1887

Abstract

The availability of next-generation sequencing techniques provides an unprecedented opportunity for the assignment of gene function. subspecies is the causative agent of strangles in horses, one of the most prevalent and important diseases of equids worldwide. However, the live attenuated vaccines that are utilized to control this disease cause adverse reactions in some animals. Here, we employ transposon-directed insertion-site sequencing (TraDIS) to identify genes that are required for the fitness of in whole equine blood or in the presence of HO to model selective pressures exerted by the equine immune response during infection. We report the fitness values of 1503 and 1471 genes, representing 94.5 and 92.5 % of non-essential genes in , following incubation in whole blood and in the presence of HO, respectively. Of these genes, 36 and 15 were identified as being important to the fitness of in whole blood or HO, respectively, with 14 genes being important in both conditions. Allelic replacement mutants were generated to validate the fitness results. Our data identify genes that are important for to resist aspects of the immune response , which can be exploited for the development of safer live attenuated vaccines to prevent strangles.

Funding
This study was supported by the:
  • Australian Research Council Discovery Early Career Research Award (Award DE180100929)
    • Principle Award Recipient: Amy K. Cain
  • Biotechnology and Biological Sciences Research Counci (Award 1503883)
    • Principle Award Recipient: Duncan J. Maskell
  • Horse Trust (Award G4104)
    • Principle Award Recipient: Andrew S. Waller
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License.
Loading

Article metrics loading...

/content/journal/mgen/10.1099/mgen.0.000362
2020-03-31
2024-03-28
Loading full text...

Full text loading...

/deliver/fulltext/mgen/6/4/mgen000362.html?itemId=/content/journal/mgen/10.1099/mgen.0.000362&mimeType=html&fmt=ahah

