1887

Abstract

Tyrosine phosphorylation has long been recognized as a crucial post-translational regulatory mechanism in eukaryotes. However, only in the past decade has recognition been given to the crucial importance of bacterial tyrosine phosphorylation as an important regulatory feature of pathogenesis. This study describes the effect of tyrosine phosphorylation on the activity of a major virulence factor of the pneumococcus, the autolysin LytA, and a possible connection to the capsule synthesis regulatory proteins (CpsB, CpsC and CpsD). We show that pneumococcal tyrosine kinase, CpsD, and the protein tyrosine phosphatase, CpsB, act to phosphorylate and dephosphorylate LytA. Furthermore, this modulates LytA function with phosphorylated LytA binding more strongly to the choline analogue DEAE. A phospho-mimetic (Y264E) mutation of the LytA phosphorylation site displayed similar phenotypes as well as an enhanced dimerization capacity. Similarly, tyrosine phosphorylation increased LytA amidase activity, as evidenced by a turbidometric amidase activity assay. Similarly, when the phospho-mimetic mutation was introduced in the chromosomal of , autolysis occurred earlier and at an enhanced rate. This study thus describes, to our knowledge, the first functional regulatory effect of tyrosine phosphorylation on a non-capsule-related protein in the pneumococcus, and suggests a link between the regulation of LytA-dependent autolysis of the cell and the biosynthesis of capsular polysaccharide.

Funding
This study was supported by the:
  • NHMRC (Award 1048749)
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.080747-0
2014-12-01
2021-08-05
Loading full text...

Full text loading...

/deliver/fulltext/micro/160/12/2745.html?itemId=/content/journal/micro/10.1099/mic.0.080747-0&mimeType=html&fmt=ahah

