Phosphoproteomic analysis of the elementary body and reticulate body forms Free

Abstract

are Gram-negative, obligate intracellular bacteria responsible for significant diseases in humans and economically important domestic animals. These pathogens undergo a unique biphasic developmental cycle transitioning between the environmentally stable elementary body (EB) and the replicative intracellular reticulate body (RB), a conversion that appears to require extensive regulation of protein synthesis and function. However, possess a limited number of canonical mechanisms of transcriptional regulation. Ser/Thr/Tyr phosphorylation of proteins in bacteria has been increasingly recognized as an important mechanism of post-translational control of protein function. We utilized 2D gel electrophoresis coupled with phosphoprotein staining and MALDI-TOF/TOF analysis to map the phosphoproteome of the EB and RB forms of Forty-two non-redundant phosphorylated proteins were identified (some proteins were present in multiple locations within the gels). Thirty-four phosphorylated proteins were identified in EBs, including proteins found in central metabolism and protein synthesis, -specific hypothetical proteins and virulence-related proteins. Eleven phosphorylated proteins were identified in RBs, mostly involved in protein synthesis and folding and a single virulence-related protein. Only three phosphoproteins were found in both EB and RB phosphoproteomes. Collectively, 41 of 42 phosphoproteins were present across species, consistent with the existence of a conserved chlamydial phosphoproteome. The abundance of stage-specific phosphoproteins suggests that protein phosphorylation may play a role in regulating the function of developmental-stage-specific proteins and/or may function in concert with other factors in directing EB–RB transitions.

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2015-08-01
2024-03-29
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References

  1. AbdelRahman Y.M., Belland R.J. 2005; The chlamydial developmental cycle. FEMS Microbiol Rev 29:949–959 [View Article][PubMed]
    [Google Scholar]
  2. Archambaud C., Gouin E., Pizarro-Cerda J., Cossart P., Dussurget O. 2005; Translation elongation factor EF-Tu is a target for Stp, a serine-threonine phosphatase involved in virulence of Listeria monocytogenes . Mol Microbiol 56:383–396 [View Article][PubMed]
    [Google Scholar]
  3. Attwood P.V., Piggott M.J., Zu X.L., Besant P.G. 2007; Focus on phosphohistidine. Amino Acids 32:145–156 [View Article][PubMed]
    [Google Scholar]
  4. Bastidas R.J., Elwell C.A., Engel J.N., Valdivia R.H. 2013; Chlamydial intracellular survival strategies. Cold Spring Harb Perspect Med 3:a010256 [View Article][PubMed]
    [Google Scholar]
  5. Belland R.J., Zhong G., Crane D.D., Hogan D., Sturdevant D., Sharma J., Beatty W.L., Caldwell H.D. 2003; Genomic transcriptional profiling of the developmental cycle of Chlamydia trachomatis . Proc Natl Acad Sci U S A 100:8478–8483 [View Article][PubMed]
    [Google Scholar]
  6. Binet R., Bowlin A.K., Maurelli A.T., Rank R.G. 2010; Impact of azithromycin resistance mutations on the virulence and fitness of Chlamydia caviae in guinea pigs. Antimicrob Agents Chemother 54:1094–1101 [View Article][PubMed]
    [Google Scholar]
  7. Clifton D.R., Fields K.A., Grieshaber S.S., Dooley C.A., Fischer E.R., Mead D.J., Carabeo R.A., Hackstadt T. 2004; A chlamydial type III translocated protein is tyrosine-phosphorylated at the site of entry and associated with recruitment of actin. Proc Natl Acad Sci U S A 101:10166–10171 [View Article][PubMed]
    [Google Scholar]
  8. Dautry-Varsat A., Subtil A., Hackstadt T. 2005; Recent insights into the mechanisms of Chlamydia entry. Cell Microbiol 7:1714–1722[PubMed]
    [Google Scholar]
  9. Dworkin J. 2015; Ser/Thr phosphorylation as a regulatory mechanism in bacteria. Curr Opin Microbiol 24:47–52 [View Article][PubMed]
    [Google Scholar]
  10. Fiuza M., Canova M.J., Patin D., Letek M., Zanella-Cléon I., Becchi M., Mateos L.M., Mengin-Lecreulx D., Molle V., Gil J.A. 2008; The MurC ligase essential for peptidoglycan biosynthesis is regulated by the serine/threonine protein kinase PknA in Corynebacterium glutamicum . J Biol Chem 283:36553–36563 [View Article][PubMed]
    [Google Scholar]
  11. Gnad F., Forner F., Zielinska D.F., Birney E., Gunawardena J., Mann M. 2010; Evolutionary constraints of phosphorylation in eukaryotes, prokaryotes, and mitochondria. Mol Cell Proteomics 9:2642–2653 [View Article][PubMed]
