Archives of Microbiology

, Volume 196, Issue 1, pp 9–16 | Cite as

Deciphering the role of Burkholderia cenocepacia membrane proteins in antimicrobial properties of chitosan

  • Muhammad Ibrahim
  • Zhongyun Tao
  • Annam Hussain
  • Yang Chunlan
  • Mehmoona Ilyas
  • Abdul Waheed
  • Fang Yuan
  • Bin Li
  • Guan-Lin XieEmail author
Original Paper


Chitosan, a versatile derivative of chitin, is widely used as an antimicrobial agent either alone or mixed with other natural polymers. Burkholderia cenocepacia is a multidrug-resistant bacteria and difficult to eradicate. Our previous studies shown that chitosan had strong antibacterial activity against B. cenocepacia. In the current study, we have investigated the molecular aspects for the susceptibility of B. cenocepacia in response to chitosan antibacterial activity. We have conducted RNA expression analysis of drug efflux system by RT-PCR, membrane protein profiling by SDS–PAGE, and by LC-MS/MS analysis following the validation of selected membrane proteins by real-time PCR analysis. By RT-PCR analysis, it was found that orf3, orf9, and orf13 were expressed at detectable levels, which were similar to control, while rest of the orf did not express. Moreover, shotgun proteomics analysis revealed 21 proteins in chitosan-treated cells and 16 proteins in control. Among them 4 proteins were detected as shared proteins under control and chitosan-treated cells and 17 proteins as uniquely identified proteins under chitosan-treated cells. Among the catalog of uniquely identified proteins, there were proteins involved in electron transport chain and ATP synthase, metabolism of carbohydrates and adaptation to atypical conditions proteins which indicate that utilization and pattern of chitosan is diverse which might be responsible for its antibacterial effects on bacteria. Moreover, our results showed that RND drug efflux system, which display the ability to transport a variety of structurally unrelated drugs from a cell and consequently are capable of conferring resistance to a diverse range of chemotherapeutic agents, was not determined to play its role in response to chitosan. It might be lipopolysaccharides interaction with chitosan resulted in the destabilization of membrane protein to membrane lyses to cell death. Membrane proteome analysis were also validated by RT-qPCR analysis, which corroborated our results that of membrane proteins.


Chitosan Antibacterial activity RND efflux Membrane proteins 



This project was supported by the Special Fund for Agro-scientific Research in the Public Interest (201303015, 201003066) and Zhejiang Provincial Natural Science Foundation of China (LY12C14007).


