전체메뉴
검색
Article Search

JMB Journal of Microbiolog and Biotechnology

QR Code QR Code

Research article

Food Microbiology and Biotechnology (FMB)  |  Probiotics and Foodborne Microorganisms

Role of eptC in Biofilm Formation by Campylobacter jejuni NCTC11168 on Polystyrene and Glass Surfaces

Eun Seob Lim 1 and Joo-Sung Kim 1, 2*

1Department of Food Biotechnology, Korea University of Science and Technology, Daejeon 34113, Republic of Korea, 2Korea Food Research Institute, Seongnam 13539, Republic of Korea

Received: October 18, 2016; Accepted: July 5, 2017

J. Microbiol. Biotechnol. 2017; 27(9): 1609-1616

Published September 28, 2017 https://doi.org/10.4014/jmb.1610.10046

Copyright © The Korean Society for Microbiology and Biotechnology.

Abstract

The complex roles of cell surface modification in the biofilm formation of Campylobacter jejuni, a major cause of worldwide foodborne diarrheal disease, are poorly understood. In a screen of mutants from random transposon mutagenesis, an insertional mutation in the eptC gene (cj0256) resulted in a significant decrease in C. jejuni NCTC11168 biofilm formation (<20%) on major food contact surfaces, such as polystyrene and borosilicate glass, when compared with wild-type cells (p < 0.05). In C. jejuni strain 81-176, the protein encoded by eptC modified cell surface structures, such as lipid A, the inner core of lipooligosaccharide, and the flagellar rod protein (FlgG), by attaching phosphoethanolamine. To assess the role of eptC in C. jejuni NCTC11168, adherence and motility tests were performed. In adhesion assays with glass surfaces, the eptC mutant exhibited a 0.77 log CFU/cm2 decrease in adherence compared with wild-type cells during the initial 2 h of the assay (p < 0.05). These results support the hypothesis that the modification of cell surface structures by eptC affects the initial adherence in biofilm formation of C. jejuni NCTC11168. In motility tests, the eptC mutant demonstrated reduced motility when compared with wild-type cells, but wild-type cells with the transposon inserted in a gene irrelevant to biofilm formation (cj1111c) also exhibited decreased motility to a similar extent as the eptC mutant. This suggests that although eptC affects motility, it does not significantly affect biofilm formation. This study demonstrates that eptC is essential for initial adherence, and plays a significant role in the biofilm formation of C. jejuni NCTC11168.

