#PAGE_PARAMS# #ADS_HEAD_SCRIPTS# #MICRODATA#

Lactobacillus rhamnosus Lcr35 as an effective treatment for preventing Candida albicans infection in the invertebrate model Caenorhabditis elegans: First mechanistic insights


Autoři: Cyril Poupet aff001;  Taous Saraoui aff001;  Philippe Veisseire aff001;  Muriel Bonnet aff001;  Caroline Dausset aff002;  Marylise Gachinat aff001;  Olivier Camarès aff001;  Christophe Chassard aff001;  Adrien Nivoliez aff002;  Stéphanie Bornes aff001
Působiště autorů: Université Clermont Auvergne, INRA, VetAgro Sup, Aurillac, France aff001;  Biose Industrie, Aurillac, France aff002
Vyšlo v časopise: PLoS ONE 14(11)
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.pone.0216184

Souhrn

The increased recurrence of Candida albicans infections is associated with greater resistance to antifungal drugs. This involves the establishment of alternative therapeutic protocols, such as probiotic microorganisms whose antifungal potential has already been demonstrated using preclinical models (cell cultures, laboratory animals). Understanding the mechanisms of action of probiotic microorganisms has become a strategic need for the development of new therapeutics for humans. In this study, we investigated the prophylactic anti-C. albicans properties of Lactobacillus rhamnosus Lcr35® using the in vitro Caco-2 cell model and the in vivo Caenorhabditis elegans model. In Caco-2 cells, we showed that the strain Lcr35® significantly inhibited the growth (~2 log CFU.mL-1) and adhesion (150 to 6,300 times less) of the pathogen. Moreover, in addition to having a pro-longevity activity in the nematode (+42.9%, p = 3.56.10−6), Lcr35® protects the animal from the fungal infection (+267% of survival, p < 2.10−16) even if the yeast is still detectable in its intestine. At the mechanistic level, we noticed the repression of genes of the p38 MAPK signalling pathway and genes involved in the antifungal response induced by Lcr35®, suggesting that the pathogen no longer appears to be detected by the worm immune system. However, the DAF-16/FOXO transcription factor, implicated in the longevity and antipathogenic response of C. elegans, is activated by Lcr35®. These results suggest that the probiotic strain acts by stimulating its host via DAF-16 but also by suppressing the virulence of the pathogen.

Klíčová slova:

Caco-2 cells – Caenorhabditis elegans – Candida albicans – Fungal pathogens – Gastrointestinal tract – Nematode infections – Probiotics – Yeast infections


Zdroje

1. Cauchie M, Desmet S, Lagrou K. Candida and its dual lifestyle as a commensal and a pathogen. Res Microbiol [Internet]. 2017 Nov [cited 2018 Sep 5];168(9–10):802–10. Available from: https://linkinghub.elsevier.com/retrieve/pii/S0923250817300402 28263903

2. Neville BA, D’enfert C, Bougnoux M-E. Candida albicans commensalism in the gastrointestinal tract. FEMS Yeast Res [Internet]. 2015 [cited 2018 Sep 5];15:81. Available from: https://unite.ut.ee/

3. Mayer FL, Wilson D, Hube B. Candida albicans pathogenicity mechanisms. Vol. 4, Virulence. 2013. p. 119–28.

4. Kadosh D, Antonio S. Control of Candida albicans morphology and pathogenicity by post-transcriptional mechanisms. Cell Mol Life Sci. 2017;73(22):4265–78.

5. Wächtler B, Wilson D, Haedicke K, Dalle F, Hube B. From attachment to damage: Defined genes of Candida albicans mediate adhesion, invasion and damage during interaction with oral epithelial cells. Munro C, editor. PLoS One [Internet]. 2011 Feb 23 [cited 2018 Sep 10];6(2):e17046. Available from: http://www.ncbi.nlm.nih.gov/pubmed/21407800 21407800

6. Farmakiotis D, Kontoyiannis DP. Epidemiology of antifungal resistance in human pathogenic yeasts: current viewpoint and practical recommendations for management. Int J Antimicrob Agents [Internet]. 2017 Sep [cited 2018 Sep 5];50(3):318–24. Available from: https://linkinghub.elsevier.com/retrieve/pii/S0924857917302364 28669831