References

  1. Boyle AG, Timoney JF, Newton JR, Hines MT, Waller AS et al. Streptococcus equi infections in horses: guidelines for treatment, control, and prevention of strangles – revised consensus statement. J Vet Intern Med 2018; 32:633–647 [View Article][PubMed]
    [Google Scholar]
  2. Timoney JF, Kumar P. Early pathogenesis of equine Streptococcus equi infection (strangles). Equine Vet J 2008; 40:637–642 [View Article][PubMed]
    [Google Scholar]
  3. Harrington DJ, Sutcliffe IC, Chanter N. The molecular basis of Streptococcus equi infection and disease. Microbes Infect 2002; 4:501–510 [View Article][PubMed]
    [Google Scholar]
  4. Bazeley PL, Battle J. Studies with equine streptococci 1. Aust Vet J 1940; 16:140–146 [View Article]
    [Google Scholar]
  5. Bazeley PL. Studies with equine streptococci 2. Aust Vet J 1940; 16:243–259 [View Article]
    [Google Scholar]
  6. Bazeley PL. Studies with equine streptococci 3. Aust Vet J 1942; 18:141–155 [View Article]
    [Google Scholar]
  7. Bazeley PL. Studies with equine streptococci 4. Aust Vet J 1942; 18:189–194 [View Article]
    [Google Scholar]
  8. Bazeley PL. Studies with equine streptococci 5. Aust Vet J 1943; 19:62–85 [View Article]
    [Google Scholar]
  9. Hoffman AM, Staempfli HR, Prescott JF, Viel L. Field evaluation of a commercial M-protein vaccine against Streptococcus equi infection in foals. Am J Vet Res 1991; 52:589–592[PubMed]
    [Google Scholar]
  10. Guss B, Flock M, Frykberg L, Waller AS, Robinson C et al. Getting to grips with strangles: an effective multi-component recombinant vaccine for the protection of horses from Streptococcus equi infection. PLoS Pathog 2009; 5:e1000584 [View Article][PubMed]
    [Google Scholar]
  11. Robinson C, Frykberg L, Flock M, Guss B, Waller AS et al. Strangvac: a recombinant fusion protein vaccine that protects against strangles, caused by Streptococcus equi . Vaccine 2018; 36:1484–1490 [View Article][PubMed]
    [Google Scholar]
  12. Walker JA, Timoney JF. Construction of a stable non-mucoid deletion mutant of the Streptococcus equi Pinnacle vaccine strain. Vet Microbiol 2002; 89:311–321 [View Article][PubMed]
    [Google Scholar]
  13. Jacobs AA, Goovaerts D, Nuijten PJ, Theelen RP, Hartford OM et al. Investigations towards an efficacious and safe strangles vaccine: submucosal vaccination with a live attenuated Streptococcus equi . Vet Rec 2000; 147:563–567 [View Article][PubMed]
    [Google Scholar]
  14. Cursons R, Patty O, Steward KF, Waller AS. Strangles in horses can be caused by vaccination with Pinnacle I. N. Vaccine 2015; 33:3440–3443 [View Article][PubMed]
    [Google Scholar]
  15. Kelly C, Bugg M, Robinson C, Mitchell Z, Davis-Poynter N et al. Sequence variation of the SeM gene of Streptococcus equi allows discrimination of the source of strangles outbreaks. J Clin Microbiol 2006; 44:480–486 [View Article][PubMed]
    [Google Scholar]
  16. Kemp-Symonds J, Kemble T, Waller A. Modified live Streptococcus equi ('strangles') vaccination followed by clinically adverse reactions associated with bacterial replication. Equine Vet J 2007; 39:284–286 [View Article][PubMed]
    [Google Scholar]
  17. Livengood JL, Lanka S, Maddox C, Tewari D. Detection and differentiation of wild-type and a vaccine strain of Streptococcus equi ssp. equi using pyrosequencing. Vaccine 2016; 34:3935–3937 [View Article][PubMed]
    [Google Scholar]
  18. Robinson C, Heather Z, Slater J, Potts N, Steward KF et al. Vaccination with a live multi-gene deletion strain protects horses against virulent challenge with Streptococcus equi . Vaccine 2015; 33:1160–1167 [View Article][PubMed]
    [Google Scholar]
  19. Boschwitz JS, Timoney JF. Inhibition of C3 deposition on Streptococcus equi subsp. equi by M protein: a mechanism for survival in equine blood. Infect Immun 1994; 62:3515–3520 [View Article][PubMed]
