Abstract
Full expression of the elastolytic phenotype of Pseudomonas aeruginosa depends on LasA, an extracellular protease with restricted specificity whose mode of action on elastin and biological role is not understood. LasA exhibits amino acid sequence homology to some bacteriolytic proteases and shares several physicochemical properties with the staphylolytic protease of P. aeruginosa. This led us to examine whether the two proteases are the same. Production of LasA and staphylolytic protease by prototrophic and lasA mutant strains of P. aeruginosa was investigated. The two prototrophic strains examined, PAO1 and FRD2, exhibited extracellular staphylolytic activity and secreted LasA. LasA mutants, PAO-E64 lasA1 (Ts), FRD2128 delta lasA, and FRD244 lasA::mTn10, did not exhibit staphylolytic activity. A low level of the LasA protein was detected in the culture filtrate of the temperature-sensitive lasA mutant PAO-E64, but none was detectable in those of the deletion and insertion mutants, FRD2128, and FRD244, respectively. The staphylolytic protease was purified from the culture filtrate of P. aeruginosa strain FRD2 by DEAE-cellulose chromatography. The purified enzyme hydrolyzed pentaglycine into the respective di- and tripeptides and reacted specifically with antibodies against a synthetic peptide identical in sequence to positions 77-98 in LasA. The amino-terminal sequence of the first 15 amino acid residues of the staphylolytic protease was found to be identical with that of the secreted LasA. These results clearly indicate that LasA is a staphylolytic protease. In addition to lysing staphylococci, it may enhance elastolysis by cleaving Gly-Gly bonds, which are abundant in elastin.
Highlights
Full expressionof the elastolyticphenotype of Pseu- with the virulence of this opportunistic pathogen [1,2]
The elastase structural gene h 3 indicated that these genes are staphylolytic protease was purified from the culture distinct [13]
In thepresent studywe provide genetic, immunological, and biochemical evidence that this is the case, and we suggest that LasA may enhance elastolysis by cleaving Gly-Gly peptide bonds, which are abundant in elastin
Summary
Bacterial Strainsand Culture Conditions-The P. aeruginosa strains used were: PA01 (prototroph, h A + ) ; PAO-E64 ( h A I ) , a temperature-sensitive mutant of P A 0 1 with reduced elastolytic activity [12]; FRD2 ( h A + ) , a non-mucoid revertant of the prototroph FRDl [29]; and its derivatives FRD2128 ( A h A xxx::Tn501) ( X ) , FRD244 (lusA::miniTnlO xxx::Tn501) [15], and FRD240 Following selection for resistance to HgCl, (18 pg/ml) encoded by Tn501, colonies were screened for tetracycline sensitivity, indicating loss of vector sequences and gene replacement. Enzyme (0.002-0.2 units) was added to 1 mlof 0.3% azocasein (Sigma) in 0.05 M Tris-HC1, 0.5 mM CaCl2,pH 7.5 (buffer B) andincubated at 37 "C for 15 min. Reactions were stopped by adding trichloroacetic acid (lo%,0.5 ml) followedby centrifugation and measurement of absorbance at 400 nm of the clear supernatants. One unit of activity is the amount of enzyme which causes an optical density increase of 1unit/min. Staphylolytic activity was determined by measuring the rate of lysis of an S.aurew (strainNewman DzC from Sigma) cell suspension [32]. One unit of activity is the amount of enzyme which causes an Ass decrease of 1unit/min. Elastolytic activity was determined with elastin-Congo red (Sigma) as thesubstrate. GenBank accession numbers of the LasA sequence and those of L. enzymogenes and A. lyticus 8-lytic proteases are M20982, -400994, and A37151, respectively
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
