#PAGE_PARAMS# #ADS_HEAD_SCRIPTS# #MICRODATA#

The phosphorelay BarA/SirA activates the non-cognate regulator RcsB in Salmonella enterica


Autoři: Hubert Salvail aff001;  Eduardo A. Groisman aff001
Působiště autorů: Department of Microbial Pathogenesis, Yale School of Medicine, New Haven, Connecticut, United States of America aff001;  Yale Microbial Sciences Institute, West Haven, Connecticut, United States of America aff002
Vyšlo v časopise: The phosphorelay BarA/SirA activates the non-cognate regulator RcsB in Salmonella enterica. PLoS Genet 16(5): e32767. doi:10.1371/journal.pgen.1008722
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.pgen.1008722

Souhrn

To survive an environmental stress, organisms must detect the stress and mount an appropriate response. One way that bacteria do so is by phosphorelay systems that respond to a stress by activating a regulator that modifies gene expression. To ensure an appropriate response, a given regulator is typically activated solely by its cognate phosphorelay protein(s). However, we now report that the regulator RcsB is activated by both cognate and non-cognate phosphorelay proteins, depending on the condition experienced by the bacterium Salmonella enterica serovar Typhimurium. The RcsC and RcsD proteins form a phosphorelay that activates their cognate regulator RcsB in response to outer membrane stress and cell wall perturbations, conditions Salmonella experiences during infection. Surprisingly, the non-cognate phosphorelay protein BarA activates RcsB during logarithmic growth in Luria-Bertani medium in three ways. That is, BarA’s cognate regulator SirA promotes transcription of the rcsDB operon; the SirA-dependent regulatory RNAs CsrB and CsrC further increase RcsB-activated gene transcription; and BarA activates RcsB independently of the RcsC, RcsD, and SirA proteins. Activation of a regulator by multiple sensors broadens the spectrum of environments in which a set of genes is expressed without evolving binding sites for different regulators at each of these genes.

Klíčová slova:

Gene expression – Outer membrane proteins – Phosphorylation – Plasmid construction – Prisms – Regulator genes – Salmonella – Isomorphous replacement


Zdroje

1. Papon N, Stock AM. Two-component systems. Curr Biol. 2019;29(15):R724–R5. Epub 2019/08/07. doi: 10.1016/j.cub.2019.06.010 31386843.

2. Jung K, Fried L, Behr S, Heermann R. Histidine kinases and response regulators in networks. Curr Opin Microbiol. 2012;15(2):118–24. Epub 2011/12/17. doi: 10.1016/j.mib.2011.11.009 22172627.

3. Majdalani N, Gottesman S. The Rcs phosphorelay: a complex signal transduction system. Annu Rev Microbiol. 2005;59:379–405. Epub 2005/09/13. doi: 10.1146/annurev.micro.59.050405.101230 16153174.

4. Cotter PA, Jones AM. Phosphorelay control of virulence gene expression in Bordetella. Trends Microbiol. 2003;11(8):367–73. Epub 2003/08/14. doi: 10.1016/s0966-842x(03)00156-2 12915094.

5. Pernestig AK, Melefors O, Georgellis D. Identification of UvrY as the cognate response regulator for the BarA sensor kinase in Escherichia coli. J Biol Chem. 2001;276(1):225–31. Epub 2000/10/07. doi: 10.1074/jbc.M001550200 11022030.

6. Capra EJ, Laub MT. Evolution of two-component signal transduction systems. Annu Rev Microbiol. 2012;66:325–47. Epub 2012/07/04. doi: 10.1146/annurev-micro-092611-150039 22746333; PubMed Central PMCID: PMC4097194.

7. Jacob-Dubuisson F, Mechaly A, Betton JM, Antoine R. Structural insights into the signalling mechanisms of two-component systems. Nat Rev Microbiol. 2018;16(10):585–93. Epub 2018/07/17. doi: 10.1038/s41579-018-0055-7 30008469.

8. Hoch JA, Varughese KI. Keeping signals straight in phosphorelay signal transduction. J Bacteriol. 2001;183(17):4941–9. Epub 2001/08/08. doi: 10.1128/JB.183.17.4941-4949.2001 11489844; PubMed Central PMCID: PMC95367.