7. Sanguinetti M, Posteraro B, Lass-Flörl C. Antifungal drug resistance among Candida species: Mechanisms and clinical impact. Mycoses [Internet]. 2015 Jun [cited 2018 Sep 5];58(S2):2–13. Available from: http://www.ncbi.nlm.nih.gov/pubmed/26033251

8. Scorzoni L, de Paula E Silva ACA, Marcos CM, Assato PA, de Melo WCMA, de Oliveira HC, et al. Antifungal Therapy: New Advances in the Understanding and Treatment of Mycosis. Front Microbiol [Internet]. 2017 [cited 2018 Sep 5];8:36. Available from: http://www.ncbi.nlm.nih.gov/pubmed/28167935 28167935

9. Wheeler ML, Limon JJ, Bar AS, Leal CA, Gargus M, Tang J, et al. Immunological Consequences of Intestinal Fungal Dysbiosis. Cell Host Microbe [Internet]. 2016;19(6):865–73. Available from: http://dx.doi.org/10.1016/j.chom.2016.05.003 27237365

10. Hu H-J, Zhang G-Q, Zhang Q, Shakya S, Li Z-Y. Probiotics Prevent Candida Colonization and Invasive Fungal Sepsis in Preterm Neonates: A Systematic Review and Meta-Analysis of Randomized Controlled Trials. Pediatr Neonatol [Internet]. 2017 Apr [cited 2018 Sep 5];58(2):103–10. Available from: http://linkinghub.elsevier.com/retrieve/pii/S1875957216301401 27793494

11. Matsubara VH, Bandara HMHN, Mayer MPA, Samaranayake LP. Probiotics as Antifungals in Mucosal Candidiasis. 2016 [cited 2018 Sep 5]; https://academic.oup.com/cid/article-abstract/62/9/1143/1745140

12. Agrawal S, Rao S, Patole S. Probiotic supplementation for preventing invasive fungal infections in preterm neonates—a systematic review and meta-analysis. Mycoses [Internet]. 2015 Nov 1 [cited 2018 Sep 5];58(11):642–51. Available from: http://doi.wiley.com/10.1111/myc.12368 26468692

13. FAO, WHO. Health and Nutritional Properties of Probiotics in Food including Powder Milk with Live Lactic Acid Bacteria. Food Nutr Pap [Internet]. 2001 [cited 2016 Jun 14]; http://www.crcnetbase.com/doi/abs/10.1201/9781420009613.ch16

14. Fijan S. Microorganisms with Claimed Probiotic Properties: An Overview of Recent Literature. Int J Environ Res Public Heal Int J Environ Res Public Heal Int J Environ Res Public Heal [Internet]. 2014 [cited 2017 May 13];11:4745–67. Available from: www.mdpi.com/journal/ijerph

15. Olle B. Medicines from microbiota. Nat Biotechnol [Internet]. 2013 Apr 5 [cited 2017 Mar 31];31(4):309–15. Available from: http://www.nature.com/doifinder/10.1038/nbt.2548 23563425

16. Coudeyras S, Jugie G, Vermerie M, Forestier C. Adhesion of human probiotic Lactobacillus rhamnosus to cervical and vaginal cells and interaction with vaginosis-associated pathogens. Infect Dis Obstet Gynecol [Internet]. 2008 Jan 27 [cited 2018 Sep 10];2008:549640. Available from: http://www.ncbi.nlm.nih.gov/pubmed/19190778 19190778

17. Coudeyras S, Marchandin H, Fajon C, Forestier C. Taxonomic and strain-specific identification of the probiotic strain Lactobacillus rhamnosus 35 within the Lactobacillus casei group. Appl Environ Microbiol [Internet]. 2008 May [cited 2018 Sep 10];74(9):2679–89. Available from: http://www.ncbi.nlm.nih.gov/pubmed/18326671 18326671

18. Forestier C, De Champs C, Vatoux C, Joly B. Probiotic activities of Lactobacillus casei rhamnosus: in vitro adherence to intestinal cells and antimicrobial properties. Res Microbiol [Internet]. 2001 Mar [cited 2018 Sep 10];152(2):167–73. Available from: http://www.ncbi.nlm.nih.gov/pubmed/11316370 11316370