Keywords

Campylobacter jejuni, eptC, biofilm

References

  1. Luber P, Brynestad S, Topsch D, Scherer K, Bartelt E. 2006. Quantification of Campylobacter species cross-contamination during handling of contaminated fresh chicken parts in kitchens. Appl. Environ. Microbiol. 72: 66-70.
    Pubmed PMC CrossRef
  2. Olson CK, Ethelberg S, Pelt W van, Tauxe RV. 2008. Epidemiology of Campylobacter jejuni infections in industrialized nations. In Nachamkin I, Szymanski CM, Blaser MJ (eds), Campylobacter, 3rd Ed. American Society of Microbiology, Washington, DC.
    CrossRef
  3. MFDS. Ministry of Food and Drug Safety. 2015. Occurrence of foodborne disease from 2003-2014. Available from http://www.foodsafetykorea.go.kr/portal/healthyfoodlife/foodPoisoningStat.do?menu_no=5 19&menu_grp=MENU_GRP02.
  4. Costerton J W, S tewart P S, G reenberg EP. 1 999. B acterial biofilms: a common cause of persistent infections. Science 284: 1318-1322.
    Pubmed CrossRef
  5. Bridier A, Sanchez-Vizuete P, Guilbaud M, Piard JC, Naïtali M, Briandet R. 2015. Biofilm-associated persistence of food-borne pathogens. Food Microbiol. 45: 167-178.
    Pubmed CrossRef
  6. Gunther NW, Chen CY. 2009. The biofilm forming potential of bacterial species in the genus Campylobacter. Food Microbiol. 26: 44-51.
    Pubmed CrossRef
  7. Joshua GWP, Guthrie-Irons C, Karlyshev AV, Wren BW. 2006. Biofilm formation in Campylobacter jejuni. Microbiology 152: 387-396.
    Pubmed CrossRef
  8. Kalmokoff M, Lanthier P, Tremblay TL, Foss M, Lau PC, Sanders G, et al. 2006. Proteomic analysis of Campylobacter jejuni 11168 biofilms reveals a role for the motility complex in biofilm formation. J. Bacteriol. 188: 4312-4320.
    Pubmed PMC CrossRef
  9. Kim JS, Park CW, Kim YJ. 2015. Role of flgA for flagellar biosynthesis and biofilm formation of Campylobacter jejuni NCTC11168. J. Microbiol. Biotechnol. 25: 1871-1879.
    Pubmed CrossRef
  10. Fields JA, Thompson SA. 2008. Campylobacter jejuni CsrA mediates oxidative stress responses, biofilm formation, and host cell invasion. J. Bacteriol. 190: 3411-3416.
    Pubmed PMC CrossRef
  11. Bronowski C, James CE, Winstanley C. 2014. Role of environmental survival in transmission of Campylobacter jejuni. FEMS Microbiol. Lett. 356: 8-19.
    Pubmed CrossRef
  12. Lin J, Wang Y, Hoang K Van. 2009. Systematic identification of genetic loci required for polymyxin resistance in Campylobacter jejuni using an efficient in vivo transposon mutagenesis system. Foodborne Pathog. Dis. 6: 173-185.
    Pubmed CrossRef
  13. Cullen TW, Trent MS. 2010. A link between the assembly of flagella and lipooligosaccharide of the gram-negative bacterium Campylobacter jejuni. Proc. Natl. Acad. Sci. USA 107: 5160-5165.
    Pubmed PMC CrossRef
  14. Watnick P, Kolter R. 2000. Biofilm, city of microbes. J. Bacteriol. 182: 2675-2679.
    Pubmed PMC CrossRef
  15. Abee T, Kovács ÁT, Kuipers OP, Veen SVD. 2011. Biofilm formation and dispersal in gram-positive bacteria. Curr. Opin. Biotechnol. 22: 172-179.
    Pubmed CrossRef
  16. Stoodley P, Sauer K, Davies DG, Costerton JW. 2002. Biofilms as complex differentiated communities. Annu. Rev. Microbiol. 56: 187-209.
    Pubmed CrossRef
  17. Scott NE, Nothaft H, Edwards AVG, Labbate M, Djordjevic SP, Larsen MR, et al. 2012. Modification of the Campylobacter jejuni N-linked glycan by EptC protein-mediated addition of phosphoethanolamine. J. Biol. Chem. 287: 29384-29396.
    Pubmed PMC CrossRef
  18. Cullen TW, O’Brien JP, Hendrixson DR, Giles DK, Hobb RI, Thompson SA, et al. 2013. EptC of Campylobacter jejuni mediates phenotypes involved in host interactions and virulence. Infect. Immun. 81: 430-440.
    Pubmed PMC CrossRef