    [Google Scholar]
  12. Hatch T.P., Allan I., Pearce J.H. 1984; Structural and polypeptide differences between envelopes of infective and reproductive life cycle forms of Chlamydia spp. J Bacteriol 157:13–20[PubMed]
    [Google Scholar]
  13. Horn M. 2008; Chlamydiae as symbionts in eukaryotes. Annu Rev Microbiol 62:113–131 [View Article][PubMed]
    [Google Scholar]
  14. Horn M., Collingro A., Schmitz-Esser S., Beier C.L., Purkhold U., Fartmann B., Brandt P., Nyakatura G.J., Droege M., other authors. 2004; Illuminating the evolutionary history of chlamydiae. Science 304:728–730 [CrossRef]
    [Google Scholar]
  15. Hua L., Hefty P.S., Lee Y.J., Lee Y.M., Stephens R.S., Price C.W. 2006; Core of the partner switching signalling mechanism is conserved in the obligate intracellular pathogen Chlamydia trachomatis . Mol Microbiol 59:623–636 [View Article][PubMed]
    [Google Scholar]
  16. Jers C., Pedersen M.M., Paspaliari D.K., Schütz W., Johnsson C., Soufi B., Macek B., Jensen P.R., Mijakovic I. 2010; Bacillus subtilis BY-kinase PtkA controls enzyme activity and localization of its protein substrates. Mol Microbiol 77:287–299 [View Article][PubMed]
    [Google Scholar]
  17. Johnson D.L., Mahony J.B. 2007; Chlamydophila pneumoniae PknD exhibits dual amino acid specificity and phosphorylates Cpn0712, a putative type III secretion YscD homolog. J Bacteriol 189:7549–7555 [View Article][PubMed]
    [Google Scholar]
  18. Johnson D.L., Stone C.B., Bulir D.C., Coombes B.K., Mahony J.B. 2009; A novel inhibitor of Chlamydophila pneumoniae protein kinase D (PknD) inhibits phosphorylation of CdsD and suppresses bacterial replication. BMC Microbiol 9:218 [View Article][PubMed]
    [Google Scholar]
  19. Kalman S., Mitchell W., Marathe R., Lammel C., Fan J., Hyman R.W., Olinger L., Grimwood J., Davis R.W., Stephens R.S. 1999; Comparative genomes of Chlamydia pneumoniae C. trachomatis . Nat Genet 21:385–389 [View Article][PubMed]
    [Google Scholar]
  20. Koo I.C., Stephens R.S. 2003; A developmentally regulated two-component signal transduction system in Chlamydia . J Biol Chem 278:17314–17319 [View Article][PubMed]
    [Google Scholar]
  21. Kumar C.M., Khare G., Srikanth C.V., Tyagi A.K., Sardesai A.A., Mande S.C. 2009; Facilitated oligomerization of mycobacterial GroEL: evidence for phosphorylation-mediated oligomerization. J Bacteriol 191:6525–6538 [View Article][PubMed]
    [Google Scholar]
  22. Lundemose A.G., Birkelund S., Larsen P.M., Fey S.J., Christiansen G. 1990; Characterization and identification of early proteins in Chlamydia trachomatis serovar L2 by two-dimensional gel electrophoresis. Infect Immun 58:2478–2486[PubMed]
    [Google Scholar]
  23. Matsumoto A., Izutsu H., Miyashita N., Ohuchi M. 1998; Plaque formation by and plaque cloning of Chlamydia trachomatis biovar trachoma. J Clin Microbiol 36:3013–3019[PubMed]
    [Google Scholar]
  24. Mäurer A.P., Mehlitz A., Mollenkopf H.J., Meyer T.F. 2007; Gene expression profiles of Chlamydophila pneumoniae during the developmental cycle and iron depletion-mediated persistence. PLoS Pathog 3:e83 [View Article][PubMed]
    [Google Scholar]
  25. Mijakovic I., Macek B. 2012; Impact of phosphoproteomics on studies of bacterial physiology. FEMS Microbiol Rev 36:877–892 [View Article][PubMed]
    [Google Scholar]
  26. Mukhopadhyay S., Miller R.D., Summersgill J.T. 2004; Analysis of altered protein expression patterns of Chlamydia pneumoniae by an integrated proteome-works system. J Proteome Res 3:878–883 [View Article][PubMed]
    [Google Scholar]
  27. Mukhopadhyay S., Miller R.D., Sullivan E.D., Theodoropoulos C., Mathews S.A., Timms P., Summersgill J.T. 2006a; Protein expression profiles of Chlamydia pneumoniae in models of persistence versus those of heat shock stress response. Infect Immun 74:3853–3863 [View Article][PubMed]
    [Google Scholar]
  28. Mukhopadhyay S., Good D., Miller R.D., Graham J.E., Mathews S.A., Timms P., Summersgill J.T. 2006b; Identification of Chlamydia pneumoniae proteins in the transition from reticulate to elementary body formation. Mol Cell Proteomics 5:2311–2318 [View Article][PubMed]
    [Google Scholar]
  29. Nicholson T.L., Olinger L., Chong K., Schoolnik G., Stephens R.S. 2003; Global stage-specific gene regulation during the developmental cycle of Chlamydia trachomatis . J Bacteriol 185:3179–3189 [View Article][PubMed]