19. de Champs C, Maroncle N, Balestrino D, Rich C, Forestier C. Persistence of colonization of intestinal mucosa by a probiotic strain, Lactobacillus casei subsp. rhamnosus Lcr35, after oral consumption. J Clin Microbiol [Internet]. 2003 Mar [cited 2018 Sep 10];41(3):1270–3. Available from: http://www.ncbi.nlm.nih.gov/pubmed/12624065 12624065

20. Petricevic L, Witt A. The role of Lactobacillus casei rhamnosus Lcr35 in restoring the normal vaginal flora after antibiotic treatment of bacterial vaginosis. BJOG An Int J Obstet Gynaecol [Internet]. 2008 Oct [cited 2016 Jun 14];115(11):1369–74. Available from: http://doi.wiley.com/10.1111/j.1471-0528.2008.01882.x

21. Muller C, Mazel V, Dausset C, Busignies V, Bornes S, Nivoliez A, et al. Study of the Lactobacillus rhamnosus Lcr35® properties after compression and proposition of a model to predict tablet stability. Eur J Pharm Biopharm. 2014;88(3):787–94. doi: 10.1016/j.ejpb.2014.07.014 25128853

22. Nivoliez A, Veisseire P, Alaterre E, Dausset C, Baptiste F, Camarès O, et al. Influence of manufacturing processes on cell surface properties of probiotic strain Lactobacillus rhamnosus Lcr35®. Appl Microbiol Biotechnol [Internet]. 2015 [cited 2017 Jan 1];99(1):399–411. Available from: http://link.springer.com/10.1007/s00253-014-6110-z 25280746

23. Dausset C, Patrier S, Gajer P, Thoral C, Lenglet Y, Cardot JM, et al. Comparative phase I randomized open-label pilot clinical trial of Gynophilus® (Lcr regenerans®) immediate release capsules versus slow release muco-adhesive tablets. Eur J Clin Microbiol Infect Dis [Internet]. 2018 [cited 2019 Apr 2];37(10):1869–80. Available from: https://doi.org/10.1007/s10096-018-3321-8 30032443

24. Nivoliez A, Camares O, Paquet-Gachinat M, Bornes S, Forestier C, Veisseire P. Influence of manufacturing processes on in vitro properties of the probiotic strain Lactobacillus rhamnosus Lcr35®. J Biotechnol. 2012;160(3–4):236–41. doi: 10.1016/j.jbiotec.2012.04.005 22542933

25. Isolauri E, Kirjavainen P V, Salminen S. Probiotics: a role in the treatment of intestinal infection and inflammation? Gut [Internet]. 2002;50(Supplement 3):iii54–9. Available from: http://gut.bmj.com/cgi/doi/10.1136/gut.50.suppl_3.iii54

26. do Carmo MS, Santos C itapary dos, Araújo MC, Girón JA, Fernandes ES, Monteiro-Neto V. Probiotics, mechanisms of action, and clinical perspectives for diarrhea management in children. Food Funct [Internet]. 2018;9(10):5074–95. Available from: http://dx.doi.org/10.1039/c8fo00376a 30183037

27. Coudeyras S, Forestier C. Microbiote et probiotiques: impact en santé humaine. Can J Microbiol [Internet]. 2010 [cited 2018 Jan 30];56(8):611–50. Available from: http://www.nrcresearchpress.com/doi/pdfplus/10.1139/W10-052 20725126

28. Lacroix C, de Wouters T, Chassard C. Integrated multi-scale strategies to investigate nutritional compounds and their effect on the gut microbiota. Curr Opin Biotechnol [Internet]. 2015 [cited 2017 Apr 30];32:149–55. Available from: http://dx.doi.org/10.1016/j.copbio.2014.12.009 25562815

29. Vinderola G, Gueimonde M, Gomez-Gallego C, Delfederico L, Salminen S. Correlation between in vitro and in vivo assays in selection of probiotics from traditional species of bacteria. Trends Food Sci Technol [Internet]. 2017;68:83–90. Available from: http://dx.doi.org/10.1016/j.tifs.2017.08.005

30. Montoro BP, Benomar N, Lerma LL, Gutiérrez SC, Gálvez A, Abriouel H. Fermented aloreña table olives as a source of potential probiotic Lactobacillus pentosus strains. Front Microbiol. 2016;7(OCT).

