Skip to main content
SpringerLink
Log in

Characterization of a S-layer protein from Lactobacillus crispatus K313 and the domains responsible for binding to cell wall and adherence to collagen

  • Biotechnologically relevant enzymes and proteins
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

It was previously shown that the surface (S)-layer proteins covering the cell surface of Lactobacillus crispatus K313 were involved in the adherence of this strain to human intestinal cell line HT-29. To further elucidate the structures and functions of S-layers, three putative S-layer protein genes (slpA, slpB, and slpC) of L. crispatus K313 were amplified by PCR, sequenced, and characterized in detail. Quantitative real-time PCR analysis reveals that slpA was silent under the tested conditions; whereas slpB and slpC, the putative amino acid sequences which exhibited minor similarities to the previously reported S-layer proteins in L. crispatus, were actively expressed. slpB, which was predominantly expressed in L. crispatus K313, was further investigated for its functional domains. Genetic truncation of the untranslated leader sequence (UTLS) of slpB results in a reduction in protein production, indicating that the UTLS contributed to the efficient S-layer protein expression. By producing a set of N- and C-terminally truncated recombinant SlpB proteins in Escherichia coli, the cell wall-binding region was mapped to the C terminus, where rSlpB380–501 was sufficient for binding to isolated cell wall fragments. Moreover, the binding ability of the C terminus was variable among the Lactobacillus species (S-layer- and non-S-layer-producing strains), and teichoic acid may be acting as the receptor of SlpB. To determine the adhesion region of SlpB to extracellular matrix proteins, ELISA was performed. Binding to immobilized types I and IV collagen was observed with the His-SlpB1–379 peptides, suggesting that the extracellular matrix protein-binding domain was located in the N terminus.

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

Access this article

Log in via an institution

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

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  • Abbot EL, Smith WD, Siou GP, Chiriboga C, Smith RJ, Wilson JA, Hirst BH, Kehoe MA (2007) Pili mediate specific adhesion of Streptococcus pyogenes to human tonsil and skin. Cell Microbiol 9:1822–1833

    Article  CAS  Google Scholar 

  • Ames BN, Dubin DT (1960) The role of polyamines in the neutralization of bacteriophage deoxyribonucleic acid. J Biol Chem 235:769–775

    CAS  Google Scholar 

  • Antikainen J, Anton L, Sillanpaa J, Korhonen TK (2002) Domains in the S-layer protein CbsA of Lactobacillus crispatus involved in adherence to collagens, laminin and lipoteichoic acids and in self-assembly. Mol Microbiol 46:381–394

    Article  CAS  Google Scholar 

  • Avall-Jaaskelainen S, Hynonen U, Ilk N, Pum D, Sleytr UB, Palva A (2008) Identification and characterization of domains responsible for self-assembly and cell wall binding of the surface layer protein of Lactobacillus brevis ATCC 8287. BMC Microbiol 8:165

    Article  Google Scholar 

  • Avall-Jaaskelainen S, Palva A (2005) Lactobacillus surface layers and their applications. FEMS Microbiol Rev 29:511–529

    Article  Google Scholar 

  • Barbosa AS, Abreu PA, Neves FO, Atzingen MV, Watanabe MM, Vieira ML, Morais ZM, Vasconcellos SA, Nascimento AL (2006) A newly identified leptospiral adhesin mediates attachment to laminin. Infect Immun 74:6356–6364

    Article  CAS  Google Scholar 

  • Boot HJ, Kolen CP, Andreadaki FJ, Leer RJ, Pouwels PH (1996a) The Lactobacillus acidophilus S-layer protein gene expression site comprises two consensus promoter sequences, one of which directs transcription of stable mRNA. J Bacteriol 178:5388–5394

    CAS  Google Scholar 

  • Boot HJ, Kolen CP, Pot B, Kersters K, Pouwels PH (1996b) The presence of two S-layer-protein-encoding genes is conserved among species related to Lactobacillus acidophilus. Microbiology 142(Pt 9):2375–2384

    Article  CAS  Google Scholar 

  • Boot HJ, Kolen CP, van Noort JM, Pouwels PH (1993) S-layer protein of Lactobacillus acidophilus ATCC 4356: purification, expression in Escherichia coli, and nucleotide sequence of the corresponding gene. J Bacteriol 175:6089–6096

    CAS  Google Scholar 

  • Buck BL, Altermann E, Svingerud T, Klaenhammer TR (2005) Functional analysis of putative adhesion factors in Lactobacillus acidophilus NCFM. Appl Environ Microbiol 71:8344–8351