  1. Allan CR, Hardwiger LA (1979) The fungicidal effect of chitosan on fungi of varying cell wall composition. Exp Mycol 3:285–287CrossRefGoogle Scholar
  2. Bazzini S, Udine C, Sass A, Pasca MR, Longo F, Emiliani G, Fondi M, Perrin E, Decorosi F, Viti C, Giovannetti L, Leoni L, Fani R, Riccardi G, Mahenthiralingam E, Buroni S (2011) Deciphering the role of RND efflux transporters in Burkholderia cenocepacia. PLoS One 6(4):e18902PubMedCentralPubMedCrossRefGoogle Scholar
  3. Caroff M, Karibian D (2003) Structure of bacterial lipopolysaccharides. Carbohydr Res 338:2431–2447PubMedCrossRefGoogle Scholar
  4. Chang W, Small DA, Toghrol F, Bentley WE (2006) Global transcriptome analysis of Staphylococcus aureus response to hydrogen peroxide. J Bacteriol 188:1648–1659PubMedCentralPubMedCrossRefGoogle Scholar
  5. Chen CZS, Cooper SL (2002) Interactions between dendrimer biocides and bacterial membranes. Biomaterials 23:3359–3368PubMedCrossRefGoogle Scholar
  6. Dina Raafat, Bargen K, Haas A, Sahl HG (2008) Insights into the mode of action of chitosan as an antibacterial compound. Appl Environ Microbial 12:3764–3773Google Scholar
  7. Dodane V, Vilivalam VD (1998) Pharmaceutical applications of chitosan. Pharm Sci Technol Today 1:246–253CrossRefGoogle Scholar
  8. Eaton P, Fernandes JC, Pereira E, Pintado ME, Xavier Malcata F (2008) Atomic force microscopy study of the antibacterial effects of chitosans on Escherichia coli and Staphylococcus aureus. Ultramicroscopy 108:1128–1134PubMedCrossRefGoogle Scholar
  9. Fang Y, Lou MM, Li B, Xie GL, Wang F, Zhang LX, Luo YC (2010) Characterization of Burkholderia cepacia complex from cystic fibrosis patients in China and their chitosan susceptibility. World J Microbiol Biotechnol 26:443–450CrossRefGoogle Scholar
  10. Farag RK, Mohamed RR (2012) Synthesis and characterization of carboxymethyl chitosan nanogels for swelling studies and antimicrobial activity. Molecules 18:190–203PubMedCrossRefGoogle Scholar
  11. Franklin TJ, Snow GA (1981) Biochemistry of antimicrobial action, 3rd edn. Chapman and Hall, London, p 175Google Scholar
  12. Fuchs S, Pané-Farré J, Kohler C, Hecker M, Engelmann S (2007) Anaerobic gene expression in Staphylococcus aureus. J Bacteriol 189:4275–4289PubMedCentralPubMedCrossRefGoogle Scholar
  13. Gaffer HE, Gouda M, Abdel-Latif E (2013) Antibacterial activity of cotton fabrics treated with sulfadimidine azo dye/chitosan colloid. J Ind Text 42:392–399CrossRefGoogle Scholar
  14. Guglierame P, Pasca MR, De Rossi E, Buroni S, Arrigo P, Manina G, Riccardi G (2006) Effluc Pump genes of resistance nodulation divisions family in Burkholderia cenocepacia genome. BMC Microbiol 6:66PubMedCentralPubMedCrossRefGoogle Scholar
  15. Hofhaus G, Weiss H, Leonard K (1991) Electron microscopic analysis of the peripheral and the membrane parts of mitochondrial NADH dehydrogenase (Complex I). J Mol Biol 221:1027–1043PubMedCrossRefGoogle Scholar
  16. Ibrahim M, Tang QM, Shi Yu, Almoneafy AW, Fang Y, Xu LH, Li W, Li B, Xie GL (2012) Diversity of potential pathogenicity and biofilm formation among Burkholderia cepacia complex water clinical and agricultural isolates in China. World J Microbiol Biotechnol 528:2113–2121CrossRefGoogle Scholar
  17. Jagannadham MV, Chowdhury C (2012) Differential expression of membrane proteins helps Antarctic Pseudomonas syringae to acclimatize upon temperature variations. J Proteomics 75:2488–2499PubMedCrossRefGoogle Scholar
  18. Kohler C, von Eiff C, Peters G, Proctor RA, Hecker M, Engelmann S (2003) Physiological characterization of a heme-deficient mutant of Staphylococcus aureus by a proteomic approach. J Bacteriol 185:6928–6937PubMedCentralPubMedCrossRefGoogle Scholar
  19. Kong M, Chen XG, Liu CS, Liu CG, Meng XH, le Yu J (2008) Antibacterial mechanism of chitosan microspheres in a solid dispersing system against E. coli. Colloids Surf B Biointerfaces 65:197–202PubMedCrossRefGoogle Scholar
  20. Kong M, Chen XG, Xing K, Park HJ (2010) Antimicrobial properties of chitosan and mode of action: a state of the art review. Int J Food Microbiol 144:51–63PubMedCrossRefGoogle Scholar
  21. Li B, Wang X, Chen RX, Huangfu WG, Xie GL (2008) Antibacterial activity of chitosan solution against Xanthomonas pathogenic bacteria isolated from Eurphorbia pulcherrima. Carbohydr Polym 72:287–292CrossRefGoogle Scholar
  22. Lou MM, Zho B, Ibrahim M, Xie GL (2011) Antibacterial activity and mechanism of chitosan solutions against aprocot fruit rot pathogen. Carbohydr. Res 346: 1296–1301CrossRefGoogle Scholar