31. Roselli M, Finamore A, Britti MS, Mengheri E. Probiotic bacteria Bifidobacterium animalis MB5 and Lactobacillus rhamnosus GG protect intestinal Caco-2 cells from the inflammation-associated response induced by enterotoxigenic Escherichia coli K88. Br J Nutr [Internet]. 2006;95(06):1177. Available from: http://www.journals.cambridge.org/abstract_S0007114506001589

32. Papadimitriou K, Zoumpopoulou G, Foligné B, Alexandraki V, Kazou M, Pot B, et al. Discovering probiotic microorganisms: In vitro, in vivo, genetic and omics approaches. Front Microbiol. 2015;6(FEB):1–28.

33. Lai CH, Chou CY, Ch’ang LY, Liu CS, Lin W. Identification of novel human genes evolutionarily conserved in Caenorhabditis elegans by comparative proteomics. Genome Res [Internet]. 2000 May [cited 2018 Sep 10];10(5):703–13. Available from: http://www.ncbi.nlm.nih.gov/pubmed/10810093 10810093

34. Pukkila-Worley R, Peleg AY, Tampakakis E, Mylonakis E. Candida albicans hyphal formation and virulence assessed using a Caenorhabditis elegans infection model. Eukaryot Cell [Internet]. 2009 [cited 2018 Feb 7];8(11):1750–8. Available from: http://ec.asm.org/content/8/11/1750.full.pdf 19666778

35. Pukkila-Worley R, Ausubel FM, Mylonakis E. Candida albicans infection of Caenorhabditis elegans induces antifungal immune defenses. PLoS Pathog. 2011;7(6).

36. Alves V de S, Mylonakis E. The eIF2 kinase Gcn2 modulates Candida albicans virulence to Caenorhabditis elegans. Clin Microbiol Infect Dis [Internet]. 2018;3(2):1–4. Available from: https://www.oatext.com/the-eif2-kinase-gcn2-modulates-candida-albicans-virulence-to-caenorhabditis-elegans.php

37. Tan X, Fuchs BB, Wang Y, Chen W, Yuen GJ, Chen RB, et al. The role of Candida albicans SPT20 in filamentation, biofilm formation and pathogenesis. PLoS One. 2014;9(4):1–10.

38. de Barros PP, Scorzoni L, Ribeiro F de C, Fugisaki LR de O, Fuchs BB, Mylonakis E, et al. Lactobacillus paracasei 28.4 reduces in vitro hyphae formation of Candida albicans and prevents the filamentation in an experimental model of Caenorhabditis elegans. Microb Pathog [Internet]. 2018;117(November 2017):80–7. Available from: https://doi.org/10.1016/j.micpath.2018.02.019

39. Pinto M, Robineleon S, Appay MD, Kedinger M, Triadou N, Dussaulx E, et al. Enterocyte-like differentiation and polarization of the human colon carcinoma cell line Caco-2 in culture. Biol Cell [Internet]. 1983 Jan 1 [cited 2018 Oct 10];47:323–30. Available from: https://www.scienceopen.com/document?vid=07f3fdcd-c23c-47d4-ad63-105346ef5453

40. Brenner S. The genetics of Caenorhabditis elegans. Genetics. 1974;77(1):71–94. 4366476

41. Hellemans J, Mortier G, De Paepe A, Speleman F, Vandesompele J. qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. 2007 [cited 2017 Jun 14];8(2). Available from: http://download.springer.com/static/pdf/804/art%253A10.1186%252Fgb-2007-8-2-r19.pdf?originUrl=http%3A%2F%2Fgenomebiology.biomedcentral.com%2Farticle%2F10.1186%2Fgb-2007-8-2-r19&token2=exp=1497424427~acl=%2Fstatic%2Fpdf%2F804%2Fart%25253A10.1186%25252Fgb-2

42. Semple JI, Garcia-Verdugo R, Lehner B. Rapid selection of transgenic C. elegans using antibiotic resistance. Nat Methods [Internet]. 2010 Sep 22 [cited 2017 Apr 13];7(9):725–7. Available from: http://www.ncbi.nlm.nih.gov/pubmed/20729840 20729840

43. Hoogewijs D, Houthoofd K, Matthijssens F, Vandesompele J, Vanfleteren JR. Selection and validation of a set of reliable reference genes for quantitative sod gene expression analysis in C. elegans. BMC Mol Biol [Internet]. 2008 Jan 22 [cited 2017 Apr 13];9:9. Available from: http://www.ncbi.nlm.nih.gov/pubmed/18211699 18211699