    [Google Scholar]
  20. Galán JE, Timoney JF. Molecular analysis of the M protein of Streptococcus equi and cloning and expression of the M protein gene in Escherichia coli . Infect Immun 1987; 55:3181–3187 [View Article][PubMed]
    [Google Scholar]
  21. Tiwari R, Qin A, Artiushin S, Timoney JF. Se18.9, an anti-phagocytic factor H binding protein of Streptococcus equi . Vet Microbiol 2007; 121:105–115 [View Article][PubMed]
    [Google Scholar]
  22. Flock M, Frykberg L, Sköld M, Guss B, Flock J-I. Antiphagocytic function of an IgG glycosyl hydrolase from Streptococcus equi subsp. equi and its use as a vaccine component. Infect Immun 2012; 80:2914–2919 [View Article][PubMed]
    [Google Scholar]
  23. Hulting G, Flock M, Frykberg L, Lannergård J, Flock J-I et al. Two novel IgG endopeptidases of Streptococcus equi . FEMS Microbiol Lett 2009; 298:44–50 [View Article][PubMed]
    [Google Scholar]
  24. Lannergård J, Flock M, Johansson S, Flock J-I, Guss B. Studies of fibronectin-binding proteins of Streptococcus equi. Infect Immun 2005; 73:7243–7251 [View Article][PubMed]
    [Google Scholar]
  25. Holden MTG, Heather Z, Paillot R, Steward KF, Webb K et al. Genomic evidence for the evolution of Streptococcus equi: host restriction, increased virulence, and genetic exchange with human pathogens. PLoS Pathog 2009; 5:e1000346 [View Article][PubMed]
    [Google Scholar]
  26. von Beek C, Waern I, Eriksson J, Melo FR, Robinson C et al. Streptococcal sagA activates a proinflammatory response in mast cells by a sublytic mechanism. Cell Microbiol 2019; 21:e13064 [View Article][PubMed]
    [Google Scholar]
  27. Langridge GC, Phan MD, Turner DJ, Perkins TT, Parts L et al. Simultaneous assay of every Salmonella Typhi gene using one million transposon mutants. Genome Res 2009; 19:2308–2316 [View Article][PubMed]
    [Google Scholar]
  28. van Opijnen T, Bodi KL, Camilli A. Tn-seq: high-throughput parallel sequencing for fitness and genetic interaction studies in microorganisms. Nat Methods 2009; 6:767–772 [View Article][PubMed]
    [Google Scholar]
  29. Goodman AL, McNulty NP, Zhao Y, Leip D, Mitra RD et al. Identifying genetic determinants needed to establish a human gut symbiont in its habitat. Cell Host Microbe 2009; 6:279–289 [View Article][PubMed]
    [Google Scholar]
  30. Gawronski JD, Wong SMS, Giannoukos G, Ward DV, Akerley BJ. Tracking insertion mutants within libraries by deep sequencing and a genome-wide screen for Haemophilus genes required in the lung. Proc Natl Acad Sci USA 2009; 106:16422–16427 [View Article][PubMed]
    [Google Scholar]
  31. Blanchard AM, Egan SA, Emes RD, Warry A, Leigh JA. PIMMS (Pragmatic Insertional Mutation Mapping System) laboratory methodology a readily accessible tool for identification of essential genes in Streptococcus . Front Microbiol 2016; 7:1645 [View Article][PubMed]
    [Google Scholar]
  32. Charbonneau ARL, Forman OP, Cain AK, Newland G, Robinson C et al. Defining the ABC of gene essentiality in streptococci. BMC Genomics 2017; 18:426 [View Article][PubMed]
    [Google Scholar]
  33. Barquist L, Boinett CJ, Cain AK. Approaches to querying bacterial genomes with transposon-insertion sequencing. RNA Biol 2013; 10:1161–1169 [View Article][PubMed]
    [Google Scholar]
  34. van Opijnen T, Camilli A. A fine scale phenotype-genotype virulence map of a bacterial pathogen. Genome Res 2012; 22:2541–2551 [View Article][PubMed]
    [Google Scholar]
  35. Dembek M, Barquist L, Boinett CJ, Cain AK, Mayho M et al. High-throughput analysis of gene essentiality and sporulation in Clostridium difficile . mBio 2015; 6:e02383 [View Article][PubMed]
    [Google Scholar]
  36. Zhu L, Charbonneau ARL, Waller AS, Olsen RJ, Beres SB et al. Novel genes required for the fitness of Streptococcus pyogenes in human saliva. mSphere 2017; 2:e00460-17 [View Article][PubMed]
    [Google Scholar]
