GroEL of Lactobacillus johnsonii La1 (NCC 533) Is Cell Surface Associated: Potential Role in Interactions with the Host and the Gastric Pathogen Helicobacter pylori

ABSTRACT Heat shock proteins of the GroEL or Hsp60 class are highly conserved proteins essential to all living organisms. Even though GroEL proteins are classically considered intracellular proteins, they have been found at the surface of several mucosal pathogens and have been implicated in cell attachment and immune modulation. The purpose of the present study was to investigate the GroEL protein of a gram-positive probiotic bacterium, Lactobacillus johnsonii La1 (NCC 533). Its presence at the bacterial surface was demonstrated using a whole-cell enzyme-linked immunosorbent assay and could be detected in bacterial spent culture medium by immunoblotting. To assess binding of La1 GroEL to mucins and intestinal epithelial cells, the La1 GroEL protein was expressed in Escherichia coli. We report here that La1 recombinant GroEL (rGroEL) binds to mucins and epithelial cells and that this binding is pH dependent. Immunomodulation studies showed that La1 rGroEL stimulates interleukin-8 secretion in macrophages and HT29 cells in a CD14-dependent mechanism. This property is common to rGroEL from other gram-positive bacteria but not to the rGroEL of the gastric pathogen Helicobacter pylori. In addition, La1 rGroEL mediates the aggregation of H. pylori but not that of other intestinal pathogens. Our in vitro results suggest that GroEL proteins from La1 and other lactic acid bacteria might play a role in gastrointestinal homeostasis due to their ability to bind to components of the gastrointestinal mucosa and to aggregate H. pylori.

[1]  A. Gobert,et al.  Helicobacter pylori Heat Shock Protein 60 Mediates Interleukin-6 Production by Macrophages via a Toll-like Receptor (TLR)-2-, TLR-4-, and Myeloid Differentiation Factor 88-independent Mechanism* , 2004, Journal of Biological Chemistry.

[2]  T. Klein,et al.  Bacterial heat shock proteins directly induce cytokine mRNA and interleukin-1 secretion in macrophage cultures , 1994, Infection and immunity.

[3]  P. Conway,et al.  Colonization by lactobacilli of piglet small intestinal mucus. , 1996, The Journal of applied bacteriology.

[4]  A. Blum,et al.  Favourable effect of regular intake of fermented milk containing Lactobacillus johnsonii on Helicobacter pylori associated gastritis , 2003, Alimentary pharmacology & therapeutics.

[5]  D. Ito,et al.  Rapid and Sensitive Detection of Hiochi Bacteria by Amplification of Hiochi Bacterial Common Antigen Gene by PCR Method and Characterization of the Antigen , 1997 .

[6]  S. Salminen,et al.  The ability of probiotic bacteria to bind to human intestinal mucus. , 1998, FEMS microbiology letters.

[7]  A. Labigne,et al.  Evidence for Specific Secretion Rather than Autolysis in the Release of Some Helicobacter pyloriProteins , 1998, Infection and Immunity.

[8]  M. Affolter,et al.  Innate Recognition of Bacteria in Human Milk Is Mediated by a Milk-Derived Highly Expressed Pattern Recognition Receptor, Soluble Cd14 , 2000, The Journal of experimental medicine.

[9]  L. Fay,et al.  Characterization of lactosylated proteins of infant formula powders using two‐dimensional gel electrophoresis and nanoelectrospray mass spectrometry , 2002, Electrophoresis.

[10]  P. Munkholm.  Review article: the incidence and prevalence of colorectal cancer in inflammatory bowel disease , 2003, Alimentary pharmacology & therapeutics.

[11]  P. Hoffman,et al.  Surface-associated heat shock proteins of Legionella pneumophila and Helicobacter pylori: roles in pathogenesis and immunity. , 1999, Infectious diseases in obstetrics and gynecology.

[12]  M. Kitajima,et al.  Induction of secretion of interleukin-8 from human gastric epithelial cells by heat-shock protein 60 homologue of Helicobacter pylori. , 1999, Journal of medical microbiology.

[13]  G. Oliver,et al.  Study of adhesion of Lactobacillus casei CRL 431 to ileal intestinal cells of mice. , 1999, Journal of food protection.