44. Nakagawa H, Shiozaki T, Kobatake E, Hosoya T, Moriya T, Sakai F, et al. Effects and mechanisms of prolongevity induced by Lactobacillus gasseri SBT2055 in Caenorhabditis elegans. Aging Cell. 2016;15(2):227–36. doi: 10.1111/acel.12431 26710940

45. R Core Team. R: A language and Environment for Statistical Computing [Internet]. Vienna, Austria: R Foundation for Statistical Computing; 2018. https://www.r-project.org/

46. Therneau TM. _A Package for Survival Analysis in S_. 2015.

47. Kassambara A, Kosinski M. survminer: Drawing Survival Curves using “ggplot2.” 2017.

48. Fatima S, Haque R, Jadiya P, Shamsuzzama, Kumar L, Nazir A. Ida-1, the Caenorhabditis elegans orthologue of mammalian diabetes autoantigen IA-2, potentially acts as a common modulator between Parkinson’s disease and diabetes: Role of Daf-2/Daf-16 insulin like signalling pathway. PLoS One. 2014;9(12).

49. Jankowska A, Laubitz D, Antushevich H, Zabielski R, Grzesiuk E. Competition of Lactobacillus paracasei with Salmonella enterica for adhesion to Caco-2 cells. J Biomed Biotechnol. 2008;2008(1).

50. Nowak A, Motyl I, Śliżewska K, Libudzisz Z, Klewicka E. Adherence of probiotic bacteria to human colon epithelial cells and inhibitory effect against enteric pathogens–In vitro study. Int J Dairy Technol. 2016;69(4):532–9.

51. Allonsius CN, van den Broek MFL, De Boeck I, Kiekens S, Oerlemans EFM, Kiekens F, et al. Interplay between Lactobacillus rhamnosus GG and Candida and the involvement of exopolysaccharides. Microb Biotechnol. 2017;10(6):1753–63. doi: 10.1111/1751-7915.12799 28772020

52. Ruas-Madiedo P, Gueimonde M, Margolles A, de los Reyes-Gavilan CG, Salminen S. Exopolysaccharides Produced by Probiotic Strains Modify the Adhesion of Probiotics and Enteropathogens to Human Intestinal Mucus. J Food Prot [Internet]. 2006;69(8):2011–5. Available from: http://jfoodprotection.org/doi/abs/10.4315/0362-028X-69.8.2011 16924934

53. Irazoqui JE, Troemel ER, Feinbaum RL, Luhachack LG, Cezairliyan BO, Ausubel FM. Distinct pathogenesis and host responses during infection of C. elegans by P. aeruginosa and S. aureus. PLoS Pathog. 2010;6(7):1–24.

54. Wu K, Conly J, McClure JA, Elsayed S, Louie T, Zhang K. Caenorhabditis elegans as a host model for community-associated methicillin-resistant Staphylococcus aureus. Clin Microbiol Infect. 2010;16(3):245–54. doi: 10.1111/j.1469-0691.2009.02765.x 19456837

55. Souza ACR, Fuchs BB, Alves V de S, Jayamani E, Colombo AL, Mylonakis E. Pathogenesis of the Candida parapsilosis complex in the model host Caenorhabditis elegans. Genes (Basel). 2018;9(8).

56. Park MR, Ryu S, Maburutse BE, Oh NS, Kim SH, Oh S, et al. Probiotic Lactobacillus fermentum strain JDFM216 stimulates the longevity and immune response of Caenorhabditis elegans through a nuclear hormone receptor. Sci Rep [Internet]. 2018 [cited 2019 Jan 3];8(1):7441. Available from: www.nature.com/scientificreports/ 29748542

57. Kim Y, Mylonakis E. Caenorhabditis elegans immune conditioning with the probiotic bacterium Lactobacillus acidophilus strain ncfm enhances gram-positive immune responses. Infect Immun. 2012;80(7):2500–8. doi: 10.1128/IAI.06350-11 22585961

