of bacterial virulence genes by negative selection. Science 269: 400–403.
6. SheaJE, HenselM, GleesonC, HoldenDW (1996) Identification of a virulence locus encoding a second type III secretion system in Salmonella typhimurium. Proc Natl Acad Sci U S A 93: 2593–2597.
7. HenselM (2000) Salmonella pathogenicity island 2. Mol Microbiol 36: 1015–1023.
8. GalanJE, CurtissR3rd (1989) Cloning and molecular characterization of genes whose products allow Salmonella typhimurium to penetrate tissue culture cells. Proc Natl Acad Sci U S A 86: 6383–6387.
9. BisphamJ, TripathiBN, WatsonPR, WallisTS (2001) Salmonella pathogenicity island 2 influences both systemic salmonellosis and Salmonella-induced enteritis in calves. Infect Immun 69: 367–377.
10. CarnellSC, BowenA, MorganE, MaskellDJ, WallisTS, et al. (2007) Role in virulence and protective efficacy in pigs of Salmonella enterica serovar Typhimurium secreted components identified by signature-tagged mutagenesis. Microbiology 153: 1940–1952.
11. LichtensteigerCA, VimrER (2003) Systemic and enteric colonization of pigs by a hilA signature-tagged mutant of Salmonella choleraesuis. Microb Pathog 34: 149–154.
12. MorganE, CampbellJD, RoweSC, BisphamJ, StevensMP, et al. (2004) Identification of host-specific colonization factors of Salmonella enterica serovar Typhimurium. Mol Microbiol 54: 994–1010.
13. ShahDH, LeeMJ, ParkJH, LeeJH, EoSK, et al. (2005) Identification of Salmonella gallinarum virulence genes in a chicken infection model using PCR-based signature-tagged mutagenesis. Microbiology 151: 3957–3968.
14. TsolisRM, TownsendSM, MiaoEA, MillerSI, FichtTA, et al. (1999) Identification of a putative Salmonella enterica serotype Typhimurium host range factor with homology to IpaH and YopM by signature-tagged mutagenesis. Infect Immun 67: 6385–6393.
15. LangridgeGC, PhanM-D, TurnerDJ, PerkinsTT, PartsL, et al. (2009) Simultaneous assay of every Salmonella Typhi gene using one million transposon mutants. Genome Research 19: 2308–2316.
16. BentleyDR, BalasubramanianS, SwerdlowHP, SmithGP, MiltonJ, et al. (2008) Accurate whole human genome sequencing using reversible terminator chemistry. Nature 456: 53–59.
17. van OpijnenT, BodiKL, CamilliA (2009) Tn-seq: high-throughput parallel sequencing for fitness and genetic interaction studies in microorganisms. Nat Methods 6: 767–772.
18. GawronskiJD, WongSMS, GiannoukosG, WardDV, AkerleyBJ (2009) Tracking insertion mutants within libraries by deep sequencing and a genome-wide screen for Haemophilus genes required in the lung. Proceedings of the National Academy of Sciences 106: 16422–16427.
19. van OpijnenT, CamilliA (2012) A fine scale phenotype-genotype virulence map of a bacterial pathogen. Genome Research 22: 2541–2551.
20. GoodmanAL, McNultyNP, ZhaoY, LeipD, MitraRD, et al. (2009) Identifying genetic determinants needed to establish a human gut symbiont in its habitat. Cell Host Microbe 6: 279–289.
21. ChaudhuriRR, PetersSE, PleasanceSJ, NorthenH, WillersC, et al. (2009) Comprehensive identification of Salmonella enterica serovar Typhimurium genes required for infection of BALB/c mice. PLoS Pathog 5: e1000529 doi:10.1371/journal.ppat.1000529.
22. LawleyTD, ChanK, ThompsonLJ, KimCC, GovoniGR, et al. (2006) Genome-wide screen for Salmonella genes required for long-term systemic infection of the mouse. PLoS Pathog 2: e11 doi:10.1371/journal.ppat.0020011.