    [Google Scholar]
  30. Olsen J.V., Vermeulen M., Santamaria A., Kumar C., Miller M.L., Jensen L.J., Gnad F., Cox J., Jensen T.S., other authors. 2010; Quantitative phosphoproteomics reveals widespread full phosphorylation site occupancy during mitosis. Sci Signal 3:ra3[PubMed] [CrossRef]
    [Google Scholar]
  31. Parikh A., Verma S.K., Khan S., Prakash B., Nandicoori V.K. 2009; PknB-mediated phosphorylation of a novel substrate, N-acetylglucosamine-1-phosphate uridyltransferase, modulates its acetyltransferase activity. J Mol Biol 386:451–464 [View Article][PubMed]
    [Google Scholar]
  32. Peters J., Wilson D.P., Myers G., Timms P., Bavoil P.M. 2007; Type III secretion à la Chlamydia . Trends Microbiol 15:241–251 [View Article][PubMed]
    [Google Scholar]
  33. Rank R.G., Hough A.J., Jr, Jacobs R.F., Cohen C., Barron A.L. 1985; Chlamydial pneumonitis induced in newborn guinea pigs. Infect Immun 48:153–158[PubMed]
    [Google Scholar]
  34. Rank R.G., Sanders M.M., Patton D.L. 1995; Increased incidence of oviduct pathology in the guinea pig after repeat vaginal inoculation with the chlamydial agent of guinea pig inclusion conjunctivitis. Sex Transm Dis 22:48–54 [View Article][PubMed]
    [Google Scholar]
  35. Rank R.G., Bowlin A.K., Reed R.L., Darville T. 2003; Characterization of chlamydial genital infection resulting from sexual transmission from male to female guinea pigs and determination of infectious dose. Infect Immun 71:6148–6154 [View Article][PubMed]
    [Google Scholar]
  36. Read T.D., Myers G.S., Brunham R.C., Nelson W.C., Paulsen I.T., Heidelberg J., Holtzapple E., Khouri H., Federova N.B., other authors. 2003; Genome sequence of Chlamydophila caviae (Chlamydia psittaci GPIC): examining the role of niche-specific genes in the evolution of the Chlamydiaceae . Nucleic Acids Res 31:2134–2147 [View Article][PubMed]
    [Google Scholar]
  37. Rockey D.D., Grosenbach D., Hruby D.E., Peacock M.G., Heinzen R.A., Hackstadt T. 1997; Chlamydia psittaci IncA is phosphorylated by the host cell and is exposed on the cytoplasmic face of the developing inclusion. Mol Microbiol 24:217–228 [View Article][PubMed]
    [Google Scholar]
  38. Saka H.A., Thompson J.W., Chen Y.S., Kumar Y., Dubois L.G., Moseley M.A., Valdivia R.H. 2011; Quantitative proteomics reveals metabolic and pathogenic properties of Chlamydia trachomatis developmental forms. Mol Microbiol 82:1185–1203 [View Article][PubMed]
    [Google Scholar]
  39. Shaw A.C., Christiansen G., Birkelund S. 1999; Effects of interferon gamma on Chlamydia trachomatis serovar A and L2 protein expression investigated by two-dimensional gel electrophoresis. Electrophoresis 20:775–780 [View Article][PubMed]
    [Google Scholar]
  40. Shaw A.C., Gevaert K., Demol H., Hoorelbeke B., Vandekerckhove J., Larsen M.R., Roepstorff P., Holm A., Christiansen G., Birkelund S. 2002; Comparative proteome analysis of Chlamydia trachomatis serovar A, D and L2. Proteomics 2:164–186 [View Article][PubMed]
    [Google Scholar]
  41. Skipp P., Robinson J., O'Connor C.D., Clarke I.N. 2005; Shotgun proteomic analysis of Chlamydia trachomatis . Proteomics 5:1558–1573 [View Article][PubMed]
    [Google Scholar]
  42. Stülke J. 2010; More than just activity control: phosphorylation may control all aspects of a protein's properties. Mol Microbiol 77:273–275 [View Article][PubMed]
    [Google Scholar]
  43. Vandahl B.B., Birkelund S., Demol H., Hoorelbeke B., Christiansen G., Vandekerckhove J., Gevaert K. 2001; Proteome analysis of the Chlamydia pneumoniae elementary body. Electrophoresis 22:1204–1223 [View Article][PubMed]
    [Google Scholar]
  44. Vandahl B.B., Birkelund S., Christiansen G. 2002; Proteome analysis of Chlamydia pneumoniae . Methods Enzymol 358:277–288[PubMed] [CrossRef]
    [Google Scholar]
  45. Verma A., Maurelli A.T. 2003; Identification of two eukaryote-like serine/threonine kinases encoded by Chlamydia trachomatis serovar L2 and characterization of interacting partners of Pkn1. Infect Immun 71:5772–5784 [View Article][PubMed]
    [Google Scholar]
  46. Zhao X., León I.R., Bak S., Mogensen M., Wrzesinski K., Højlund K., Jensen O.N. 2011; Phosphoproteome analysis of functional mitochondria isolated from resting human muscle reveals extensive phosphorylation of inner membrane protein complexes and enzymes. Mol Cell Proteomics 10:M110000299 [View Article][PubMed]
    [Google Scholar]
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