Further studies on Pseudomonas aeruginosa LasA: analysis of specificity

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.     

Efficient production and processing of elastase and LasA by Pseudomonas aeruginosa require zinc and calcium ions.

The ability of Pseudomonas aeruginosa to degrade elastin, a major component of connective tissue, likely contributes to its pathogenicity and multiplication in human tissues. Two extracellular enzymes are required for P. aeruginosa elastolytic activity: elastase and LasA. Elastase is a zinc metalloprotease, but little is known about the structure of LasA. When grown under metal ion-deficient conditions, P. aeruginosa culture supernatants were found to exhibit a low level of elastolytic activity, which coincided with production of low levels of the 51-kDa proelastase and no detectable LasA. By using this fact to identify factors that promote elastolytic activity, P. aeruginosa PAO1, FRD2, and DG1 were grown in metal ion-deficient medium supplemented with zinc (10(-4) M ZnCl2), calcium (2.5 x 10(-3) M CaCl2), or iron (10(-4) M FeCl3). High levels of proteolytic and elastolytic activity were exhibited by all strains when cultured in the presence of both zinc and calcium, and this was associated with the production of mature 33-kDa elastase and 21-kDa LasA. Supplementing DG1 and PAO1 cultures with zinc alone stimulated the production of 33-kDa elastase, which, because of the calcium-deficient conditions, exhibited low proteolytic and elastolytic activities. Zinc also stimulated the production of a 41-kDa form of LasA in DG1 and PAO1 culture supernatants. Elastase production by FRD2 cultured in the presence of zinc alone differed from that by the other two strains in that supernatants contained 33-kDa elastase, a 21-kDa form of LasA, and exhibited high proteolytic and elastolytic activities. Such strain-associated differences in LasA processing and elastase activity can be explained by differences in metal ion-scavenging mechanisms adapted by the strains. Supplementing cultures with calcium stimulated the production of elastase but had no effect on LasA production. The elastase produced exhibited variable sizes, possibly resulting from aberrant processing reactions, and showed little proteolytic activity. Proteolytic activity could be recovered from 33-kDa elastase produced in the presence of calcium by inclusion of zinc in the enzymatic assay. Although iron was previously found to exert a repressive effect on P. aeruginosa elastolytic activity, iron exerted little effect on elastolytic activity when added to cultures containing both zinc and calcium. These studies support the conclusion that elastase production and processing are promoted by both zinc and calcium. LasA production, in comparison, is stimulated by zinc, with both zinc and calcium facilitating its processing. The association of 41-kDa LasA with a low level of elastolytic activity and of 21-kDa LasA with a high level of activity supports the conclusion that lasA encodes a larger, precursor protein which is processed to an active 21-kDa form during secretion.     

Purification and characterization of an active fragment of the LasA protein from Pseudomonas aeruginosa: enhancement of elastase activity.

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. 170:2784-2789, 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.     

A continuous spectrophotometric assay for Pseudomonas aeruginosa LasA protease (staphylolysin) using a two-stage enzymatic reaction

Pseudomonas aeruginosa LasA protease is a secreted metalloendopeptidase that can lyse Staphylococcus aureus cells by cleaving the pentaglycine bridges of their peptidoglycan. It can also degrade elastin and stimulate shedding of cell-surface proteoglycans, activities implicated in pathogenesis of P. aeruginosa infections. The activity of LasA protease can be assayed spectrophotometrically by following the reduction in turbidity of S. aureus cell suspensions. This assay, however, does not permit kinetic studies and its reproducibility is poor. Here we describe a two-stage enzymatic reaction for the continuous measurement of LasA protease activity using a defined substrate, succinyl-Gly-Gly-Phe-4-nitroanilide, supplemented with Streptomyces griseus aminopeptidase. Cleavage of the Gly-Phe bond by LasA protease is followed by hydrolysis of the product Phe-4-nitroanilide by the aminopeptidase and the rate of release of the chromophore (4-nitroaniline) is measured spectrophotometrically using a 96-well microplate reader. Activity of nanogram amounts of LasA protease could be determined within a few minutes. Furthermore, this assay permitted the determination of Km and kcat values for LasA protease, which were 0.46 mM and 11.8s(-1), respectively. Pseudomonas elastase was also active in the assay. However, it was less effective than LasA protease and its activity was inhibited by phosphoramidon. The assay is highly sensitive and reproducible, providing a convenient tool for further studies of LasA protease function(s) and mechanism of action.     

