Skip to main content

Advertisement

Log in

CadF expression in Campylobacter jejuni strains incubated under low-temperature water microcosm conditions which induce the viable but non-culturable (VBNC) state

  • Original Paper
  • Published:
Antonie van Leeuwenhoek Aims and scope Submit manuscript

Abstract

Campylobacter jejuni is a major gastrointestinal pathogen that colonizes host mucosa via interactions with extracellular matrix proteins such as fibronectin. The aim of this work was to study in vitro the adhesive properties of C. jejuni ATCC 33291 and C. jejuni 241 strains, in both culturable and viable but non-culturable (VBNC) forms. To this end, the expression of the outer-membrane protein CadF, which mediates C. jejuni binding to fibronectin, was evaluated. VBNC bacteria were obtained after 46–48 days of incubation in freshwater at 4 °C. In both cellular forms, the expression of the cadF gene, assessed at different time points by RT-PCR, was at high levels until the third week of VBNC induction, while the intensity of the signal declined during the last stage of incubation. CadF protein expression by the two C. jejuni strains was analysed using 2-dimensional electrophoresis and mass spectrometry; the results indicated that the protein, although at low levels, is also present in the VBNC state. Adhesion assays with culturable and VBNC cells, evaluated on Caco-2 monolayers, showed that non-culturable bacteria retain their ability to adhere to intestinal cells, though at a reduced rate. Our results demonstrate that the C. jejuni VBNC population maintains an ability to adhere and this may thus have an important role in the pathogenicity of this microorganism.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Altekruse SF, Swerdlow DL, Stern NJ (1998) Campylobacter jejuni. Vet Clin North Am Food Anim Pract 14:31–40

    PubMed  CAS  Google Scholar 

  • Baffone W, Casaroli A, Citterio B, Pierfelici L, Campana R, Vittoria E, Guaglianone E, Donelli G (2006) Campylobacter jejuni loss of culturability in aqueous microcosms and ability to resuscitate in a mouse model. Int J Food Microbiol 107:83–91

    Article  PubMed  Google Scholar 

  • Barer MR, Harwood CR (1999) Bacterial viability and culturability. Adv Microb Physiol 41:93–137

    Article  PubMed  CAS  Google Scholar 

  • Bini L, Sanchez-Campillo M, Cantucci A, Magi B, Marzocchi B, Comanducci M, Christiansen G, Birkelund S, Cevenini R, Vretou E, Ratti G, Pallini V (1996) Mapping of Chlamydia trachomatis proteins by immobiline-polyacrylamide two-dimensional electrophoresis: spot identification by N-terminal sequencing and immunoblotting. Electrophoresis 17:185–190

    Article  PubMed  CAS  Google Scholar 

  • Bradford M (1976) Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  PubMed  CAS  Google Scholar 

  • Butzler JP (2004) Campylobacter, from obscurity to celebrity. Clin Microbiol Infect 10:868–876

    Article  PubMed  Google Scholar 

  • Cappelier JM, Lazaro B, Rossero A, Fernandez-Astorga A, Federighi M (1997) Double staining (CTC-DAPI) for detection and enumeration of viable but non-culturable Campylobacter jejuni cells. Vet Res 28:547–555

    PubMed  CAS  Google Scholar 

  • Cappelier JM, Minet J, Magras C, Colwell RR, Federighi M (1999) Recovery in embryonated eggs of viable but nonculturable Campylobacter jejuni cells and maintenance of ability to adhere to HeLa cells after resuscitation. Appl Environ Microbiol 65:5154–5157

    PubMed  CAS  Google Scholar 

  • Chaisowwong W, Kusumoto A, Hashimoto M, Harada T, Maklon K, Kawamoto K (2012) Physiological characterization of Campylobacter jejuni under cold stresses conditions: its potential for public threat. J Vet Med Sci 74(1):43–50