[14]  T. Yamamoto,et al.  Heat-shock protein 60 homologue of Helicobacter pylori is associated with adhesion of H. pylori to human gastric epithelial cells. , 1997, Journal of medical microbiology.

[15]  A. Pfeifer,et al.  Non-pathogenic bacteria elicit a differential cytokine response by intestinal epithelial cell/leucocyte co-cultures , 2000, Gut.

[16]  R. Schmitt,et al.  TnMax--a versatile mini-transposon for the analysis of cloned genes and shuttle mutagenesis. , 1993, Gene.

[17]  B E Dunn,et al.  Surface localization of Helicobacter pylori urease and a heat shock protein homolog requires bacterial autolysis , 1996, Infection and immunity.

[18]  J. Greene,et al.  Factors involved in adherence of lactobacilli to human Caco-2 cells , 1994, Applied and environmental microbiology.

[19]  D. Kapczynski,et al.  Adherence of Lactobacillus to Intestinal 407 Cells in Culture Correlates with Fibronectin Binding , 2000, Current Microbiology.

[20]  A. Barbat,et al.  Growth adaptation to methotrexate of HT-29 human colon carcinoma cells is associated with their ability to differentiate into columnar absorptive and mucus-secreting cells. , 1990, Cancer research.

[21]  R. Rolfe.  The role of probiotic cultures in the control of gastrointestinal health. , 2000, The Journal of nutrition.

[22]  T. Malvar,et al.  Regulation of insecticidal crystal protein production in Bacillus thuringiensis , 1995, Molecular microbiology.

[23]  N. Vats,et al.  Immunolocalization of Hsp60 inLegionella pneumophila , 1998, Journal of bacteriology.

[24]  Radhey S. Gupta.  Evolution of the chaperonin families (HSP60, HSP 10 and TCP‐1) of proteins and the origin of eukaryotic cells , 1995, Molecular microbiology.

[25]  P. Conway,et al.  Association of Lactobacillus spp. with Peyer's Patches in Mice , 2001, Clinical Diagnostic Laboratory Immunology.

[26]  P. Libby,et al.  Cutting Edge: Heat Shock Protein (HSP) 60 Activates the Innate Immune Response: CD14 Is an Essential Receptor for HSP60 Activation of Mononuclear Cells1 , 2000, The Journal of Immunology.

[27]  S. Salminen,et al.  Adhesion of some probiotic and dairy Lactobacillus strains to Caco-2 cell cultures. , 1998, International journal of food microbiology.

[28]  M. Inouye,et al.  Nine amino acid residues at the NH2-terminal of lipoprotein are sufficient for its modification, processing, and localization in the outer membrane of Escherichia coli. , 1984, The Journal of biological chemistry.

[29]  M. Loos,et al.  A 66-kilodalton heat shock protein of Salmonella typhimurium is responsible for binding of the bacterium to intestinal mucus , 1992, Infection and immunity.

[30]  P. Libby,et al.  Chlamydial and human heat shock protein 60s activate human vascular endothelium, smooth muscle cells, and macrophages. , 1999, The Journal of clinical investigation.

[31]  G. Bergonzelli,et al.  Cell Surface-Associated Elongation Factor Tu Mediates the Attachment of Lactobacillus johnsonii NCC533 (La1) to Human Intestinal Cells and Mucins , 2004, Infection and Immunity.

[32]  R. Miller,et al.  Immunochemical characterization of a protein associated with Mycobacterium leprae cell wall , 1985, Infection and immunity.

[33]  A. Donnet-Hughes,et al.  Lipoteichoic Acids from Lactobacillus johnsonii Strain La1 and Lactobacillus acidophilus Strain La10 Antagonize the Responsiveness of Human Intestinal Epithelial HT29 Cells to Lipopolysaccharide and Gram-Negative Bacteria , 2002, Infection and Immunity.

[34]  S. Salminen,et al.  Chemical, physical and enzymatic pre-treatments of probiotic lactobacilli alter their adhesion to human intestinal mucus glycoproteins. , 2000, International journal of food microbiology.

[35]  T. Saito,et al.  New binding assay and preparative trial of cell-surface lectin from Lactobacillus acidophilus group lactic acid bacteria. , 1999, Journal of dairy science.

[36]  H. Pakrasi,et al.  Investigation of the Functional Role of Ctp Proteins in the Cyanobacterium Synechocystis sp. PCC 6803 , 2002, Microbiology.