23. SantiviagoCA, ReynoldsMM, PorwollikS, ChoiSH, LongF, et al. (2009) Analysis of pools of targeted Salmonella deletion mutants identifies novel genes affecting fitness during competitive infection in mice. PLoS Pathog 5: e1000477 doi:10.1371/journal.ppat.1000477.
24. KanehisaM, GotoS, HattoriM, Aoki-KinoshitaKF, ItohM, et al. (2006) From genomics to chemical genomics: new developments in KEGG. Nucleic Acids Res 34: D354–357.
25. BjurE, Eriksson-YgbergS, AslundF, RhenM (2006) Thioredoxin 1 promotes intracellular replication and virulence of Salmonella enterica serovar Typhimurium. Infect Immun 74: 5140–5151.
26. ValdiviaRH, CirilloDM, LeeAK, BouleyDM, FalkowS (2000) mig-14 is a horizontally acquired, host-induced gene required for salmonella enterica lethal infection in the murine model of typhoid fever. Infect Immun 68: 7126–7131.
27. DetweilerCS, MonackDM, BrodskyIE, MathewH, FalkowS (2003) virK, somA and rcsC are important for systemic Salmonella enterica serovar Typhimurium infection and cationic peptide resistance. Mol Microbiol 48: 385–400.
28. LambertMA, SmithSG (2009) The PagN protein mediates invasion via interaction with proteoglycan. FEMS Microbiol Lett 297: 209–216.
29. Gal-MorO, GibsonDL, BalutaD, VallanceBA, FinlayBB (2008) A novel secretion pathway of Salmonella enterica acts as an antivirulence modulator during salmonellosis. PLoS Pathog 4: e1000036 doi:10.1371/journal.ppat.1000036.
30. DatsenkoKA, WannerBL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97: 6640–6645.
31. WangQ, MaricondaS, SuzukiA, McClellandM, HarsheyRM (2006) Uncovering a large set of genes that affect surface motility in Salmonella enterica serovar Typhimurium. J Bacteriol 188: 7981–7984.
32. ChenLM, KanigaK, GalanJE (1996) Salmonella spp. are cytotoxic for cultured macrophages. Mol Microbiol 21: 1101–1115.
33. FuY, GalanJE (1998) The Salmonella typhimurium tyrosine phosphatase SptP is translocated into host cells and disrupts the actin cytoskeleton. Mol Microbiol 27: 359–368.
34. KanigaK, UralilJ, BliskaJB, GalanJE (1996) A secreted protein tyrosine phosphatase with modular effector domains in the bacterial pathogen Salmonella typhimurium. Mol Microbiol 21: 633–641.
35. MiaoEA, BrittnacherM, HaragaA, JengRL, WelchMD, et al. (2003) Salmonella effectors translocated across the vacuolar membrane interact with the actin cytoskeleton. Mol Microbiol 48: 401–415.
36. HenselM (2004) Evolution of pathogenicity islands of Salmonella enterica. Int J Med Microbiol 294: 95–102.
37. RetamalP, Castillo-RuizM, VillagraNA, MorgadoJ, MoraGC (2010) Modified intracellular-associated phenotypes in a recombinant Salmonella Typhi expressing S. Typhimurium SPI-3 sequences. PLoS ONE 5: e9394 doi:10.1371/journal.pone.0009394.
38. MorganE, BowenAJ, CarnellSC, WallisTS, StevensMP (2007) SiiE is secreted by the Salmonella enterica serovar Typhimurium pathogenicity island 4-encoded secretion system and contributes to intestinal colonization in cattle. Infect Immun 75: 1524–1533.
39. FolkessonA, LofdahlS, NormarkS (2002) The Salmonella enterica subspecies I specific centisome 7 genomic island encodes novel protein families present in bacteria living in close contact with eukaryotic cells. Res Microbiol 153: 537–545.