58. Ikeda T, Yasui C, Hoshino K, Arikawa K, Nishikawa Y. Influence of lactic acid bacteria on longevity of Caenorhabditis elegans and host defense against Salmonella enterica serovar Enteritidis. Appl Environ Microbiol. 2007;73(20):6404–9. doi: 10.1128/AEM.00704-07 17704266

59. Zhao L, Zhao Y, Liu R, Zheng X, Zhang M, Guo H, et al. The transcription factor DAF-16 is essential for increased longevity in C. elegans Exposed to Bifidobacterium longum BB68. Sci Rep [Internet]. 2017;7(1):7408. Available from: http://www.nature.com/articles/s41598-017-07974-3 28785042

60. Zanni E, Laudenzi C, Schifano E, Palleschi C, Perozzi G, Uccelletti D, et al. Impact of a complex food microbiota on energy metabolism in the model organism Caenorhabditis elegans. Biomed Res Int. 2015;2015.

61. Guantario B, Zinno P, Schifano E, Roselli M, Perozzi G, Palleschi C, et al. In Vitro and in Vivo selection of potentially probiotic lactobacilli from nocellara del belice table olives. Front Microbiol. 2018;9(MAR):595.

62. Phelan JP, Rose MR. Why dietary restriction substantially increases longevity in animal models but won’t in humans. Ageing Res Rev. 2005;4(3):339–50. doi: 10.1016/j.arr.2005.06.001 16046282

63. Smith ED, Kaeberlein TL, Lydum BT, Sager J, Welton KL, Kennedy BK, et al. Age- and calorie-independent life span extension from dietary restriction by bacterial deprivation in Caenorhabditis elegans. BMC Dev Biol. 2008;8:1–13.

64. Heestand BN, Shen Y, Liu W, Magner DB, Storm N, Meharg C, et al. Dietary Restriction Induced Longevity Is Mediated by Nuclear Receptor NHR-62 in Caenorhabditis elegans. PLoS Genet. 2013;9(7).

65. Komura T, Ikeda T, Yasui C, Saeki S, Nishikawa Y. Mechanism underlying prolongevity induced by bifidobacteria in Caenorhabditis elegans. Biogerontology. 2013;14(1):73–87. doi: 10.1007/s10522-012-9411-6 23291976

66. Tullet JMA. DAF-16 target identification in C. elegans: past, present and future. Biogerontology [Internet]. 2015;16(2):221–34. Available from: http://link.springer.com/10.1007/s10522-014-9527-y 25156270

67. Grompone G, Martorell P, Llopis S, González N, Genovés S, Mulet AP, et al. Anti-Inflammatory Lactobacillus rhamnosus CNCM I-3690 Strain Protects against Oxidative Stress and Increases Lifespan in Caenorhabditis elegans. PLoS One. 2012;7(12).

68. Breger J, Fuchs BB, Aperis G, Moy TI, Ausubel FM, Mylonakis E. Antifungal chemical compounds identified using a C. elegans pathogenicity assay. PLoS Pathog. 2007;3(2):0168–78.

69. Evrard B, Coudeyras S, Dosgilbert A, Charbonnel N, Alamé J, Tridon A, et al. Dose-dependent immunomodulation of human dendritic cells by the probiotic Lactobacillus rhamnosus Lcr35. PLoS One. 2011;6(4):1–12.

70. Singh V, Aballay A. Regulation of DAF-16-mediated Innate Immunity in Caenorhabditis elegans. J Biol Chem [Internet]. 2009 Dec 18 [cited 2018 Dec 14];284(51):35580–7. Available from: http://www.ncbi.nlm.nih.gov/pubmed/19858203 19858203


Článek vyšel v časopise

PLOS One


2019 Číslo 11
Nejčtenější tento týden
Nejčtenější v tomto čísle
Kurzy

Zvyšte si kvalifikaci online z pohodlí domova

plice
INSIGHTS from European Respiratory Congress
nový kurz

Současné pohledy na riziko v parodontologii
Autoři: MUDr. Ladislav Korábek, CSc., MBA

Svět praktické medicíny 3/2024 (znalostní test z časopisu)

Kardiologické projevy hypereozinofilií
Autoři: prof. MUDr. Petr Němec, Ph.D.

Střevní příprava před kolonoskopií
Autoři: MUDr. Klára Kmochová, Ph.D.

Všechny kurzy
Kurzy Podcasty Doporučená témata Časopisy
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#