  19. Cullen TW, Madsen JA, Ivanov PL, Brodbelt JS, Trent MS. 2012. Characterization of unique modification of flagellar rod protein FlgG by Campylobacter jejuni lipid A phosphoethanolamine transferase, linking bacterial locomotion and antimicrobial peptide resistance. J. Biol. Chem. 287: 3326-3336.
    Pubmed PMC CrossRef
  20. Donlan RM. 2002. Biofilms: microbial life on surfaces. Emerg. Infect. Dis. 8: 881-890.
    Pubmed PMC CrossRef
  21. O’Toole GA, Kolter R. 1998. Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signalling pathways: a genetic analysis. Mol. Microbiol. 28: 449-461.
    Pubmed CrossRef
  22. O’Toole GA, Kolter R. 1998. Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development. Mol. Microbiol. 30: 295-304.
    Pubmed CrossRef
  23. Svensson SL, Pryjma M, Gaynor EC. 2014. Flagella-mediated adhesion and extracellular DNA release contribute to biofilm formation and stress tolerance of Campylobacter jejuni. PLoS One 9: e106063.
    Pubmed PMC CrossRef
  24. Moe KK, Mimura J, Ohnishi T, Wake T, Yamazaki W, Nakai M, et al. 2010. The mode of biofilm formation on smooth surfaces by Campylobacter jejuni. J. Vet. Med. Sci. 72: 411-416.
    Pubmed CrossRef
  25. Reeser RJ, Medler RT, Billington SJ, Jost BH, Joens LA. 2007. Characterization of Campylobacter jejuni biofilms under defined growth conditions. Appl. Environ. Microbiol. 73: 1908-1913.
    Pubmed PMC CrossRef
  26. Golden NJ, Acheson DWK. 2002. Identification of motility and autoagglutination Campylobacter jejuni mutants by random transposon mutagenesis. Infect. Immun. 70: 1761-1771.
    Pubmed PMC CrossRef
  27. Grant CCR, Konkel ME, Cieplak WJ, Tompkins LS. 1993. Role of flagella in adherence, internalization, and translocation of Campylobacter jejuni in nonpolarized and polarized epithelial cell cultures. Infect. Immun. 61: 1764-1771.
    Pubmed PMC
  28. Young NM, B risson J R, K elly J , Watson D C, T essier L , Lanthier PH, et al. 2002. Structure of the N-linked glycan present on multiple glycoproteins in the gram-negative bacterium, Campylobacter jejuni. J. Biol. Chem. 277: 42530-42539.
    Pubmed CrossRef
  29. Nguyen VT, Turner MS, Dykes GA. 2011. Influence of cell surface hydrophobicity on attachment of Campylobacter to abiotic surfaces. Food Microbiol. 28: 942-950.
    Pubmed CrossRef
  30. Martino PD, Cafferini N, Joly B, Darfeuille-Michaud A. 2003. Klebsiella pneumoniae type 3 pili facilitate adherence and biofilm formation on abiotic surfaces. Res. Microbiol. 154: 9-16.
    CrossRef
  31. Moser I, Schröder W. 1997. Hydrophobic characterization of thermophilic Campylobacter species and adhesion to INT 407 cell membranes and fibronectin. Microb. Pathog. 22: 155-164.
    Pubmed CrossRef
  32. Raetz CRH, Reynolds CM, Trent MS, Bishop RE. 2007. Lipid A modification systems in gram-negative bacteria. Annu. Rev. Biochem. 76: 295-329.
    Pubmed PMC CrossRef
  33. Kannenberg EL, C arlson RW. 2 001. L ipid A a nd O-chain modifications cause Rhizobium lipopolysaccharides to become hydrophobic during bacteroid development. Mol. Microbiol. 39: 379-391.
    Pubmed CrossRef
  34. Hankins JV, Madsen JA, Giles DK, Brodbelt JS, Trent MS. 2012. Amino acid addition to Vibrio cholerae LPS establishes a link between surface remodeling in gram-positive and gramnegative bacteria. Proc. Natl. Acad. Sci. USA 109: 8722-8727.
    Pubmed PMC CrossRef
  35. Alemka A, Nothaft H, Zheng J, Szymanski CM. 2013. Nglycosylation of Campylobacter jejuni surface proteins promotes bacterial fitness. Infect. Immun. 81: 1674-1682.
    Pubmed PMC CrossRef
  36. Karlyshev AV, Everest P, Linton D, Cawthraw S, Newell DG, Wren BW. 2004. The Campylobacter jejuni general glycosylation system is important for attachment to human epithelial cells and in the colonization of chicks. Microbiology. 150: 1957-1964.
    Pubmed CrossRef