  23. Mohamed RR, Seoudi RS, Sabaa MW (2012) Synthesis and characterization of antibacterial semi-interpenetrating carboxymethyl chitosan/poly (acrylonitrile) hydrogels. Cellulose 19:947–958CrossRefGoogle Scholar
  24. Muzzarelli RAA (2010) Chitins and chitosans as immunoadjuvants and non-allergenic drug carriers. Mar Drugs 8:292–312PubMedCentralPubMedCrossRefGoogle Scholar
  25. Muzzarelli RAA, Boudrant J, Meyer D, Manno N, DeMarchis M, Paoletti MG (2012) Current views on fungal chitin/chitosan, human chitinases, food preservation, glucans, pectins and inulin: a tribute to Henri Braconnot, precursor of the carbohydrate polymers science, on the chitin bicentennial. Carbohydr Polym 87:995–1012CrossRefGoogle Scholar
  26. Nikaido H (1996) Outer membrane. In: Neidhardt FC, Curtiss R, Ingraham JL, Brooks Low K, Magasanik B, Reznikoff WS, Riley M, Schaechter M, Umbarger HE (Eds.), Escherichia coli and Salmonella typhimurium: cellular and molecular biology (2nd ed.) (Vol. 1, pp. 29–47). edition. Washington DC: American Society for MicrobiologyGoogle Scholar
  27. Nikaido H, Takatsuka Y (2009) Mechanisms of RND multidrug efflux pumps. Biochim Biophys Acta Protein Proteomics 1794:769–781CrossRefGoogle Scholar
  28. No HK, Meyers SPJ (1995) Aquat Food Prod Technol 4:27–52Google Scholar
  29. Raafat D, Sahl HG (2009) Chitosan and its antimicrobial potential: a critical literature survey. Microb Biotechnol 2:184–219CrossRefGoogle Scholar
  30. Rabea EI, Badawy MET, Stevens CV, Smagghe G, Steurbaut W (2003) Biomacromolecules 4:1457–1465Google Scholar
  31. Rietschel ET, Kirikae T, Schade FU, Mamat U, Schmidt G, Loppnow H, Ulmer AJ, Zähringer U, Seydel U, Di Padova F et al (1994) Bacterial endotoxin: molecular relationships of structure to activity and function. FASEB J. 8:217–225PubMedGoogle Scholar
  32. Sajomsang W, Tantayanon S, Tangpasuthadol V, Daly WH (2007) Synthesis of methylated chitosan containing aromatic moieties: chemoselectivity and effect on molecular weight. Carbohydr Polym 72:740–750CrossRefGoogle Scholar
  33. Shimamoto T, Izawa H, Daimon H, Ishiguro N, Shinagawa M, Sakano Y, Tsuda M, Tsuchiya T (1991) Cloning and nucleotide sequence of the gene (citA) encoding a citrate carrier from Salmonella typhimurium. J Biochem 110:22–28PubMedGoogle Scholar
  34. Solov'eva T, Davydova V, Krasikova I, Yermak I (2013) Marine compounds with therapeutics potential in Gram negative species. Mar Drugs 11:2216–2229Google Scholar
  35. Takemono K, Sunamoto J, Askasi M (1989) Polymers and medical care. Mita, Tokyo Chapter IVGoogle Scholar
  36. Tanaka I, Appelt K, Dijk J, White SW, Wilson KS (1984) 3-A resolution structure of a protein with histone-like properties in prokaryotes. Nature 310:376–378PubMedCrossRefGoogle Scholar
  37. Tharanathan RN, Kittur FS (2003) Chitin-the undisputed biomolecule of great potential. Crit Rev Food Sci Nutr 43:61–87PubMedCrossRefGoogle Scholar
  38. Tikhonov VE, Stepnova EA, Babak VG, Yamskov IA, Palma-Guerrero J, Jansson HB, Lopez-Llorca LV, Salinas J, Gerasimenko DV, Avdienko ID, Varlamov VP (2006) Bactericidal and antifungal activities of a low molecular weight chitosan and its N-/2(3)-(dodec-2-enyl) succinoyl/-derivatives. Carbohydr Polym 64:66–72CrossRefGoogle Scholar
  39. Yermak IM, Davidova V, Gorbach N, Luk’yanov VIP, Solovèva A, Ulmer TF et al (2006) Forming and immunological properties of some lipopolysaccharide–chitosan complexes. Biochimie 88:23–30PubMedCrossRefGoogle Scholar
  40. Young DH, Kohle H, Kauss H (1982) Effect of chitosan on membrane permeability of suspension-cultured Glycine max and Phaseolus vulgaris cells. Plant Physiol 70:1449–1454PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Muhammad Ibrahim
    • 1
    • 3
  • Zhongyun Tao
    • 1
  • Annam Hussain
    • 3
  • Yang Chunlan
    • 1
  • Mehmoona Ilyas
    • 4
  • Abdul Waheed
    • 3
  • Fang Yuan
    • 2
  • Bin Li
    • 1
  • Guan-Lin Xie
    • 1
    Email author
  1. 1.State Key Laboratory of Rice Biology, Key Laboratory of Molecular Biology of Crop Pathogens and Insects, Ministry of Agriculture, Institute of BiotechnologyZhejiang UniversityHangzhouPeople’s Republic of China
  2. 2.College of Chemistry and Life SciencesZhejiang Normal UniversityJinhuaChina
  3. 3.Department of BiosciencesCOMSATS Institute of Information TechnologySahiwalPakistan
  4. 4.Department of BotanyPMAS Arid Agriculture UniversityRawalpindiPakistan

Personalised recommendations