  37. Chaudhuri RR, Morgan E, Peters SE, Pleasance SJ, Hudson DL et al. Comprehensive assignment of roles for Salmonella Typhimurium genes in intestinal colonization of food-producing animals. PLoS Genet 2013; 9:e1003456 [View Article][PubMed]
    [Google Scholar]
  38. Subashchandrabose S, Smith S, DeOrnellas V, Crepin S, Kole M et al. Acinetobacter baumannii genes required for bacterial survival during bloodstream infection. mSphere 2016; 1:e00013-15 [View Article][PubMed]
    [Google Scholar]
  39. Grant AJ, Oshota O, Chaudhuri RR, Mayho M, Peters SE et al. Genes required for the fitness of Salmonella enterica serovar Typhimurium during infection of immunodeficient gp91-/- phox mice. Infect Immun 2016; 84:989–997 [View Article][PubMed]
    [Google Scholar]
  40. Moule MG, Spink N, Willcocks S, Lim J, Guerra-Assunção JA et al. Characterization of new virulence factors involved in the intracellular growth and survival of Burkholderia pseudomallei . Infect Immun 2015; 84:701–710 [View Article][PubMed]
    [Google Scholar]
  41. Gutierrez MG, Yoder-Himes DR, Warawa JM. Comprehensive identification of virulence factors required for respiratory melioidosis using Tn-seq mutagenesis. Front Cell Infect Microbiol 2015; 5:78 [View Article][PubMed]
    [Google Scholar]
  42. Zhu L, Olsen R, Beres S, Eraso J, Saavedra MO et al. Gene fitness landscape of group A Streptococcus during necrotizing myositis. J Clin Investig 2019; 129:887–901
    [Google Scholar]
  43. Barquist L, Mayho M, Cummins C, Cain AK, Boinett CJ et al. The TraDIS toolkit: sequencing and analysis for dense transposon mutant libraries. Bioinformatics 2016; 32:1109–1111 [View Article][PubMed]
    [Google Scholar]
  44. Heather Z, Holden MTG, Steward KF, Parkhill J, Song L et al. A novel streptococcal integrative conjugative element involved in iron acquisition. Mol Microbiol 2008; 70:1274–1292 [View Article][PubMed]
    [Google Scholar]
  45. Hamilton A, Robinson C, Sutcliffe IC, Slater J, Maskell DJ et al. Mutation of the maturase lipoprotein attenuates the virulence of Streptococcus equi to a greater extent than does loss of general lipoprotein lipidation. Infect Immun 2006; 74:6907–6919 [View Article][PubMed]
    [Google Scholar]
  46. Chédin F, Kowalczykowski SC. A novel family of regulated helicases/nucleases from Gram-positive bacteria: insights into the initiation of DNA recombination. Mol Microbiol 2002; 43:823–834 [View Article][PubMed]
    [Google Scholar]
  47. Halpern D, Gruss A, Claverys J-P, El Karoui M. rexAB mutants in Streptococcus pneumoniae . Microbiology 2004; 150:2409–2414 [View Article][PubMed]
    [Google Scholar]
  48. Yeeles JTP, Gwynn EJ, Webb MR, Dillingham MS. The AddAB helicase-nuclease catalyses rapid and processive DNA unwinding using a single superfamily 1A motor domain. Nucleic Acids Res 2011; 39:2271–2285 [View Article][PubMed]
    [Google Scholar]
  49. Whitby MC, Vincent SD, Lloyd RG. Branch migration of Holliday junctions: identification of RecG protein as a junction specific DNA helicase. Embo J 1994; 13:5220–5228 [View Article][PubMed]
    [Google Scholar]
  50. Hong X, Cadwell GW, Kogoma T. Escherichia coli RecG and RecA proteins in R-loop formation. Embo J 1995; 14:2385–2392 [View Article][PubMed]
    [Google Scholar]
  51. Vincent SD, Mahdi AA, Lloyd RG. The RecG branch migration protein of Escherichia coli dissociates R-loops. J Mol Biol 1996; 264:713–721 [View Article][PubMed]
    [Google Scholar]
  52. Martinussen J, Schallert J, Andersen B, Hammer K. The pyrimidine operon pyrRPB-carA from Lactococcus lactis . J Bacteriol 2001; 183:2785–2794 [View Article][PubMed]
    [Google Scholar]
  53. Moukadiri I, Prado S, Piera J, Velázquez-Campoy A, Björk GR et al. Evolutionarily conserved proteins MnmE and GidA catalyze the formation of two methyluridine derivatives at tRNA wobble positions. Nucleic Acids Res 2009; 37:7177–7193 [View Article][PubMed]
