A 22-kilodalton protein purified from the culture supernatant fraction of Pseudomonas aeruginosa (strains PA220 and PAO1) was found to enhance the elastolytic activity of purified P. aeruginosa elastase. N-terminal sequence analysis identified the protein as a fragment of the lasA gene product (P. A. Schad and B. H. Iglewski, J. Bacteriol. 170:2784Bacteriol. 170: -2789Bacteriol. 170: , 1988. However, comparative analysis with the reported LasA sequence indicated that the purified LasA fragment is longer than the deduced sequence reported. The purified LasA fragment had minimal elastolytic and proteolytic activity and did not enhance the proteolytic activity of purified elastase, yet enhanced the elastolytic activity more than 25-fold. The LasA fragment was found to also enhance the elastolytic activities of thermolysin, human neutrophil elastase, and proteinase K. The results presented here suggest that the LasA protein interacts with the elastin substrate rather than modifying elastase.

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Full elastolytic activity in Pseudomonas aeruginosa is a result of the combined activities of elastase, alkaline proteinase, and the lasA gene product, LasA. The results of this study demonstrate that an active fragment of the LasA protein which is isolated from the culture supernatant fraction is capable of degrading elastin in the absence of elastase, thus showing that LasA is a second elastase produced by this organism. In addition, it is shown that LasA-mediated enhancement of elastolysis results from the separate activities of LasA and elastase upon elastin. The LasA protein does not affect the secretion or activation of a proelastase as previously proposed in other studies. Furthermore, LasA has specific proteolytic capability, as demonstrated by its ability to cleave beta-casein. Preliminary analysis of beta-casein cleavage in the presence of various protease inhibitors suggests that LasA may be classified as a modified serine protease.

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Two ceftazidime-resistant variants of Pseudomonas aeruginosa (PA-48, PA-60), obtained from cardiac vegetations of rabbits with endocarditis receiving ceftazidime therapy, were studied for mechanisms of resistance. Both resistant variants were stably derepressed for the type Id I-lactamase, which was ceftazidime inducible in the parental strain (PA-96) used to initially infect the rabbits. There was no evidence of ceftazidime bioinactivation by the resistant strains, and their outer membrane permeabilities were comparable to those of the parental strain. No alterations were observed in patterns of outer membrane proteins or membrane lipopolysaccharides in the resistant variants as compared with the parental strain. Penicillin-binding protein patterns of the resistant variants revealed the absence of penicillin-binding protein 4 in both, with acquisition of a new protein of higher apparent molecular weight in PA-60. Calculation of the rate of appearance of ceftazidime in the periplasm at sub-MICs suggested that slow enzymatic hydrolysis of the P-lactam, rather than nonhydrolytic trapping, was the major explanation for the induced resistance in vivo in strains PA-48 and PA-60.,-Lactam antibiotics are of primary use in the therapy of gram-negative bacterial infections. However, the appearance of bacterial resistance to successive generations of P-lactams (30,36) has necessitated the continuous development and assessment of new compounds. The major impediments to ,-lactam therapy have been the high intrinsic resistance of some organisms, including Pseudomonas aeruginosa due to the intrinsic barrier properties of their outer membranes (21, 39) and the acquisition of high levels of P-lactamase due either to the presence of a plasmid or to the derepression of a formerly inducible, chromosomally encoded , B-lactamase (30, 34, 36). Mutational alterations in the 3-lactam targets, penicillin-binding proteins (PBPs), have also been observed (26) but seem to occur with somewhat less frequency. One novel approach taken by pharmaceutical companies in the development of newer ,-lactam antibiotics has been to search for compounds that are refractory to ,-lactamase-mediated hydrolysis. Recently, however, it has been observed that, despite the extremely low rates at which these compounds are hydrolyzed, clinical isolates of P. aeruginosa and Enterobacter cloacae that are resistant to these so-called P-lactamase-stable P-lactams often have much-increased levels of the chromosomal P-lactamase (4,30,31,(34)(35)(36). The mechanism of resistance has been suggested to involve nonhydrolytic binding of the ,-lactam by molecules of ,-lactamase, thus reducing the periplasmic concentration (i.e., the concentration in the vicinity of the target PBPs) to subinhibitory levels (34). Alternatively, Vu and Nikaido (35) have argued convincingly that a combination of low-level hydrolysis by ,-lactamase (low Km. low Vmax) and reduced outer membrane permeability, thus di-* Corresponding author. t Present address: