title: Recombinant expression and characterization of a Xylella fastidiosa cysteine protease differentially expressed in a nonpathogenic strain.
abstract: Xylella fastidiosa is a xylem-limited, Gram-negative bacterium responsible for citrus variegated chlorosis (CVC) in sweet oranges. In the present study, we present the recombinant expression, purification and characterization of an X. fastidiosa cysteine protease (dubbed Xylellain). The recombinant Xylellain ((HIS)Xylellain) was able to hydrolyze carbobenzoxy-Phe-Arg-7-amido-4-methylcoumarin (Z-FR-MCA) and carbobenzoxy-Arg-Arg-7-amido-4-methylcoumarin (Z-RR-MCA) with similar catalytic efficiencies, suggesting that this enzyme presents substrate specificity requirements similar to cathepsin B. The immunization of mice with (HIS)Xylellain provided us with antibodies, which recognized a protein of c. 31 kDa in the X. fastidiosa pathogenic strains 9a5c, and X. fastidiosa isolated from coffee plants. However, these antibodies recognized no protein in the nonpathogenic X. fastidiosa J1a12, suggesting the absence or low expression of this protein in the strain. These findings enabled us to identify Xylellain as a putative target for combating CVC and other diseases caused by X. fastidiosa strains.
authors.affiliation: Laboratory of Molecular Biology, Department of Genetics and Evolution, Federal University of São Carlos, SP, Brazil.
authors.forename: Viviane
authors.initials: V
authors.lastname: Nogaroto
authors.forename: Sandraa
authors.initials: S
authors.lastname: Tagliavini
authors.forename: Andréia
authors.initials: A
authors.lastname: Gianotti
authors.forename: Angela
authors.initials: A
authors.lastname: Mikawa
authors.forename: Nilanam T
authors.initials: NT
authors.lastname: Barros
authors.forename: Luciano
authors.initials: L
authors.lastname: Puzer
authors.forename: Adriana K
authors.initials: AK
authors.lastname: Carmona
authors.forename: Pauloi
authors.initials: P
authors.lastname: Costa
authors.forename: Flávio
authors.initials: F
authors.lastname: Henrique-Silva
mesh_terms: D000595:Amino Acid Sequence; D015153:Blotting, Western; D032084:Citrus sinensis / Q000382:microbiology; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D010935:Plant Diseases; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D044167:Xylella / Q000201:enzymology* / Q000235:genetics / Q000472:pathogenicity*
chemical_list: D011994:Recombinant Proteins; D003546:Cysteine Endopeptidases