9. Stephenson K, Hoch JA. Evolution of signalling in the sporulation phosphorelay. Mol Microbiol. 2002;46(2):297–304. Epub 2002/10/31. doi: 10.1046/j.1365-2958.2002.03186.x 12406209.

10. Wall E, Majdalani N, Gottesman S. The Complex Rcs Regulatory Cascade. Annu Rev Microbiol. 2018;72:111–39. Epub 2018/06/14. doi: 10.1146/annurev-micro-090817-062640 29897834.

11. Cho SH, Szewczyk J, Pesavento C, Zietek M, Banzhaf M, Roszczenko P, et al. Detecting envelope stress by monitoring beta-barrel assembly. Cell. 2014;159(7):1652–64. Epub 2014/12/20. doi: 10.1016/j.cell.2014.11.045 25525882.

12. Francez-Charlot A, Laugel B, Van Gemert A, Dubarry N, Wiorowski F, Castanie-Cornet MP, et al. RcsCDB His-Asp phosphorelay system negatively regulates the flhDC operon in Escherichia coli. Mol Microbiol. 2003;49(3):823–32. Epub 2003/07/17. doi: 10.1046/j.1365-2958.2003.03601.x 12864862.

13. Ferrieres L, Clarke DJ. The RcsC sensor kinase is required for normal biofilm formation in Escherichia coli K-12 and controls the expression of a regulon in response to growth on a solid surface. Mol Microbiol. 2003;50(5):1665–82. Epub 2003/12/04. doi: 10.1046/j.1365-2958.2003.03815.x 14651646.

14. Latasa C, Garcia B, Echeverz M, Toledo-Arana A, Valle J, Campoy S, et al. Salmonella biofilm development depends on the phosphorylation status of RcsB. J Bacteriol. 2012;194(14):3708–22. Epub 2012/05/15. doi: 10.1128/JB.00361-12 22582278; PubMed Central PMCID: PMC3393492.

15. Majdalani N, Hernandez D, Gottesman S. Regulation and mode of action of the second small RNA activator of RpoS translation, RprA. Mol Microbiol. 2002;46(3):813–26. Epub 2002/11/02. doi: 10.1046/j.1365-2958.2002.03203.x 12410838.

16. Pescaretti Mde L, Farizano JV, Morero R, Delgado MA. A novel insight on signal transduction mechanism of RcsCDB system in Salmonella enterica serovar typhimurium. PLoS One. 2013;8(9):e72527. Epub 2013/09/12. doi: 10.1371/journal.pone.0072527 24023746; PubMed Central PMCID: PMC3762810.

17. Kuhne C, Singer HM, Grabisch E, Codutti L, Carlomagno T, Scrima A, et al. RflM mediates target specificity of the RcsCDB phosphorelay system for transcriptional repression of flagellar synthesis in Salmonella enterica. Mol Microbiol. 2016;101(5):841–55. Epub 2016/05/21. doi: 10.1111/mmi.13427 27206164.

18. Pannen D, Fabisch M, Gausling L, Schnetz K. Interaction of the RcsB Response Regulator with Auxiliary Transcription Regulators in Escherichia coli. J Biol Chem. 2016;291(5):2357–70. Epub 2015/12/05. doi: 10.1074/jbc.M115.696815 26635367; PubMed Central PMCID: PMC4732218.

19. Papenfort K, Espinosa E, Casadesus J, Vogel J. Small RNA-based feedforward loop with AND-gate logic regulates extrachromosomal DNA transfer in Salmonella. Proc Natl Acad Sci U S A. 2015;112(34):E4772–81. Epub 2015/08/27. doi: 10.1073/pnas.1507825112 26307765; PubMed Central PMCID: PMC4553797.

20. Kroger 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. Epub 2013/12/18. doi: 10.1016/j.chom.2013.11.010 24331466.

21. Majdalani N, Heck M, Stout V, Gottesman S. Role of RcsF in signaling to the Rcs phosphorelay pathway in Escherichia coli. J Bacteriol. 2005;187(19):6770–8. Epub 2005/09/17. doi: 10.1128/JB.187.19.6770-6778.2005 16166540; PubMed Central PMCID: PMC1251585.