    [Google Scholar]
  54. Yim L, Moukadiri I, Björk GR, Armengod M-E. Further insights into the tRNA modification process controlled by proteins MnmE and GidA of Escherichia coli . Nucleic Acids Res 2006; 34:5892–5905 [View Article][PubMed]
    [Google Scholar]
  55. Prado S, Villarroya M, Medina M, Armengod M-E. The tRNA-modifying function of MnmE is controlled by post-hydrolysis steps of its GTPase cycle. Nucleic Acids Res 2013; 41:6190–6208 [View Article][PubMed]
    [Google Scholar]
  56. Le Breton Y, Belew AT, Valdes KM, Islam E, Curry P et al. Essential genes in the core genome of the human pathogen Streptococcus pyogenes . Sci Rep 2015; 5:9838 [View Article][PubMed]
    [Google Scholar]
  57. Hooven TA, Catomeris AJ, Akabas LH, Randis TM, Maskell DJ et al. The essential genome of Streptococcus agalactiae . BMC Genomics 2016; 17:406 [View Article][PubMed]
    [Google Scholar]
  58. Cho KH, Caparon MG. tRNA modification by GidA/MnmE is necessary for Streptococcus pyogenes virulence: a new strategy to make live attenuated strains. Infect Immun 2008; 76:3176–3186 [View Article][PubMed]
    [Google Scholar]
  59. Li D, Shibata Y, Takeshita T, Yamashita Y. A novel gene involved in the survival of Streptococcus mutans under stress conditions. Appl Environ Microbiol 2014; 80:97–103 [View Article][PubMed]
    [Google Scholar]
  60. Wilson AT. The relative importance of the capsule and the M-antigen in determining colony form of group A streptococci. J Exp Med 1959; 109:257–270 [View Article][PubMed]
    [Google Scholar]
  61. Woolcock JB. The capsule of Streptococcus equi . J Gen Microbiol 1974; 85:372–375 [View Article][PubMed]
    [Google Scholar]
  62. Wessels MR, Moses AE, Goldberg JB, DiCesare TJ. Hyaluronic acid capsule is a virulence factor for mucoid group A streptococci. Proc Natl Acad Sci USA 1991; 88:8317–8321 [View Article][PubMed]
    [Google Scholar]
  63. Dale JB, Washburn RG, Marques MB, Wessels MR. Hyaluronate capsule and surface M protein in resistance to opsonization of group A streptococci. Infect Immun 1996; 64:1495–1501 [View Article][PubMed]
    [Google Scholar]
  64. Harris SR, Robinson C, Steward KF, Webb KS, Paillot R et al. Genome specialization and decay of the strangles pathogen, Streptococcus equi, is driven by persistent infection. Genome Res 2015; 25:1360–1371 [View Article][PubMed]
    [Google Scholar]
  65. Willenborg J, Fulde M, de Greeff A, Rohde M, Smith HE et al. Role of glucose and CcpA in capsule expression and virulence of Streptococcus suis . Microbiology 2011; 157:1823–1833 [View Article][PubMed]
    [Google Scholar]
  66. Willenborg J, de Greeff A, Jarek M, Valentin-Weigand P, Goethe R. The CcpA regulon of Streptococcus suis reveals novel insights into the regulation of the streptococcal central carbon metabolism by binding of CcpA to two distinct binding motifs. Mol Microbiol 2014; 92:61–83 [View Article][PubMed]
    [Google Scholar]
  67. Chang JC, Federle MJ. PptAB exports Rgg quorum-sensing peptides in Streptococcus . PLoS One 2016; 11:e0168461 [View Article][PubMed]
    [Google Scholar]
  68. Jonsson I-M, Juuti JT, François P, AlMajidi R, Pietiäinen M et al. Inactivation of the Ecs ABC transporter of Staphylococcus aureus attenuates virulence by altering composition and function of bacterial wall. PLoS One 2010; 5:e14209 [View Article][PubMed]
    [Google Scholar]
  69. Van Bokhorst-van de Veen H, Bongers RS, Wels M, Bron PA, Kleerebezem M. Transcriptome signatures of class I and III stress response deregulation in Lactobacillus plantarum reveal pleiotropic adaptation. Microb Cell Fact 2013; 12:112 [View Article][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/mgen/10.1099/mgen.0.000362
Loading
/content/journal/mgen/10.1099/mgen.0.000362
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error