    Article  PubMed  CAS  Google Scholar 

  • Colwell RR, Huq A (1994) Environmental reservoir of Vibrio cholerae. The causative agent of cholera. Ann NY Acad Sci 740:44–54

    Article  PubMed  CAS  Google Scholar 

  • Coutard F, Lozach S, Pommepuy M, Hervio-Heath D (2007) Real-Time Reverse Transcription-PCR for transcriptional expression analysis of virulence and housekeeping genes in viable but nonculturable Vibrio parahaemolyticus after recovery of culturability. Appl Environ Microbiol 73:5183–5189

    Article  PubMed  CAS  Google Scholar 

  • Dorrell N, Wren BW (2007) The second century of Campylobacter research: recent advances, new opportunities and old problems. Curr Opin Infect Dis 20:514–518

    Article  PubMed  Google Scholar 

  • Duffy LL, Dykes GA (2009) The ability of Campylobacter jejuni cells to attach to stainless steel does not change as they become nonculturable. Foodborne Pathog Dis 6:631–634

    Article  PubMed  Google Scholar 

  • Ganan M, Campos G, Muñoz R, Carrascosa AV, de Pascual-Teresa S, Martinez-Rodriguez AJ (2010) Effect of growth phase on the adherence to and invasion of Caco-2 epithelial cells by Campylobacter. Int J Food Microbiol 140:14–18

    Article  PubMed  CAS  Google Scholar 

  • Guescini M, Guidolin D, Vallorani L, Casadei L, Gioacchini AM, Tibollo P, Battistelli M, Falcieri E, Battistin L, Agnati LF, Stocchi V (2010) C2C12 myoblasts release micro-vesicles containing mtDNA and proteins involved in signal transduction. Exp Cell Res 316:1977–1984

    Article  PubMed  CAS  Google Scholar 

  • Ketley JM (1997) Pathogenesis of enteric infection by Campylobacter. Microbiology 143:5–21

    Article  PubMed  CAS  Google Scholar 

  • Konkel ME, Garvis SG, Tipton SL, Anderson DE Jr, Cieplak W Jr (1997) Identification and molecular cloning of a gene encoding a fibronectin-binding protein (CadF) from Campylobacter jejuni. Mol Microbiol 24:953–963

    Article  PubMed  CAS  Google Scholar 

  • Konkel ME, Gray SA, Kim BJ, Garvis SG, Yoon J (1999) Identification of the enteropathogens Campylobacter jejuni and Campylobacter coli based on the cadF virulence gene and its product. J Clin Microbiol 37:510–517

    PubMed  CAS  Google Scholar 

  • Konkel ME, Christensen JE, Keech AM, Monteville MR, Klena JD, Garvis SG (2005) Identification of a fibronectin-binding domain within the Campylobacter jejuni CadF protein. Mol Microbiol 57:1022–1035

    Article  PubMed  CAS  Google Scholar 

  • Korhonen LK, Martikainen PJ (1991) Comparison of the survival of Campylobacter jejuni and Campylobacter coli in culturable form in surface water. Can J Microbiol 37:530–533

    Article  PubMed  CAS  Google Scholar 

  • Ma Y, Hanning I, Slavik M (2009) Stress-induced adaptive tolerance response and virulence gene expression in Campylobacter jejuni. J Food Safety 29:126–143

    Article  CAS  Google Scholar 

  • Monteville MR, Yoon JE, Konkel ME (2003) Maximal adherence and invasion of INT 407 cells by Campylobacter jejuni requires the CadF outer-membrane protein and microfilament reorganization. Microbiology 149:153–165

    Article  PubMed  CAS  Google Scholar 

  • Oliver JD (1993) Formation of viable but nonculturable cells. In: Kjelleberg S (ed) Starvation in bacteria. Plenum Press, New York, pp 239–272

    Google Scholar 

  • Pruzzo C, Tarsi R, Lleò MM, Signoretto C, Zampini M, Colwell RR, Canepari P (2002) In vitro adhesion to human cells by viable but nonculturable Enterococcus faecalis. Curr Microbiol 45:105–110