Related articles in JMB

More Related Articles

Article

Research article

J. Microbiol. Biotechnol. 2017; 27(9): 1609-1616

Published online September 28, 2017 https://doi.org/10.4014/jmb.1610.10046

Copyright © The Korean Society for Microbiology and Biotechnology.

Role of eptC in Biofilm Formation by Campylobacter jejuni NCTC11168 on Polystyrene and Glass Surfaces

Eun Seob Lim 1 and Joo-Sung Kim 1, 2*

1Department of Food Biotechnology, Korea University of Science and Technology, Daejeon 34113, Republic of Korea, 2Korea Food Research Institute, Seongnam 13539, Republic of Korea

Received: October 18, 2016; Accepted: July 5, 2017

Abstract

The complex roles of cell surface modification in the biofilm formation of Campylobacter jejuni,
a major cause of worldwide foodborne diarrheal disease, are poorly understood. In a screen of
mutants from random transposon mutagenesis, an insertional mutation in the eptC gene
(cj0256) resulted in a significant decrease in C. jejuni NCTC11168 biofilm formation (<20%) on
major food contact surfaces, such as polystyrene and borosilicate glass, when compared with
wild-type cells (p < 0.05). In C. jejuni strain 81-176, the protein encoded by eptC modified cell
surface structures, such as lipid A, the inner core of lipooligosaccharide, and the flagellar rod
protein (FlgG), by attaching phosphoethanolamine. To assess the role of eptC in C. jejuni
NCTC11168, adherence and motility tests were performed. In adhesion assays with glass
surfaces, the eptC mutant exhibited a 0.77 log CFU/cm2 decrease in adherence compared with
wild-type cells during the initial 2 h of the assay (p < 0.05). These results support the
hypothesis that the modification of cell surface structures by eptC affects the initial adherence
in biofilm formation of C. jejuni NCTC11168. In motility tests, the eptC mutant demonstrated
reduced motility when compared with wild-type cells, but wild-type cells with the transposon
inserted in a gene irrelevant to biofilm formation (cj1111c) also exhibited decreased motility to
a similar extent as the eptC mutant. This suggests that although eptC affects motility, it does
not significantly affect biofilm formation. This study demonstrates that eptC is essential for
initial adherence, and plays a significant role in the biofilm formation of C. jejuni NCTC11168.