    Article  CAS  Google Scholar 

  • Callegari ML, Riboli B, Sanders JW, Cocconcelli PS, Kok J, Venema G, Morelli L (1998) The S-layer gene of Lactobacillus helveticus CNRZ 892: cloning, sequence and heterologous expression. Microbiology 144(Pt 3):719–726

    Article  CAS  Google Scholar 

  • Chen X, Xu J, Shuai J, Chen J, Zhang Z, Fang W (2007) The S-layer proteins of Lactobacillus crispatus strain ZJ001 is responsible for competitive exclusion against Escherichia coli O157:H7 and Salmonella typhimurium. Int J Food Microbiol 115:307–312

    Article  CAS  Google Scholar 

  • Giulietti A, Overbergh L, Valckx D, Decallonne B, Bouillon R, Mathieu C (2001) An overview of real-time quantitative PCR: applications to quantify cytokine gene expression. Methods 25:386–401

    Article  CAS  Google Scholar 

  • Goh YJ, Azcarate-Peril MA, O'Flaherty S, Durmaz E, Valence F, Jardin J, Lortal S, Klaenhammer TR (2009) Development and application of a upp-based counterselective gene replacement system for the study of the S-layer protein SlpX of Lactobacillus acidophilus NCFM. Appl Environ Microbiol 75:3093–3105

    Article  CAS  Google Scholar 

  • Golowczyc MA, Mobili P, Garrote GL, Abraham AG, De Antoni GL (2007) Protective action of Lactobacillus kefir carrying S-layer protein against Salmonella enterica serovar Enteritidis. Int J Food Microbiol 118:264–273

    Article  CAS  Google Scholar 

  • Hagen KE, Guan LL, Tannock GW, Korver DR, Allison GE (2005) Detection, characterization, and in vitro and in vivo expression of genes encoding S-layer proteins in Lactobacillus gallinarum strains isolated from chicken crops. Appl Environ Microbiol 71:6633–6643

    Article  CAS  Google Scholar 

  • Hazebrouck S, Pothelune L, Azevedo V, Corthier G, Wal JM, Langella P (2007) Efficient production and secretion of bovine beta-lactoglobulin by Lactobacillus casei. Microb Cell Fact 6:6–1

    Article  Google Scholar 

  • Horie M, Kajikawa HS, Toba T (2002) Identification of Lactobacillus crispatus by polymerase chain reaction targeting S-layer protein gene. Lett Appl Microbiol 35:57–61

    Article  CAS  Google Scholar 

  • Huber C, Ilk N, Runzler D, Egelseer EM, Weigert S, Sleytr UB, Sara M (2005) The three S-layer-like homology motifs of the S-layer protein SbpA of Bacillus sphaericus CCM 2177 are not sufficient for binding to the pyruvylated secondary cell wall polymer. Mol Microbiol 55:197–205

    Article  CAS  Google Scholar 

  • Hynonen U, Avall-Jaaskelainen S, Palva A (2010) Characterization and separate activities of the two promoters of the Lactobacillus brevis S-layer protein gene. Appl Microbiol Biotechnol 87:657–668

    Article  Google Scholar 

  • Hynonen U, Westerlund-Wikstrom B, Palva A, Korhonen TK (2002) Identification by flagellum display of an epithelial cell- and fibronectin-binding function in the SlpA surface protein of Lactobacillus brevis. J Bacteriol 184:3360–3367

    Article  CAS  Google Scholar 

  • Khang YH, Park HY, Jeong YS, Kim JA, Kim YH (2009) Recombinant S-layer proteins of Lactobacillus brevis mediating antibody adhesion to calf intestine alleviated neonatal diarrhea syndrome. J Microbiol Biotechnol 19:511–519

    Article  CAS  Google Scholar 

  • Liu YG, Whittier RF (1995) Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking. Genomics 25:674–681

    Article  CAS  Google Scholar 

  • Mercenier A, Muller-Alouf H, Grangette C (2000) Lactic acid bacteria as live vaccines. Curr Issues Mol Biol 2:17–25

    CAS  Google Scholar 

  • Narita J, Ishida S, Okano K, Kimura S, Fukuda H, Kondo A (2006) Improvement of protein production in lactic acid bacteria using 5′-untranslated leader sequence of slpA from Lactobacillus acidophilus. Appl Microbiol Biotechnol 73:366–373

    Article  CAS  Google Scholar 

  • Oozeer R, Furet JP, Goupil-Feuillerat N, Anba J, Mengaud J, Corthier G (2005) Differential activities of four Lactobacillus casei promoters during bacterial transit through the gastrointestinal tracts of human-microbiota-associated mice. Appl Environ Microbiol 71:1356–1363