22. Farris C, Sanowar S, Bader MW, Pfuetzner R, Miller SI. Antimicrobial peptides activate the Rcs regulon through the outer membrane lipoprotein RcsF. J Bacteriol. 2010;192(19):4894–903. Epub 2010/08/03. doi: 10.1128/JB.00505-10 20675476; PubMed Central PMCID: PMC2944553.

23. Zere TR, Vakulskas CA, Leng Y, Pannuri A, Potts AH, Dias R, et al. Genomic Targets and Features of BarA-UvrY (-SirA) Signal Transduction Systems. PLoS One. 2015;10(12):e0145035. Epub 2015/12/18. doi: 10.1371/journal.pone.0145035 26673755; PubMed Central PMCID: PMC4682653.

24. Altier C, Suyemoto M, Ruiz AI, Burnham KD, Maurer R. Characterization of two novel regulatory genes affecting Salmonella invasion gene expression. Mol Microbiol. 2000;35(3):635–46. Epub 2000/02/15. doi: 10.1046/j.1365-2958.2000.01734.x 10672185.

25. Teplitski M, Goodier RI, Ahmer BM. Pathways leading from BarA/SirA to motility and virulence gene expression in Salmonella. J Bacteriol. 2003;185(24):7257–65. Epub 2003/12/04. doi: 10.1128/JB.185.24.7257-7265.2003 14645287; PubMed Central PMCID: PMC296259.

26. Palaniyandi S, Mitra A, Herren CD, Lockatell CV, Johnson DE, Zhu X, et al. BarA-UvrY two-component system regulates virulence of uropathogenic E. coli CFT073. PLoS One. 2012;7(2):e31348. Epub 2012/03/01. doi: 10.1371/journal.pone.0031348 22363626; PubMed Central PMCID: PMC3283629.

27. Herren CD, Mitra A, Palaniyandi SK, Coleman A, Elankumaran S, Mukhopadhyay S. The BarA-UvrY two-component system regulates virulence in avian pathogenic Escherichia coli O78:K80:H9. Infect Immun. 2006;74(8):4900–9. Epub 2006/07/25. doi: 10.1128/IAI.00412-06 16861679; PubMed Central PMCID: PMC1539585.

28. Wang Q, Zhao Y, McClelland M, Harshey RM. The RcsCDB signaling system and swarming motility in Salmonella enterica serovar typhimurium: dual regulation of flagellar and SPI-2 virulence genes. J Bacteriol. 2007;189(23):8447–57. Epub 2007/10/02. doi: 10.1128/JB.01198-07 17905992; PubMed Central PMCID: PMC2168921.

29. Holmqvist E, Wright PR, Li L, Bischler T, Barquist L, Reinhardt R, et al. Global RNA recognition patterns of post-transcriptional regulators Hfq and CsrA revealed by UV crosslinking in vivo. EMBO J. 2016;35(9):991–1011. Epub 2016/04/06. doi: 10.15252/embj.201593360 27044921; PubMed Central PMCID: PMC5207318.

30. Weilbacher T, Suzuki K, Dubey AK, Wang X, Gudapaty S, Morozov I, et al. A novel sRNA component of the carbon storage regulatory system of Escherichia coli. Mol Microbiol. 2003;48(3):657–70. Epub 2003/04/16. doi: 10.1046/j.1365-2958.2003.03459.x 12694612.

31. Vakulskas CA, Potts AH, Babitzke P, Ahmer BM, Romeo T. Regulation of bacterial virulence by Csr (Rsm) systems. Microbiol Mol Biol Rev. 2015;79(2):193–224. Epub 2015/04/03. doi: 10.1128/MMBR.00052-14 25833324; PubMed Central PMCID: PMC4394879.

32. Liu MY, Gui G, Wei B, Preston JF 3rd, Oakford L, Yuksel U, et al. The RNA molecule CsrB binds to the global regulatory protein CsrA and antagonizes its activity in Escherichia coli. J Biol Chem. 1997;272(28):17502–10. Epub 1997/07/11. doi: 10.1074/jbc.272.28.17502 9211896.