    Article  PubMed  CAS  Google Scholar 

  • Rodriguez GG, Phipps D, Ishiguro K, Ridgway HF (1992) Use of a fluorescent redox probe for direct visualisation of actively respiring bacteria. Appl Environ Microbiol 58:1801–1808

    PubMed  CAS  Google Scholar 

  • Rollins DM, Colwell RR (1986) Viable but nonculturable stage of Campylobacter jejuni and its role in survival in the natural aquatic environment. Appl Environ Microbiol 52:531–538

    PubMed  CAS  Google Scholar 

  • Saltarelli R, Ceccaroli P, Iotti M, Zambonelli A, Casadei L, Vallorani L, Stocchi V (2009) Biochemical characterization and antioxidant activity of mycelium of Ganoderma lucidum from Central Italy. Food Chem 116:143–151

    Article  CAS  Google Scholar 

  • Sestili P, Barbieri E, Martinelli C, Battistelli M, Guescini M, Vallorani L, Casadei L, D’emilio A, Falcieri E, Piccoli G, Agostani D, Annibalini G, Paolillo M, Gioacchini AM, Stocchi V (2009) Creatine supplementation prevents the inhibition of myogenic differentiation in oxidatively injured C2C12 murine myoblasts. Mol Nutr Food Res 53:1187–1204

    Article  PubMed  CAS  Google Scholar 

  • Shevchenko A, Wilm M, Vorm O, Mann M (1996) Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem 68:850–858

    Article  PubMed  CAS  Google Scholar 

  • Sinha P, Poland J, Schnölzer M, Rabilloud T (2001) A new silver staining apparatus and procedure for matrix-assisted laser desorption/ionization-time of flight analysis of proteins after two-dimensional electrophoresis. Proteomics 1:835–840

    Article  PubMed  CAS  Google Scholar 

  • Snelling WJ, Matsuda M, Moore JE, Dooley JS (2005) Campylobacter jejuni. Lett Appl Microbiol 41:297–302

    Article  PubMed  CAS  Google Scholar 

  • Tholozan JL, Cappelier JM, Tissier JP, Delattre G, Federighi M (1999) Physiological characterization of viable-but-nonculturable Campylobacter jejuni cells. Appl Environ Microbiol 65:1110–1116

    PubMed  CAS  Google Scholar 

  • Verhoeff-Bakkenes L, Hazeleger WC, Zwietering MH, De Jonge R (2008) Lack of response of INT-407 cells to the presence of non-culturable Campylobacter jejuni. Epidemiol Infect 136:1401–1406

    Article  PubMed  CAS  Google Scholar 

  • Vora GJ, Meador CE, Bird MM, Bopp CA, Andreadis JD, Stenger DA (2005) Microarray-based detection of genetic heterogeneity, antimicrobial resistance, and the viable but nonculturable state in human pathogenic Vibrio spp. Proc Natl Acad Sci USA 102:19109–19114

    Article  PubMed  CAS  Google Scholar 

  • Young KT, Davis LM, Dirita VJ (2007) Campylobacter jejuni: molecular biology and pathogenesis. Nat Rev Microbiol 5:665–679

    Article  PubMed  CAS  Google Scholar 

  • Zhang MJ, Xiao D, Zhao F, Gu YX, Meng FL, He LH, Ma GY, Zhang JZ (2009) Comparative proteomic analysis of Campylobacter jejuni cultured at 37°C and 42°C. Jpn J Infect Dis 62:356–361

    PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Wally Baffone.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Patrone, V., Campana, R., Vallorani, L. et al. CadF expression in Campylobacter jejuni strains incubated under low-temperature water microcosm conditions which induce the viable but non-culturable (VBNC) state. Antonie van Leeuwenhoek 103, 979–988 (2013). https://doi.org/10.1007/s10482-013-9877-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10482-013-9877-5

Keywords

Navigation