Pseudomonas aeruginosa LasB mutant constructed by insertional mutagenesis reveais elastolytic activity due to alkaline proteinase and the LasA fragment

The extracellularly secreted endopeptidase elastase (LasB) is regarded as an important virulence factor of Pseudomonas aeruginosa. It has also been implicated in the processing of LasA which enhances elastolytic activity of LasB. In order to investigate the role of LasB in virulence and LasA processing, a LasB-negative mutant, PAO1E, was constructed by insertional mutagenesis of the LasB structural gene, lasB, in P. aeruginosa PAO. An internal 636 bp lasB fragment of the plasmid pRB1803 was ligated into a derivative of the mobilization vector pSUP201-1. The resulting plasmid, pBRMOB-LasB, was transformed into Escherichia coli and transferred by filter matings to the LasB-positive P. aeruginosa strain, PAO1. Plasmid integration in the lasB site of the chromosome was confirmed by Southern blot analysis. Radioimmunoassay and immunoblotting of PAO1E supernatant fluids yielded no detectable LasB (less than 1 ng ml-1 LasB). The absence of LasB in PAO1E was further proven by the inability of its culture supernatant fluid to cleave transferrin or rabbit immunoglobulin G (IgG) after a 72 h incubation. The residual proteolytic activity of PAO1E culture supernatant fluid was attributed to alkaline proteinase (Apr), since it was totally inhibited by specific antibodies against Apr. Residual elastolytic activity in culture supernatant fluid of PAO1E was due to the LasA fragment and to the combined action of the LasA fragment with Apr on elastin. The sizes of purified LasA from PAO1 and PAO1E were identical (22 kDa). These results show that, besides LasB and the LasA fragment, Apr may also act on elastin in the presence of the LasA fragment and that the proteolytic processing of LasA in P. aeruginosa is independent of LasB.     

Pseudomonas aeruginosa proteases and corneal virulence

Pseudomonas aeruginosa is a common cause of corneal infections, particularly among users of soft contact lenses. Previous studies with chemically induced mutants deficient in alkaline protease (AP) or elastase (LasB) suggested that these proteases contributed to the rapid liquifactive stromal necrosis characteristic of P. aeruginosa corneal infections. Because these mutants might harbor other chromosomal changes that could affect virulence, the role of these proteases in the pathogenesis of corneal disease (as well as a second elastase, LasA protease) was reexamined by constructing isogenic mutants deficient only in these enzymes. Allelic exchange was used to construct mutants of P. aeruginosa PAO1-V deficient in AP (PAO1-V AP[ - ]), LasB and LasA protease (PDO801 LasB[ - ]), or all three proteases (PDO801 TM). These mutants were then evaluated for virulence using mouse scratch and rabbit intrastromal injection models of corneal disease. Loss of AP significantly increased disease scores in the rabbit (P < 0.030) but not the mouse (P > 0.060) model of infection. Loss of both elastases had no effect on ocular virulence in either animal model of corneal disease (P > 0.100). The loss of all three proteases significantly decreased disease scores in the rabbit (P < 0.035), but not in the mouse (P > 0.110). Taken together, these data suggest that AP, LasB, and LasA protease are not essential for initiating or maintaining a corneal infection. Furthermore, AP appears to be an important mediator of pathology depending on the location of the organism within the cornea and whether or not concomitant elastolytic activity is present.     

Purification and characterization of LasD: a second staphylolytic proteinase produced by Pseudomonas aeruginosa

We have previously described studies of a 22 kDa active fragment of the LasA proteinase. In follow-up studies of LasA, we have discovered the separate existence of a 23 kDa proteinase which shares many of the enzymatic properties of LasA, including the ability to lyse heat-killed staphylococoi. However, this apparent serine proteinase, which we designate LasD, is distinct from the 22 kDa active LasA protein for the following reasons: (i) the N-terminal sequence of LasD shares no homology with LasA or the LasA precursor sequence; (ii) Pseudomonas aeruginosa LasA mutant strains AD1825 and FRD2128 do not produce LasA yet produce LasD; and (iii) specific antibodies to each proteinase do not show any cross-reactivity. LasD appears to be produced as a 30 kDa protein, which is possibly cleaved to produce a 23 kDa active fragment. The purified LasD fragment (23 kDa) shows strong staphylolytic activity only at higher pH conditions, while LasA exhibits staphylolytic activity over a broad pH range, in addition to their ability to cleave at internal diglycine sites, both the LasD and LasA endoproteinases efficiently cleave β-casein.     