40. EdwardsRA, PuenteJL (1998) Fimbrial expression in enteric bacteria: a critical step in intestinal pathogenesis. Trends Microbiol 6: 282–287.
41. RychlikI, BarrowPA (2005) Salmonella stress management and its relevance to behaviour during intestinal colonisation and infection. FEMS Microbiol Rev 29: 1021–1040.
42. MorganRW, ChristmanMF, JacobsonFS, StorzG, AmesBN (1986) Hydrogen peroxide-inducible proteins in Salmonella typhimurium overlap with heat shock and other stress proteins. Proc Natl Acad Sci U S A 83: 8059–8063.
43. KnuthK, NiesallaH, HueckCJ, FuchsTM (2004) Large-scale identification of essential Salmonella genes by trapping lethal insertions. Mol Microbiol 51: 1729–1744.
44. KrogerC, DillonSC, CameronAD, PapenfortK, SivasankaranSK, et al. (2012) The transcriptional landscape and small RNAs of Salmonella enterica serovar Typhimurium. Proceedings of the National Academy of Sciences of the United States of America 109: E1277–1286.
45. RichardsonEJ, LimayeB, InamdarH, DattaA, ManjariKS, et al. (2011) Genome sequences of Salmonella enterica serovar typhimurium, Choleraesuis, Dublin, and Gallinarum strains of well- defined virulence in food-producing animals. J Bacteriol 193: 3162–3163.
46. MarteynB, WestNP, BrowningDF, ColeJA, ShawJG, et al. (2010) Modulation of Shigella virulence in response to available oxygen in vivo. Nature 465: 355–358.
47. Sanderson KE, Stocker BAD (1987) Salmonella typhimurium strains used in genetic analysis. In: Neidhardt FC, Ingraham JL, Low KB, Magasanik B, Schaechter M et al.., editors. Escherichia coli and Salmonella typhimurium: cellular and molecular biology. Washington D.C.: ASM Press. pp. 1220–1224.
48. SwordsWE, CannonBM, BenjaminWHJr (1997) Avirulence of LT2 strains of Salmonella typhimurium results from a defective rpoS gene. Infection and Immunity 65: 2451–2453.
49. KingsleyRA, MsefulaCL, ThomsonNR, KariukiS, HoltKE, et al. (2009) Epidemic multiple drug resistant Salmonella Typhimurium causing invasive disease in sub-Saharan Africa have a distinct genotype. Genome Res 19: 2279–2287.
50. EckertS, DzivaF, ChaudhuriRR, LangridgeGC, TurnerDJ, et al. (2011) Retrospective application of transposon-directed insertion-site sequencing to a library of signature-tagged mini-Tn5Km2 mutants of Escherichia coli O157:H7 screened in cattle. J Bacteriol 193: 1771–1776.
51. BeckerD, SelbachM, RollenhagenC, BallmaierM, MeyerTF, et al. (2006) Robust Salmonella metabolism limits possibilities for new antimicrobials. Nature 440: 303–307.
52. AndersS, HuberW (2010) Differential expression analysis for sequence count data. Genome Biol 11: R106.
Štítky
Genetika Reprodukčná medicínaČlánok vyšiel v časopise
PLOS Genetics
2013 Číslo 4
- Je „freeze-all“ pro všechny? Odborníci na fertilitu diskutovali na virtuálním summitu
- Gynekologové a odborníci na reprodukční medicínu se sejdou na prvním virtuálním summitu
Najčítanejšie v tomto čísle
- The G4 Genome
- Neutral Genomic Microevolution of a Recently Emerged Pathogen, Serovar Agona
- The Histone Demethylase Jarid1b Ensures Faithful Mouse Development by Protecting Developmental Genes from Aberrant H3K4me3
- The Tissue-Specific RNA Binding Protein T-STAR Controls Regional Splicing Patterns of Pre-mRNAs in the Brain