33. Schachterle JK, Stewart RM, Schachterle MB, Calder JT, Kang H, Prince JT, et al. Yersinia pseudotuberculosis BarA-UvrY Two-Component Regulatory System Represses Biofilms via CsrB. Front Cell Infect Microbiol. 2018;8:323. Epub 2018/10/04. doi: 10.3389/fcimb.2018.00323 30280093; PubMed Central PMCID: PMC6153318.

34. Pescaretti Mde L, Lopez FE, Morero RD, Delgado MA. Transcriptional autoregulation of the RcsCDB phosphorelay system in Salmonella enterica serovar Typhimurium. Microbiology. 2010;156(Pt 12):3513–21. Epub 2010/08/21. doi: 10.1099/mic.0.041319-0 20724387.

35. Kroger C, Dillon SC, Cameron AD, Papenfort K, Sivasankaran SK, Hokamp K, et al. The transcriptional landscape and small RNAs of Salmonella enterica serovar Typhimurium. Proc Natl Acad Sci U S A. 2012;109(20):E1277–86. Epub 2012/04/28. doi: 10.1073/pnas.1201061109 22538806; PubMed Central PMCID: PMC3356629.

36. Pescaretti Mde L, Morero R, Delgado MA. Identification of a new promoter for the response regulator rcsB expression in Salmonella enterica serovar Typhimurium. FEMS Microbiol Lett. 2009;300(2):165–73. Epub 2009/09/29. doi: 10.1111/j.1574-6968.2009.01771.x 19780840.

37. Fredericks CE, Shibata S, Aizawa S, Reimann SA, Wolfe AJ. Acetyl phosphate-sensitive regulation of flagellar biogenesis and capsular biosynthesis depends on the Rcs phosphorelay. Mol Microbiol. 2006;61(3):734–47. Epub 2006/06/17. doi: 10.1111/j.1365-2958.2006.05260.x 16776655.

38. Hu LI, Chi BK, Kuhn ML, Filippova EV, Walker-Peddakotla AJ, Basell K, et al. Acetylation of the response regulator RcsB controls transcription from a small RNA promoter. J Bacteriol. 2013;195(18):4174–86. Epub 2013/07/16. doi: 10.1128/JB.00383-13 23852870; PubMed Central PMCID: PMC3754749.

39. Laubacher ME, Ades SE. The Rcs phosphorelay is a cell envelope stress response activated by peptidoglycan stress and contributes to intrinsic antibiotic resistance. J Bacteriol. 2008;190(6):2065–74. Epub 2008/01/15. doi: 10.1128/JB.01740-07 18192383; PubMed Central PMCID: PMC2258881.

40. Huang YH, Ferrieres L, Clarke DJ. Comparative functional analysis of the RcsC sensor kinase from different Enterobacteriaceae. FEMS Microbiol Lett. 2009;293(2):248–54. Epub 2009/03/06. doi: 10.1111/j.1574-6968.2009.01543.x 19260968.

41. Romeo T, Babitzke P. Global Regulation by CsrA and Its RNA Antagonists. Microbiol Spectr. 2018;6(2). Epub 2018/03/25. doi: 10.1128/microbiolspec.RWR-0009-2017 29573256; PubMed Central PMCID: PMC5868435.

42. Babitzke P, Romeo T. CsrB sRNA family: sequestration of RNA-binding regulatory proteins. Curr Opin Microbiol. 2007;10(2):156–63. Epub 2007/03/27. doi: 10.1016/j.mib.2007.03.007 17383221.

43. Dubey AK, Baker CS, Romeo T, Babitzke P. RNA sequence and secondary structure participate in high-affinity CsrA-RNA interaction. RNA. 2005;11(10):1579–87. Epub 2005/09/01. doi: 10.1261/rna.2990205 16131593; PubMed Central PMCID: PMC1370842.

44. Potts AH, Guo Y, Ahmer BMM, Romeo T. Role of CsrA in stress responses and metabolism important for Salmonella virulence revealed by integrated transcriptomics. PLoS One. 2019;14(1):e0211430. Epub 2019/01/27. doi: 10.1371/journal.pone.0211430 30682134; PubMed Central PMCID: PMC6347204.