References

  1. Beard S. J., Salisbury V., Lewis R. J., Sharpe J. A., MacGowan A. P. ( 2002). Expression of lux genes in a clinical isolate of Streptococcus pneumoniae: using bioluminescence to monitor gemifloxacin activity. Antimicrob Agents Chemother 46:538–542 [View Article][PubMed]
    [Google Scholar]
  2. Bender M. H., Yother J. ( 2001). CpsB is a modulator of capsule-associated tyrosine kinase activity in Streptococcus pneumoniae . J Biol Chem 276:47966–47974[PubMed] [CrossRef]
    [Google Scholar]
  3. Berry A. M., Paton J. C. ( 2000). Additive attenuation of virulence of Streptococcus pneumoniae by mutation of the genes encoding pneumolysin and other putative pneumococcal virulence proteins. Infect Immun 68:133–140 [View Article][PubMed]
    [Google Scholar]
  4. Byrne J. P., Morona J. K., Paton J. C., Morona R. ( 2011). Identification of Streptococcus pneumoniae Cps2C residues that affect capsular polysaccharide polymerization, cell wall ligation, and Cps2D phosphorylation. J Bacteriol 193:2341–2346 [View Article][PubMed]
    [Google Scholar]
  5. Dalia A. B., Weiser J. N. ( 2011). Minimization of bacterial size allows for complement evasion and is overcome by the agglutinating effect of antibody. Cell Host Microbe 10:486–496 [View Article][PubMed]
    [Google Scholar]
  6. De Las Rivas B., García J. L., López R., García P. ( 2002). Purification and polar localization of pneumococcal LytB, a putative endo-β-N-acetylglucosaminidase: the chain-dispersing murein hydrolase. J Bacteriol 184:4988–5000 [View Article][PubMed]
    [Google Scholar]
  7. Eldholm V., Johnsborg O., Haugen K., Ohnstad H. S., Håvarstein L. S. ( 2009). Fratricide in Streptococcus pneumoniae: contributions and role of the cell wall hydrolases CbpD, LytA and LytC. Microbiology 155:2223–2234 [View Article][PubMed]
    [Google Scholar]
  8. Ericsson D. J., Standish A., Kobe B., Morona R. ( 2012). Wzy-dependent bacterial capsules as potential drug targets. Curr Drug Targets 13:1421–1431 [View Article][PubMed]
    [Google Scholar]
  9. Fernández-Tornero C., López R., García E., Giménez-Gallego G., Romero A. ( 2001). A novel solenoid fold in the cell wall anchoring domain of the pneumococcal virulence factor LytA. Nat Struct Biol 8:1020–1024 [View Article][PubMed]
    [Google Scholar]
  10. Fernández-Tornero C., García E., López R., Giménez-Gallego G., Romero A. ( 2002). Two new crystal forms of the choline-binding domain of the major pneumococcal autolysin: insights into the dynamics of the active homodimer. J Mol Biol 321:163–173 [View Article][PubMed]
    [Google Scholar]
  11. Fernebro J., Andersson I., Sublett J., Morfeldt E., Novak R., Tuomanen E., Normark S., Normark B. H. ( 2004). Capsular expression in Streptococcus pneumoniae negatively affects spontaneous and antibiotic-induced lysis and contributes to antibiotic tolerance. J Infect Dis 189:328–338 [View Article][PubMed]
    [Google Scholar]
  12. García E., García J. L., Ronda C., García P., López R. ( 1985). Cloning and expression of the pneumococcal autolysin gene in Escherichia coli . Mol Gen Genet 201:225–230 [View Article][PubMed]
    [Google Scholar]
  13. García P., García J. L., García E., López R. ( 1986). Nucleotide sequence and expression of the pneumococcal autolysin gene from its own promoter in Escherichia coli . Gene 43:265–272 [View Article][PubMed]
    [Google Scholar]
  14. Giudicelli S., Tomasz A. ( 1984). Attachment of pneumococcal autolysin to wall teichoic acids, an essential step in enzymatic wall degradation. J Bacteriol 158:1188–1190[PubMed]
    [Google Scholar]
  15. Goebel W. F., Avery O. T. ( 1929). A study of pneumococcus autolysis. J Exp Med 49:267–286 [View Article][PubMed]
    [Google Scholar]
  16. Hansen A. M., Chaerkady R., Sharma J., Díaz-Mejía J. J., Tyagi N., Renuse S., Jacob H. K., Pinto S. M., Sahasrabuddhe N. A. & other authors ( 2013). The Escherichia coli phosphotyrosine proteome relates to core pathways and virulence. PLoS Pathog 9:e1003403 [View Article][PubMed]
    [Google Scholar]
  17. Henriques M. X., Rodrigues T., Carido M., Ferreira L., Filipe S. R. ( 2011). Synthesis of capsular polysaccharide at the division septum of Streptococcus pneumoniae is dependent on a bacterial tyrosine kinase. Mol Microbiol 82:515–534 [View Article][PubMed]
    [Google Scholar]
  18. Kelley L. A., Sternberg M. J. ( 2009). Protein structure prediction on the Web: a case study using the Phyre server. Nat Protoc 4:363–371 [View Article][PubMed]
    [Google Scholar]
  19. Laemmli U. K. ( 1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685 [View Article][PubMed]
    [Google Scholar]
  20. Martner A., Dahlgren C., Paton J. C., Wold A. E. ( 2008). Pneumolysin released during Streptococcus pneumoniae autolysis is a potent activator of intracellular oxygen radical production in neutrophils. Infect Immun 76:4079–4087 [View Article][PubMed]
    [Google Scholar]
  21. McAllister L. J., Tseng H. J., Ogunniyi A. D., Jennings M. P., McEwan A. G., Paton J. C. ( 2004). Molecular analysis of the psa permease complex of Streptococcus pneumoniae . Mol Microbiol 53:889–901 [View Article][PubMed]
    [Google Scholar]
  22. Mellroth P., Daniels R., Eberhardt A., Rönnlund D., Blom H., Widengren J., Normark S., Henriques-Normark B. ( 2012). LytA, major autolysin of Streptococcus pneumoniae, requires access to nascent peptidoglycan. J Biol Chem 287:11018–11029 [View Article][PubMed]
