title: Suppression subtractive hybridization identifies bacterial genomic regions that are possibly involved in hBD-2 regulation by enterocytes.
abstract: METHODS
  Human β-defensin 2 (hBD-2) is an inducible antimicrobial peptide synthesized by the epithelium to counteract bacterial adherence and invasion. It has been suggested that probiotic bacteria sustain gut barrier function via induction of defensins. The goals of this study were (i) to evaluate the potential immunomodulatory effects of 11 different Lactobacillus fermentum strains isolated from Kimere, an African fermented pearl millet (Pennisetum glaucum) dough, on the hBD-2 secretion by human intestinal CaCo-2 cell line and (ii) to examine genetic differences between two strains of L. fermentum (K2-Lb4 and K11-Lb3) which differed in their effect on the production of hBD-2 in this study.
  
  
  RESULTS
  Totally, 46 strains of L. fermentum from Kimere were isolated and characterized using molecular biology methods including pulsed-field gel electrophoresis patterns. After performing time- and dose-experiments, CaCo-2 cells were incubated with or without bacteria for 12 h. L. fermentum PZ1162 was included as the positive control. Cell-free supernatants were analyzed for hBD-2 protein by enzyme-linked immunosorbent assay (ELISA). To identify potential bacterial genes associated with hBD-2 regulation, suppression subtractive hybridization (SSH) was used. Among the 11 strains tested, only two strains of bacteria, K11-Lb3 and K2-Lb6, significantly induced the production of hBD-2 by CaCo-2 cells. This effect was strain-specific, dose-dependent and particularly seems to be bacterial genomic-dependent as manifested by SSH. L. fermentum strains with and without hBD-2 inducing effect differed in genes encoding proteins involved in glycosylation of cell-wall proteins e.g. glycosyltransferase, UDP-N-acetylglucosamine 2-epimerase, rod shape-determining protein MreC, lipoprotein precursors, sugar ABC transporters, and glutamine ABC transporter ATP-binding protein.
  
  
  CONCLUSIONS
  This study implies that certain strains of L. fermentum isolated from Kimere may stimulate the intestinal innate defense through the induction of hBD-2. The molecular basis of hBD-2 induction by L. fermentum strain K11-Lb3 may be based on glycosylated cell-surface structures synthesized with the aid of glycosyltransferase, UDP-N-acetylglucosamine 2-epimerase, and rod shape-determining protein MreC.
authors.affiliation: Department of Physiology and Biochemistry of Nutrition, Max Rubner-Institut, Hermann-Weigmann, Kiel, Germany. darab.ghadimi@mri.bund.de
authors.forename: Darab
authors.initials: D
authors.lastname: Ghadimi
authors.forename: Mohamed
authors.initials: M
authors.lastname: Hassan
authors.forename: Patrisio Njiru
authors.initials: PN
authors.lastname: Njeru
authors.forename: Michael
authors.initials: M
authors.lastname: de Vrese
authors.forename: Arnold
authors.initials: A
authors.lastname: Geis
authors.forename: Samweul I
authors.initials: SI
authors.lastname: Shalabi
authors.forename: Sabah T
authors.initials: ST
authors.lastname: Abdel-Razek
authors.forename: Abd El-Al A
authors.initials: Ael-A
authors.lastname: Abdel-Khair
authors.forename: Knut J
authors.initials: KJ
authors.lastname: Heller
authors.forename: Jürgen
authors.initials: J
authors.lastname: Schrezenmeir
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D018938:Caco-2 Cells; D002238:Carbohydrate Epimerases / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D020895:Enterocytes / Q000378:metabolism* / Q000382:microbiology*; D005786:Gene Expression Regulation; D016680:Genome, Bacterial; D006031:Glycosylation; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods; D052197:Limosilactobacillus fermentum / Q000235:genetics*; D008969:Molecular Sequence Data; D031782:Pennisetum / Q000382:microbiology; D013045:Species Specificity; D023083:beta-Defensins / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; C106641:DEFB4A protein, human; C062061:MreC protein, Bacteria; D023083:beta-Defensins; C037853:glutamine transport proteins; D002238:Carbohydrate Epimerases; C100033:UDP acetylglucosamine-2-epimerase