Keywords: Campylobacter jejuni, eptC, biofilm

References

  1. Luber P, Brynestad S, Topsch D, Scherer K, Bartelt E. 2006. Quantification of Campylobacter species cross-contamination during handling of contaminated fresh chicken parts in kitchens. Appl. Environ. Microbiol. 72: 66-70.
    Pubmed KoreaMed CrossRef
  2. Olson CK, Ethelberg S, Pelt W van, Tauxe RV. 2008. Epidemiology of Campylobacter jejuni infections in industrialized nations. In Nachamkin I, Szymanski CM, Blaser MJ (eds), Campylobacter, 3rd Ed. American Society of Microbiology, Washington, DC.
    CrossRef
  3. MFDS. Ministry of Food and Drug Safety. 2015. Occurrence of foodborne disease from 2003-2014. Available from http://www.foodsafetykorea.go.kr/portal/healthyfoodlife/foodPoisoningStat.do?menu_no=5 19&menu_grp=MENU_GRP02.
  4. Costerton J W, S tewart P S, G reenberg EP. 1 999. B acterial biofilms: a common cause of persistent infections. Science 284: 1318-1322.
    Pubmed CrossRef
  5. Bridier A, Sanchez-Vizuete P, Guilbaud M, Piard JC, Naïtali M, Briandet R. 2015. Biofilm-associated persistence of food-borne pathogens. Food Microbiol. 45: 167-178.
    Pubmed CrossRef
  6. Gunther NW, Chen CY. 2009. The biofilm forming potential of bacterial species in the genus Campylobacter. Food Microbiol. 26: 44-51.
    Pubmed CrossRef
  7. Joshua GWP, Guthrie-Irons C, Karlyshev AV, Wren BW. 2006. Biofilm formation in Campylobacter jejuni. Microbiology 152: 387-396.
    Pubmed CrossRef
  8. Kalmokoff M, Lanthier P, Tremblay TL, Foss M, Lau PC, Sanders G, et al. 2006. Proteomic analysis of Campylobacter jejuni 11168 biofilms reveals a role for the motility complex in biofilm formation. J. Bacteriol. 188: 4312-4320.
    Pubmed KoreaMed CrossRef
  9. Kim JS, Park CW, Kim YJ. 2015. Role of flgA for flagellar biosynthesis and biofilm formation of Campylobacter jejuni NCTC11168. J. Microbiol. Biotechnol. 25: 1871-1879.
    Pubmed CrossRef
  10. Fields JA, Thompson SA. 2008. Campylobacter jejuni CsrA mediates oxidative stress responses, biofilm formation, and host cell invasion. J. Bacteriol. 190: 3411-3416.
    Pubmed KoreaMed CrossRef
  11. Bronowski C, James CE, Winstanley C. 2014. Role of environmental survival in transmission of Campylobacter jejuni. FEMS Microbiol. Lett. 356: 8-19.
    Pubmed CrossRef
  12. Lin J, Wang Y, Hoang K Van. 2009. Systematic identification of genetic loci required for polymyxin resistance in Campylobacter jejuni using an efficient in vivo transposon mutagenesis system. Foodborne Pathog. Dis. 6: 173-185.
    Pubmed CrossRef
  13. Cullen TW, Trent MS. 2010. A link between the assembly of flagella and lipooligosaccharide of the gram-negative bacterium Campylobacter jejuni. Proc. Natl. Acad. Sci. USA 107: 5160-5165.
    Pubmed KoreaMed CrossRef
  14. Watnick P, Kolter R. 2000. Biofilm, city of microbes. J. Bacteriol. 182: 2675-2679.
    Pubmed KoreaMed CrossRef
  15. Abee T, Kovács ÁT, Kuipers OP, Veen SVD. 2011. Biofilm formation and dispersal in gram-positive bacteria. Curr. Opin. Biotechnol. 22: 172-179.
    Pubmed CrossRef
  16. Stoodley P, Sauer K, Davies DG, Costerton JW. 2002. Biofilms as complex differentiated communities. Annu. Rev. Microbiol. 56: 187-209.
    Pubmed CrossRef
  17. Scott NE, Nothaft H, Edwards AVG, Labbate M, Djordjevic SP, Larsen MR, et al. 2012. Modification of the Campylobacter jejuni N-linked glycan by EptC protein-mediated addition of phosphoethanolamine. J. Biol. Chem. 287: 29384-29396.
    Pubmed KoreaMed CrossRef
  18. Cullen TW, O’Brien JP, Hendrixson DR, Giles DK, Hobb RI, Thompson SA, et al. 2013. EptC of Campylobacter jejuni mediates phenotypes involved in host interactions and virulence. Infect. Immun. 81: 430-440.
    Pubmed KoreaMed CrossRef