45. Potts AH, Vakulskas CA, Pannuri A, Yakhnin H, Babitzke P, Romeo T. Global role of the bacterial post-transcriptional regulator CsrA revealed by integrated transcriptomics. Nat Commun. 2017;8(1):1596. Epub 2017/11/19. doi: 10.1038/s41467-017-01613-1 29150605; PubMed Central PMCID: PMC5694010.

46. Cai SJ, Inouye M. EnvZ-OmpR interaction and osmoregulation in Escherichia coli. J Biol Chem. 2002;277(27):24155–61. Epub 2002/04/26. doi: 10.1074/jbc.M110715200 11973328.

47. Matsubara M, Kitaoka SI, Takeda SI, Mizuno T. Tuning of the porin expression under anaerobic growth conditions by his-to-Asp cross-phosphorelay through both the EnvZ-osmosensor and ArcB-anaerosensor in Escherichia coli. Genes Cells. 2000;5(7):555–69. Epub 2000/08/18. doi: 10.1046/j.1365-2443.2000.00347.x 10947842.

48. Desroy N, Denis A, Oliveira C, Atamanyuk D, Briet S, Faivre F, et al. Novel HldE-K inhibitors leading to attenuated Gram negative bacterial virulence. J Med Chem. 2013;56(4):1418–30. Epub 2013/02/16. doi: 10.1021/jm301499r 23409840.

49. Iuchi S, Matsuda Z, Fujiwara T, Lin EC. The arcB gene of Escherichia coli encodes a sensor-regulator protein for anaerobic repression of the arc modulon. Mol Microbiol. 1990;4(5):715–27. Epub 1990/05/01. doi: 10.1111/j.1365-2958.1990.tb00642.x 2201868.

50. Iuchi S, Lin EC. arcA (dye), a global regulatory gene in Escherichia coli mediating repression of enzymes in aerobic pathways. Proc Natl Acad Sci U S A. 1988;85(6):1888–92. Epub 1988/03/01. doi: 10.1073/pnas.85.6.1888 2964639; PubMed Central PMCID: PMC279886.

51. Yamamoto K, Hirao K, Oshima T, Aiba H, Utsumi R, Ishihama A. Functional characterization in vitro of all two-component signal transduction systems from Escherichia coli. J Biol Chem. 2005;280(2):1448–56. Epub 2004/11/04. doi: 10.1074/jbc.M410104200 15522865.

52. Skerker JM, Prasol MS, Perchuk BS, Biondi EG, Laub MT. Two-component signal transduction pathways regulating growth and cell cycle progression in a bacterium: a system-level analysis. PLoS Biol. 2005;3(10):e334. Epub 2005/09/24. doi: 10.1371/journal.pbio.0030334 16176121; PubMed Central PMCID: PMC1233412.

53. Groisman EA. Feedback Control of Two-Component Regulatory Systems. Annu Rev Microbiol. 2016;70:103–24. Epub 2016/09/09. doi: 10.1146/annurev-micro-102215-095331 27607549.

54. Garmendia J, Beuzon CR, Ruiz-Albert J, Holden DW. The roles of SsrA-SsrB and OmpR-EnvZ in the regulation of genes encoding the Salmonella typhimurium SPI-2 type III secretion system. Microbiology. 2003;149(Pt 9):2385–96. Epub 2003/09/02. doi: 10.1099/mic.0.26397-0 12949164.

55. Datsenko KA, Wanner BL. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A. 2000;97(12):6640–5. Epub 2000/06/01. doi: 10.1073/pnas.120163297 10829079; PubMed Central PMCID: PMC18686.

56. Watanabe T, Ogata Y, Chan RK, Botstein D. Specialized transduction of tetracycline resistance by phage P22 in Salmonella typhimurium. I. Transduction of R factor 222 by phage P22. Virology. 1972;50(3):874–82. Epub 1972/12/01. doi: 10.1016/0042-6822(72)90441-2 4565617.

57. Khetrapal V, Mehershahi K, Rafee S, Chen S, Lim CL, Chen SL. A set of powerful negative selection systems for unmodified Enterobacteriaceae. Nucleic Acids Res. 2015;43(13):e83. Epub 2015/03/25. doi: 10.1093/nar/gkv248 25800749; PubMed Central PMCID: PMC4513841.

