title: Calcium inhibits bap-dependent multicellular behavior in Staphylococcus aureus.
abstract: Bap (biofilm-associated protein) is a 254-kDa staphylococcal surface protein implicated in formation of biofilms by staphylococci isolated from chronic mastitis infections. The presence of potential EF-hand motifs in the amino acid sequence of Bap prompted us to investigate the effect of calcium on the multicellular behavior of Bap-expressing staphylococci. We found that addition of millimolar amounts of calcium to the growth media inhibited intercellular adhesion of and biofilm formation by Bap-positive strain V329. Addition of manganese, but not addition of magnesium, also inhibited biofilm formation, whereas bacterial aggregation in liquid media was greatly enhanced by metal-chelating agents. In contrast, calcium or chelating agents had virtually no effect on the aggregation of Bap-deficient strain M556. The biofilm elicited by insertion of bap into the chromosome of a biofilm-negative strain exhibited a similar dependence on the calcium concentration, indicating that the observed calcium inhibition was an inherent property of the Bap-mediated biofilms. Site-directed mutagenesis of two of the putative EF-hand domains resulted in a mutant strain that was capable of forming a biofilm but whose biofilm was not inhibited by calcium. Our results indicate that Bap binds Ca2+ with low affinity and that Ca2+ binding renders the protein noncompetent for biofilm formation and for intercellular adhesion. The fact that calcium inhibition of Bap-mediated multicellular behavior takes place in vitro at concentrations similar to those found in milk serum supports the possibility that this inhibition is relevant to the pathogenesis and/or epidemiology of the bacteria in the mastitis process.
authors.affiliation: Instituto de Agrobiotecnología y Recursos Naturales, Universidad Pública de Navarra-CSIC, Pamplona, Spain. mjesus.arrizubieta@unavarra.es
authors.forename: María Jesús
authors.initials: MJ
authors.lastname: Arrizubieta
authors.forename: Alejandro
authors.initials: A
authors.lastname: Toledo-Arana
authors.forename: Beatriz
authors.initials: B
authors.lastname: Amorena
authors.forename: José R
authors.initials: JR
authors.lastname: Penadés
authors.forename: Iñigo
authors.initials: I
authors.lastname: Lasa
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001422:Bacterial Adhesion / Q000187:drug effects; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018441:Biofilms / Q000187:drug effects* / Q000254:growth & development; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology*; D002417:Cattle; D003470:Culture Media; D020832:EF Hand Motifs; D005260:Female; D015964:Gene Expression Regulation, Bacterial; D008414:Mastitis, Bovine / Q000382:microbiology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D013203:Staphylococcal Infections / Q000382:microbiology / Q000662:veterinary; D013211:Staphylococcus aureus / Q000187:drug effects / Q000254:growth & development*
chemical_list: D001426:Bacterial Proteins; C425726:Bap protein, Staphylococcus aureus; D003470:Culture Media; D002118:Calcium