  19. Cullen TW, Madsen JA, Ivanov PL, Brodbelt JS, Trent MS. 2012. Characterization of unique modification of flagellar rod protein FlgG by Campylobacter jejuni lipid A phosphoethanolamine transferase, linking bacterial locomotion and antimicrobial peptide resistance. J. Biol. Chem. 287: 3326-3336.
    Pubmed KoreaMed CrossRef
  20. Donlan RM. 2002. Biofilms: microbial life on surfaces. Emerg. Infect. Dis. 8: 881-890.
    Pubmed KoreaMed CrossRef
  21. O’Toole GA, Kolter R. 1998. Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signalling pathways: a genetic analysis. Mol. Microbiol. 28: 449-461.
    Pubmed CrossRef
  22. O’Toole GA, Kolter R. 1998. Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development. Mol. Microbiol. 30: 295-304.
    Pubmed CrossRef
  23. Svensson SL, Pryjma M, Gaynor EC. 2014. Flagella-mediated adhesion and extracellular DNA release contribute to biofilm formation and stress tolerance of Campylobacter jejuni. PLoS One 9: e106063.
    Pubmed KoreaMed CrossRef
  24. Moe KK, Mimura J, Ohnishi T, Wake T, Yamazaki W, Nakai M, et al. 2010. The mode of biofilm formation on smooth surfaces by Campylobacter jejuni. J. Vet. Med. Sci. 72: 411-416.
    Pubmed CrossRef
  25. Reeser RJ, Medler RT, Billington SJ, Jost BH, Joens LA. 2007. Characterization of Campylobacter jejuni biofilms under defined growth conditions. Appl. Environ. Microbiol. 73: 1908-1913.
    Pubmed KoreaMed CrossRef
  26. Golden NJ, Acheson DWK. 2002. Identification of motility and autoagglutination Campylobacter jejuni mutants by random transposon mutagenesis. Infect. Immun. 70: 1761-1771.
    Pubmed KoreaMed CrossRef
  27. Grant CCR, Konkel ME, Cieplak WJ, Tompkins LS. 1993. Role of flagella in adherence, internalization, and translocation of Campylobacter jejuni in nonpolarized and polarized epithelial cell cultures. Infect. Immun. 61: 1764-1771.
    Pubmed KoreaMed
  28. Young NM, B risson J R, K elly J , Watson D C, T essier L , Lanthier PH, et al. 2002. Structure of the N-linked glycan present on multiple glycoproteins in the gram-negative bacterium, Campylobacter jejuni. J. Biol. Chem. 277: 42530-42539.
    Pubmed CrossRef
  29. Nguyen VT, Turner MS, Dykes GA. 2011. Influence of cell surface hydrophobicity on attachment of Campylobacter to abiotic surfaces. Food Microbiol. 28: 942-950.
    Pubmed CrossRef
  30. Martino PD, Cafferini N, Joly B, Darfeuille-Michaud A. 2003. Klebsiella pneumoniae type 3 pili facilitate adherence and biofilm formation on abiotic surfaces. Res. Microbiol. 154: 9-16.
    CrossRef
  31. Moser I, Schröder W. 1997. Hydrophobic characterization of thermophilic Campylobacter species and adhesion to INT 407 cell membranes and fibronectin. Microb. Pathog. 22: 155-164.
    Pubmed CrossRef
  32. Raetz CRH, Reynolds CM, Trent MS, Bishop RE. 2007. Lipid A modification systems in gram-negative bacteria. Annu. Rev. Biochem. 76: 295-329.
    Pubmed KoreaMed CrossRef
  33. Kannenberg EL, C arlson RW. 2 001. L ipid A a nd O-chain modifications cause Rhizobium lipopolysaccharides to become hydrophobic during bacteroid development. Mol. Microbiol. 39: 379-391.
    Pubmed CrossRef
  34. Hankins JV, Madsen JA, Giles DK, Brodbelt JS, Trent MS. 2012. Amino acid addition to Vibrio cholerae LPS establishes a link between surface remodeling in gram-positive and gramnegative bacteria. Proc. Natl. Acad. Sci. USA 109: 8722-8727.
    Pubmed KoreaMed CrossRef
  35. Alemka A, Nothaft H, Zheng J, Szymanski CM. 2013. Nglycosylation of Campylobacter jejuni surface proteins promotes bacterial fitness. Infect. Immun. 81: 1674-1682.
    Pubmed KoreaMed CrossRef
  36. Karlyshev AV, Everest P, Linton D, Cawthraw S, Newell DG, Wren BW. 2004. The Campylobacter jejuni general glycosylation system is important for attachment to human epithelial cells and in the colonization of chicks. Microbiology. 150: 1957-1964.
    Pubmed CrossRef