title: Identification and functional characterization of the putative polysaccharide biosynthesis protein (CapD) of Enterococcus faecium U0317.
abstract: Most bacterial species produce capsular polysaccharides that contribute to disease pathogenesis through evasion of the host innate immune system and are also involved in inhibiting leukocyte killing. In the present study, we identified a gene in Enterococcus faecium U0317 with homologies to the polysaccharide biosynthesis protein CapD that is made up of 336 amino acids and putatively catalyzes N-linked glycosylation. A capD deletion mutant was constructed and complemented by homologous recombination that was confirmed by PCR and sequencing. The mutant revealed different growth behavior and morphological changes compared to wild-type by scanning electron microscopy, also the capD mutant showed a strong hydrophobicity and that was reversed in the reconstituted mutant. For further characterization and functional analyses, in-vitro cell culture and in-vivo a mouse infection models were used. Antibodies directed against alpha lipotechoic acid (αLTA) and the peptidyl-prolyl cis-trans isomerase (αPpiC), effectively mediated the opsonophagocytic killing in the capD knock-out mutant, while this activity was not observed in the wild-type and reconstituted mutant. By comparison more than 2-fold decrease was seen in mutant colonization and adherence to both T24 and Caco2 cells. However, a significant higher bacterial colonization was observed in capD mutant during bacteremia in the animal model, while virulence in a mouse UTI (urinary tract infection) model, there were no obvious differences. Further studies are needed to elucidate the function of capsular polysaccharide synthesis gene clusters and its involvement in the disease pathogenesis with the aim to develop targeted therapies to treat multidrug-resistant E. faecium infections.
authors.affiliation: Division of Infectious Diseases, Department of Medicine, University Medical Center Freiburg, Hugstetter Straße 55, 79106 Freiburg, Germany; Faculty of Biology, Albert-Ludwigs-University Freiburg, Schänzlestraße 1, 79104 Freiburg, Germany.
authors.forename: Liaqat
authors.initials: L
authors.lastname: Ali
authors.affiliation: Division of Infectious Diseases, Department of Medicine, University Medical Center Freiburg, Hugstetter Straße 55, 79106 Freiburg, Germany.
authors.forename: Meike
authors.initials: M
authors.lastname: Spiess
authors.affiliation: Division of Infectious Diseases, Department of Medicine, University Medical Center Freiburg, Hugstetter Straße 55, 79106 Freiburg, Germany.
authors.forename: Dominique
authors.initials: D
authors.lastname: Wobser
authors.affiliation: Faculty of Biology, Albert-Ludwigs-University Freiburg, Schänzlestraße 1, 79104 Freiburg, Germany.
authors.forename: Marta
authors.initials: M
authors.lastname: Rodriguez
authors.affiliation: Division of Infectious Diseases, Department of Medicine, University Medical Center Freiburg, Hugstetter Straße 55, 79106 Freiburg, Germany.
authors.forename: Hubert E
authors.initials: HE
authors.lastname: Blum
authors.affiliation: Division of Infectious Diseases, Department of Medicine, University Medical Center Freiburg, Hugstetter Straße 55, 79106 Freiburg, Germany. Electronic address: tuerkan.sakinc@uniklinik-freiburg.de.
authors.forename: Türkân
authors.initials: T
authors.lastname: Sakιnç
mesh_terms: D000818:Animals; D001422:Bacterial Adhesion; D016667:Bacterial Capsules / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D018938:Caco-2 Cells; D002460:Cell Line; D004195:Disease Models, Animal; D016984:Enterococcus faecium / Q000254:growth & development* / Q000302:isolation & purification / Q000472:pathogenicity; D016908:Gram-Positive Bacterial Infections / Q000382:microbiology*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D051379:Mice; D009154:Mutation; D011134:Polysaccharides / Q000096:biosynthesis*
chemical_list: D011134:Polysaccharides
keywords: CapD protein; E. faecium U0317; Pathogenesis; Polysaccharides