title: Chitinases are negative regulators of Francisella novicida biofilms.
abstract: Biofilms, multicellular communities of bacteria, may be an environmental survival and transmission mechanism of Francisella tularensis. Chitinases of F. tularensis ssp. novicida (Fn) have been suggested to regulate biofilm formation on chitin surfaces. However, the underlying mechanisms of how chitinases may regulate biofilm formation are not fully determined. We hypothesized that Fn chitinase modulates bacterial surface properties resulting in the alteration of biofilm formation. We analyzed biofilm formation under diverse conditions using chitinase mutants and their counterpart parental strain. Substratum surface charges affected biofilm formation and initial attachments. Biophysical analysis of bacterial surfaces confirmed that the chi mutants had a net negative-charge. Lectin binding assays suggest that chitinase cleavage of its substrates could have exposed the concanavalin A-binding epitope. Fn biofilm was sensitive to chitinase, proteinase and DNase, suggesting that Fn biofilm contains exopolysaccharides, proteins and extracellular DNA. Exogenous chitinase increased the drug susceptibility of Fn biofilms to gentamicin while decreasing the amount of biofilm. In addition, chitinase modulated bacterial adhesion and invasion of A549 and J774A.1 cells as well as intracellular bacterial replication. Our results support a key role of the chitinase(s) in biofilm formation through modulation of the bacterial surface properties. Our findings position chitinase as a potential anti-biofilm enzyme in Francisella species.
authors.affiliation: School of Systems Biology and the National Center for Biodefense and Infectious Diseases, George Mason University, Manassas, Virginia, United States of America.
authors.forename: Myung-Chul
authors.initials: MC
authors.lastname: Chung
authors.affiliation: School of Systems Biology and the National Center for Biodefense and Infectious Diseases, George Mason University, Manassas, Virginia, United States of America.
authors.forename: Scott
authors.initials: S
authors.lastname: Dean
authors.affiliation: School of Systems Biology and the National Center for Biodefense and Infectious Diseases, George Mason University, Manassas, Virginia, United States of America.
authors.forename: Ekaterina S
authors.initials: ES
authors.lastname: Marakasova
authors.affiliation: School of Systems Biology and the National Center for Biodefense and Infectious Diseases, George Mason University, Manassas, Virginia, United States of America.
authors.forename: Albert O
authors.initials: AO
authors.lastname: Nwabueze
authors.affiliation: School of Systems Biology and the National Center for Biodefense and Infectious Diseases, George Mason University, Manassas, Virginia, United States of America.
authors.forename: Monique L
authors.initials: ML
authors.lastname: van Hoek
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001422:Bacterial Adhesion / Q000187:drug effects / Q000502:physiology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D018441:Biofilms / Q000187:drug effects / Q000254:growth & development*; D002460:Cell Line; D002688:Chitinases / Q000235:genetics / Q000378:metabolism*; D005603:Francisella / Q000502:physiology*; D005839:Gentamicins / Q000494:pharmacology; D006801:Humans; D009154:Mutation
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D005839:Gentamicins; D002688:Chitinases