Molecular analysis of Pseudomonas aeruginosa protease IV expressed in Pseudomonas putida.
OBJECTIVE
In this study, the protease IV gene of Pseudomonas aeruginosa was expressed in the nonocular pathogenic host, Pseudomonas putida, to elucidate the molecular properties and virulence contribution of the enzyme. Recent determination of the protease IV gene sequence suggests that the protein of 463 amino acids contains a signal sequence, a propeptide domain, and a mature protease. The only form of this protein that has been detected previously is the extracellular mature protease.


METHODS
The protease IV gene was cloned and expressed in a protease IV-negative Pseudomonas species, P. putida. The cloned protease IV gene product was analyzed to identify biochemical, enzymatic, and immunologic properties and its contribution to corneal virulence.


RESULTS
P. putida expressing the cloned protease IV gene had significantly greater extracellular enzyme activity than P. aeruginosa. These P. putida cell extracts produced a protein with the same molecular mass as mature protease IV and two other polypeptides representing larger precursors, all of which were recognized by protease IV-specific antibodies. P. putida producing protease IV, relative to P. putida with the vector alone, caused a threefold increase in ocular inflammation and tissue damage when intrastromally injected into rabbit corneas.


CONCLUSIONS
The present study demonstrates for the first time that protease IV is synthesized as a large precursor that is processed intracellularly through an intermediate form and secreted into the extracellular milieu as a mature protease. The results also confirm a significant correlation between production of protease IV and corneal virulence.
Traidej
Mullika
M
Department of Microbiology, Immunology, and Parasitology, Louisiana State University Health Sciences Center, 1901 Perdido Street, New Orleans, LA 70112, USA.
Caballero
Armando R
AR
Marquart
Mary E
ME
Thibodeaux
Brett A
BA
O'Callaghan
Richard J
RJ
D000595:Amino Acid Sequence; D000818:Animals; D003319:Corneal Stroma / Q000382:microbiology; D004269:DNA, Bacterial / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D015818:Eye Infections, Bacterial / Q000382:microbiology*; D015971:Gene Expression Regulation, Enzymologic; D007634:Keratitis / Q000382:microbiology; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000235:genetics* / Q000302:isolation & purification; D010957:Plasmids; D011552:Pseudomonas Infections / Q000382:microbiology; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D016958:Pseudomonas putida / Q000201:enzymology* / Q000472:pathogenicity*; D011817:Rabbits; D012697:Serine Endopeptidases / Q000235:genetics* / Q000302:isolation & purification; D014774:Virulence
D004269:DNA, Bacterial; D010447:Peptide Hydrolases; C038495:Pseudomonas serine proteinase; D012697:Serine Endopeptidases; C030732:protease IV