58. 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. Epub 2013/07/06. doi: 10.1016/j.cell.2013.06.004 23827679; PubMed Central PMCID: PMC3736803.

59. Valdivia RH, Falkow S. Bacterial genetics by flow cytometry: rapid isolation of Salmonella typhimurium acid-inducible promoters by differential fluorescence induction. Mol Microbiol. 1996;22(2):367–78. Epub 1996/10/01. doi: 10.1046/j.1365-2958.1996.00120.x 8930920.

60. Chang AC, Cohen SN. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978;134(3):1141–56. Epub 1978/06/01. 149110; PubMed Central PMCID: PMC222365.

61. Way JC, Davis MA, Morisato D, Roberts DE, Kleckner N. New Tn10 derivatives for transposon mutagenesis and for construction of lacZ operon fusions by transposition. Gene. 1984;32(3):369–79. Epub 1984/12/01. doi: 10.1016/0378-1119(84)90012-x 6099322.

62. Chun KT, Edenberg HJ, Kelley MR, Goebl MG. Rapid amplification of uncharacterized transposon-tagged DNA sequences from genomic DNA. Yeast. 1997;13(3):233–40. Epub 1997/03/15. doi: 10.1002/(SICI)1097-0061(19970315)13:3<233::AID-YEA88>3.0.CO;2-E 9090052.

63. Choi J, Groisman EA. Activation of master virulence regulator PhoP in acidic pH requires the Salmonella-specific protein UgtL. Sci Signal. 2017;10(494). Epub 2017/08/31. doi: 10.1126/scisignal.aan6284 28851823; PubMed Central PMCID: PMC5966036.

64. Barbieri CM, Stock AM. Universally applicable methods for monitoring response regulator aspartate phosphorylation both in vitro and in vivo using Phos-tag-based reagents. Anal Biochem. 2008;376(1):73–82. Epub 2008/03/11. doi: 10.1016/j.ab.2008.02.004 18328252; PubMed Central PMCID: PMC2504525.

65. Aiba H, Adhya S, de Crombrugghe B. Evidence for two functional gal promoters in intact Escherichia coli cells. J Biol Chem. 1981;256(22):11905–10. Epub 1981/11/25. 6271763.

66. Prevost K, Salvail H, Desnoyers G, Jacques JF, Phaneuf E, Masse E. The small RNA RyhB activates the translation of shiA mRNA encoding a permease of shikimate, a compound involved in siderophore synthesis. Mol Microbiol. 2007;64(5):1260–73. Epub 2007/06/05. doi: 10.1111/j.1365-2958.2007.05733.x 17542919.

67. Mouslim C, Delgado M, Groisman EA. Activation of the RcsC/YojN/RcsB phosphorelay system attenuates Salmonella virulence. Mol Microbiol. 2004;54(2):386–95. Epub 2004/10/08. doi: 10.1111/j.1365-2958.2004.04293.x 15469511.


Článek vyšel v časopise

PLOS Genetics


2020 Číslo 5
Nejčtenější tento týden
Nejčtenější v tomto čísle
Kurzy

Zvyšte si kvalifikaci online z pohodlí domova

Důležitost adherence při depresivním onemocnění
nový kurz
Autoři: MUDr. Eliška Bartečková, Ph.D.

Koncepce osteologické péče pro gynekology a praktické lékaře
Autoři: MUDr. František Šenk

Sekvenční léčba schizofrenie
Autoři: MUDr. Jana Hořínková, Ph.D.

Hypertenze a hypercholesterolémie – synergický efekt léčby
Autoři: prof. MUDr. Hana Rosolová, DrSc.

Multidisciplinární zkušenosti u pacientů s diabetem
Autoři: Prof. MUDr. Martin Haluzík, DrSc., prof. MUDr. Vojtěch Melenovský, CSc., prof. MUDr. Vladimír Tesař, DrSc.

Všechny kurzy
Přihlášení
Zapomenuté heslo

Zadejte e-mailovou adresu, se kterou jste vytvářel(a) účet, budou Vám na ni zaslány informace k nastavení nového hesla.

Přihlášení

Nemáte účet?  Registrujte se

#ADS_BOTTOM_SCRIPTS#