    [Google Scholar]
  23. Mellroth P., Sandalova T., Kikhney A., Vilaplana F., Hesek D., Lee M., Mobashery S., Normark S., Svergun D. & other authors ( 2014). Structural and functional insights into peptidoglycan access for the lytic amidase LytA of Streptococcus pneumoniae . MBio 5:e01120-13 [View Article][PubMed]
    [Google Scholar]
  24. Morona R., van den Bosch L., Manning P. A. ( 1995). Molecular, genetic, and topological characterization of O-antigen chain length regulation in Shigella flexneri . J Bacteriol 177:1059–1068[PubMed]
    [Google Scholar]
  25. Morona J. K., Paton J. C., Miller D. C., Morona R. ( 2000). Tyrosine phosphorylation of CpsD negatively regulates capsular polysaccharide biosynthesis in Streptococcus pneumoniae . Mol Microbiol 35:1431–1442 [View Article][PubMed]
    [Google Scholar]
  26. Morona J. K., Morona R., Miller D. C., Paton J. C. ( 2002). Streptococcus pneumoniae capsule biosynthesis protein CpsB is a novel manganese-dependent phosphotyrosine-protein phosphatase. J Bacteriol 184:577–583 [View Article][PubMed]
    [Google Scholar]
  27. Morona J. K., Miller D. C., Morona R., Paton J. C. ( 2004). The effect that mutations in the conserved capsular polysaccharide biosynthesis genes cpsA, cpsB, and cpsD have on virulence of Streptococcus pneumoniae . J Infect Dis 189:1905–1913 [View Article][PubMed]
    [Google Scholar]
  28. Morona J. K., Morona R., Paton J. C. ( 2006). Attachment of capsular polysaccharide to the cell wall of Streptococcus pneumoniae type 2 is required for invasive disease. Proc Natl Acad Sci U S A 103:8505–8510 [View Article][PubMed]
    [Google Scholar]
  29. Olivares-Illana V., Meyer P., Bechet E., Gueguen-Chaignon V., Soulat D., Lazereg-Riquier S., Mijakovic I., Deutscher J., Cozzone A. J. & other authors ( 2008). Structural basis for the regulation mechanism of the tyrosine kinase CapB from Staphylococcus aureus . PLoS Biol 6:e143 [View Article][PubMed]
    [Google Scholar]
  30. Romero P., López R., García E. ( 2007). Key role of amino acid residues in the dimerization and catalytic activation of the autolysin LytA, an important virulence factor in Streptococcus pneumoniae . J Biol Chem 282:17729–17737 [View Article][PubMed]
    [Google Scholar]
  31. Rosenow C., Ryan P., Weiser J. N., Johnson S., Fontan P., Ortqvist A., Masure H. R. ( 1997). Contribution of novel choline-binding proteins to adherence, colonization and immunogenicity of Streptococcus pneumoniae . Mol Microbiol 25:819–829 [View Article][PubMed]
    [Google Scholar]
  32. Sanz J. M., Lopez R., Garcia J. L. ( 1988). Structural requirements of choline derivatives for ‘conversion’ of pneumococcal amidase. A new single-step procedure for purification of this autolysin. FEBS Lett 232:308–312 [View Article][PubMed]
    [Google Scholar]
  33. Soulat D., Jault J. M., Duclos B., Geourjon C., Cozzone A. J., Grangeasse C. ( 2006). Staphylococcus aureus operates protein-tyrosine phosphorylation through a specific mechanism. J Biol Chem 281:14048–14056 [View Article][PubMed]
    [Google Scholar]
  34. Standish A. J., Morona R. ( 2014). The role of bacterial protein tyrosine phosphatases in the regulation of the biosynthesis of secreted polysaccharides. Antioxid Redox Signal 20:2274–2289 [View Article][PubMed]
    [Google Scholar]
  35. Standish A. J., Stroeher U. H., Paton J. C. ( 2005). The two-component signal transduction system RR06/HK06 regulates expression of cbpA in Streptococcus pneumoniae . Proc Natl Acad Sci U S A 102:7701–7706 [View Article][PubMed]
    [Google Scholar]
  36. Standish A. J., Salim A. A., Zhang H., Capon R. J., Morona R. ( 2012). Chemical inhibition of bacterial protein tyrosine phosphatase suppresses capsule production. PLoS ONE 7:e36312 [View Article][PubMed]
    [Google Scholar]
  37. Standish A. J., Salim A. A., Capon R. J., Morona R. ( 2013). Dual inhibition of DNA polymerase PolC and protein tyrosine phosphatase CpsB uncovers a novel antibiotic target. Biochem Biophys Res Commun 430:167–172 [View Article][PubMed]
    [Google Scholar]
  38. Sun X., Ge F., Xiao C. L., Yin X. F., Ge R., Zhang L. H., He Q. Y. ( 2010). Phosphoproteomic analysis reveals the multiple roles of phosphorylation in pathogenic bacterium Streptococcus pneumoniae . J Proteome Res 9:275–282 [View Article][PubMed]
    [Google Scholar]
  39. Sung C. K., Li H., Claverys J. P., Morrison D. A. ( 2001). An rpsL cassette, Janus, for gene replacement through negative selection in Streptococcus pneumoniae . Appl Environ Microbiol 67:5190–5196 [View Article][PubMed]
    [Google Scholar]
  40. Tomasz A., Westphal M. ( 1971). Abnormal autolytic enzyme in a pneumococcus with altered teichoic acid composition. Proc Natl Acad Sci U S A 68:2627–2630 [View Article][PubMed]
    [Google Scholar]
  41. Trappetti C., Potter A. J., Paton A. W., Oggioni M. R., Paton J. C. ( 2011). LuxS mediates iron-dependent biofilm formation, competence, and fratricide in Streptococcus pneumoniae . Infect Immun 79:4550–4558 [View Article][PubMed]
    [Google Scholar]
  42. Whitmore S. E., Lamont R. J. ( 2012). Tyrosine phosphorylation and bacterial virulence. Int J Oral Sci 4:1–6 [View Article][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.080747-0
Loading
/content/journal/micro/10.1099/mic.0.080747-0
Loading

Data & Media loading...

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