Recombinant expression and characterization of a Xylella fastidiosa cysteine protease differentially expressed in a nonpathogenic strain.
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.
Nogaroto
Viviane
V
Laboratory of Molecular Biology, Department of Genetics and Evolution, Federal University of São Carlos, SP, Brazil.
Tagliavini
Sandraa
S
Gianotti
Andréia
A
Mikawa
Angela
A
Barros
Nilanam T
NT
Puzer
Luciano
L
Carmona
Adriana K
AK
Costa
Pauloi
P
Henrique-Silva
Flávio
F
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*
D011994:Recombinant Proteins; D003546:Cysteine Endopeptidases