    Article  CAS  Google Scholar 

  • Ribeiro LA, Azevedo V, Le Loir Y, Oliveira SC, Dieye Y, Piard JC, Gruss A, Langella P (2002) Production and targeting of the Brucella abortus antigen L7/L12 in Lactococcus lactis: a first step towards food-grade live vaccines against brucellosis. Appl Environ Microbiol 68:910–916

    Article  CAS  Google Scholar 

  • Sawa N, Zendo T, Kiyofuji J, Fujita K, Himeno K, Nakayama J, Sonomoto K (2009) Identification and characterization of lactocyclicin Q, a novel cyclic bacteriocin produced by Lactococcus sp. strain QU 12. Appl Environ Microbiol 75:1552–1558

    Article  CAS  Google Scholar 

  • Sillanpaa J, Martinez B, Antikainen J, Toba T, Kalkkinen N, Tankka S, Lounatmaa K, Keranen J, Hook M, Westerlund-wikstrom B, Pouwels PH, Korhonen TK (2000) Characterization of the collagen-binding S-layer protein CbsA of Lactobacillus crispatus. J Bacteriol 182:6440–6450

    Article  CAS  Google Scholar 

  • Sleytr UB, Beveridge TJ (1999) Bacterial S-layers. Trends Microbiol 7:253–260

    Article  CAS  Google Scholar 

  • Smit E, Oling F, Demel R, Martinez B, Pouwels PH (2001) The S-layer protein of Lactobacillus acidophilus ATCC 4356: identification and characterisation of domains responsible for S-protein assembly and cell wall binding. J Mol Biol 305:245–257

    Article  CAS  Google Scholar 

  • Steidler L, Hans W, Schotte L, Neirynck S, Obermeier F, Falk W, Fiers W, Remaut E (2000) Treatment of murine colitis by Lactococcus lactis secreting interleukin-10. Science 289:1352–1355

    Article  CAS  Google Scholar 

  • Sun Z, Kong J, Kong W (2010) Characterization of a cryptic plasmid pD403 from Lactobacillus plantarum and construction of shuttle vectors based on its replicon. Mol Biotechnol 45:24–33

    Article  CAS  Google Scholar 

  • Toba T, Virkola R, Westerlund B, Bjorkman Y, Sillanpaa J, Vartio T, Kalkkinen N, Korhonen TK (1995) A collagen-binding S-Layer protein in Lactobacillus crispatus. Appl Environ Microbiol 61:2467–2471

    CAS  Google Scholar 

  • Van de Guchte M, Penaud S, Grimaldi C, Barbe V, Bryson K, Nicolas P, Robert C, Oztas S, Mangenot S, Couloux A, Loux V, Dervyn R, Bossy R, Bolotin A, Batto JM, Walunas T, Gibrat JF, Bessières P, Weissenbach J, Ehrlich SD, Maguin E (2006) The complete genome sequence of Lactobacillus bulgaricus reveals extensive and ongoing reductive evolution. Proc Natl Acad Sci U S A 103:9274–9279

    Article  Google Scholar 

  • Walter J, Heng NC, Hammes WP, Loach DM, Tannock GW, Hertel C (2003) Identification of Lactobacillus reuteri genes specifically induced in the mouse gastrointestinal tract. Appl Environ Microbiol 69:2044–2051

    Article  CAS  Google Scholar 

  • Westerlund B, Korhonen TK (1993) Bacterial proteins binding to the mammalian extracellular matrix. Mol Microbiol 9:687–694

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We would like to thank M. van de Guchte and S. Hazebrouck for their generous gifts of L. delbrueckii subsp. bulgaricus ATCC 11842 and L. casei BL23, respectively. This research was supported by the National Natural Science Foundation of China (31070091), and the 863 Hi-Tech Research and Development Program of China (2011AA100902).

Author information

Authors and Affiliations

  1. State Key Laboratory of Microbial Technology, Shandong University, Jinan, 250100, People’s Republic of China

    Zhilan Sun, Jian Kong, Shumin Hu, Wentao Kong, Wenwei Lu & Wei Liu

  2. Scientific Research Center, Tsingtao Brewery Co.LTD, Qingdao, People’s Republic of China

    Shumin Hu

Authors
  1. Zhilan Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jian Kong
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shumin Hu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Wentao Kong
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Wenwei Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Wei Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jian Kong.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sun, Z., Kong, J., Hu, S. et al. Characterization of a S-layer protein from Lactobacillus crispatus K313 and the domains responsible for binding to cell wall and adherence to collagen. Appl Microbiol Biotechnol 97, 1941–1952 (2013). https://doi.org/10.1007/s00253-012-4044-x

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-012-4044-x

Keywords

Search

Navigation