[37]  S. Salminen,et al.  The effect of probiotic bacteria on the adhesion of pathogens to human intestinal mucus. , 1999, FEMS immunology and medical microbiology.

[38]  B. Dunn,et al.  Localization of Helicobacter pylori urease and heat shock protein in human gastric biopsies , 1997, Infection and immunity.

[39]  H. Kolb,et al.  Different heat shock protein 60 species share pro‐inflammatory activity but not binding sites on macrophages , 2003, FEBS letters.

[40]  M. Labéta,et al.  Soluble CD14 in human breast milk and its role in innate immune responses , 2001, Acta odontologica Scandinavica.

[41]  H. Jonsson,et al.  A high-molecular-mass cell-surface protein from Lactobacillus reuteri 1063 adheres to mucus components. , 2002, Microbiology.

[42]  L. Madoff,et al.  Subcellular fractionation of group B Streptococcus. , 1999, BioTechniques.

[43]  A. Pfeifer,et al.  Probiotics and immune response , 2002, Clinical reviews in allergy & immunology.

[44]  Francesca Patrignani,et al.  Use of natural aroma compounds to improve shelf-life and safety of minimally processed fruits , 2004 .

[45]  F. Ascencio,et al.  Purification and Characterization of a Surface Protein from Lactobacillus fermentum 104R That Binds to Porcine Small Intestinal Mucus and Gastric Mucin , 2002, Applied and Environmental Microbiology.

[46]  T. Klaenhammer,et al.  Protein-mediated adhesion of Lactobacillus acidophilus BG2FO4 on human enterocyte and mucus-secreting cell lines in culture , 1992, Applied and environmental microbiology.

[47]  E. Craig,et al.  Heat shock proteins: molecular chaperones of protein biogenesis , 1993, Microbiological reviews.

[48]  T. Lagergård,et al.  GroEL Heat Shock Protein of Haemophilus ducreyi: Association with Cell Surface and Capacity To Bind to Eukaryotic Cells , 1998, Infection and Immunity.

[49]  J. Havránková,et al.  Simple method for development of sensitive and specific antiinsulin antisera for laboratory use. , 1984, Journal of immunoassay.

[50]  A. Blum,et al.  Effect of Whey-Based Culture Supernatant of Lactobacillus acidophilus (johnsonii) La1 on Helicobacter pylori Infection in Humans , 1999, Digestion.

[51]  S. Salminen,et al.  Human ileostomy glycoproteins as a model for small intestinal mucus to investigate adhesion of probiotics , 1999, Letters in applied microbiology.

[52]  M. Gasson,et al.  Plasmid complements of Streptococcus lactis NCDO 712 and other lactic streptococci after protoplast-induced curing , 1983, Journal of bacteriology.

[53]  A. Miller,et al.  Homogeneous Escherichia coli chaperonin 60 induces IL-1 beta and IL-6 gene expression in human monocytes by a mechanism independent of protein conformation. , 1998, Journal of immunology.

[54]  L. Morelli,et al.  Adhesion studies for probiotics : need for validation and refinement , 1999 .

[55]  A. Servin,et al.  Adhesion of human Lactobacillus acidophilus strain LB to human enterocyte-like Caco-2 cells. , 1992, Journal of general microbiology.

[56]  D. Brassart,et al.  Cell Surface-Associated Lipoteichoic Acid Acts as an Adhesion Factor for Attachment of Lactobacillus johnsoniiLa1 to Human Enterocyte-Like Caco-2 Cells , 1999, Applied and Environmental Microbiology.

[57]  N. D. de Roos,et al.  Effects of probiotic bacteria on diarrhea, lipid metabolism, and carcinogenesis: a review of papers published between 1988 and 1998. , 2000, The American journal of clinical nutrition.

[58]  M. Tsan,et al.  Cytokine function of heat shock proteins. , 2004, American journal of physiology. Cell physiology.

[59]  P. Bourlioux,et al.  GroEL (Hsp60) of Clostridium difficile is involved in cell adherence. , 2001, Microbiology.

[60]  T. Klaenhammer,et al.  The groESL Chaperone Operon ofLactobacillus johnsonii , 1999, Applied and Environmental Microbiology.