Characterization of a proton pumping transmembrane protein incorporated into a supported three-dimensional matrix of proteoliposomes

A highly sensitive assay based on new internally quenched fluorogenic peptide substrates has been developed for monitoring protease activities. These novel substrates comprise an Edans (5-(2-aminoethylamino)-1-naphthalenesulfonic acid) group at the C terminus and a Dabsyl (4-(dimethylamino)azobenzene-4'-sulfonyl chloride) fluorophore at the N terminus of the peptide chains. The Edans fluorescence increases upon peptide hydrolysis by Pseudomonas aeruginosa proteases, and this increase is directly proportional to the amount of substrate cleaved, i.e., protease activity. The substrates Dabsyl-Ala-Ala-Phe-Ala-Edans and Dabsyl-Leu-Gly-Gly-Gly-Ala-Edans were used for testing the peptidasic activities of P. aeruginosa elastase and LasA protease, respectively. Elastase and LasA kinetic parameters were calculated and a sensitive assay was designed for the detection of P. aeruginosa proteases in bacterial supernatants. The sensitivity and the small sample requirements make the assay suitable for high-throughput screening of biological samples. Furthermore, this P. aeruginosa protease assay improves upon existing assays because it is simple, it requires only one step, and even more significantly it is enzyme specific.     

A substitution at His-120 in the LasA protease of Pseudomonas aeruginosa blocks enzymatic activity without affecting propeptide processing or extracellular secretion.

The LasA protease of Pseudomonas aeruginosa can degrade elastin and is an important contributor to the pathogenesis of this organism. LasA (20 kDa) is a member of the beta-lytic endopeptidase family of extracellular bacterial proteases, and it shows high-level staphylolytic activity. We sequenced the lasA gene from strain FRD1 and overexpressed it in Escherichia coli. The lasA gene encodes a precursor, known as pre-proLasA, of 45,582 Da. Amino-terminal sequence analysis allowed the identification of the signal peptidase cleavage site and revealed that the 31-amino-acid signal peptide was removed in E. coli. The remaining proLasA (42 kDa) did not undergo autoproteolytic processing and showed little staphylolytic activity. However, it was readily processed to a 20-kDa active staphylolytic protease by incubation with trypsin or with the culture filtrate of a P. aeruginosa lasAdelta mutant. Thus, removal of the propeptide (22 kDa) was required to convert proLasA into an active protease. Although LasA protease was critical for staphylolytic activity, other proteases like elastase were found to enhance staphylolysis. Under the control of an inducible trc promoter, lasA was overexpressed in P. aeruginosa and the processing intermediates were examined. Compared with wild-type cells, the overproducing cells accumulated more 42-kDa proLasA species, and the culture supernatants of the overproducing cells showed increased levels of active 20-kDa LasA protease. Small amounts of a 25-kDa extracellular LasA-related protein, which could represent a potential processing intermediate, were also observed. To better understand the structure-function relationships in LasA protease, we tested whether His-120-X-His-122 in the mature portion of LasA plays a role in activity. This motif and surrounding sequences are conserved in the related beta-lytic protease of Achromobacter lyticus. Oligonucleotide-directed mutagenesis was used to change His-120 to Ala-120, thus forming the lasA5 allele. The product of lasA5 expressed from the chromosome of P. aeruginosa was processed to a stable, secreted 20-kDa protein (designated LasA-H120A) which was devoid of staphylolytic activity. This suggests that His-120 is essential for LasA activity and favors the possibility that proLasA processing and secretion in P. aeruginosa can proceed via mechanisms which do not involve autoproteolysis.     

A novel secreted protease from Pseudomonas aeruginosa activates NF-kappaB through protease-activated receptors

The Pseudomonas aeruginosa -derived alkaline protease (AprA), elastase A (LasA), elastase B (LasB) and protease IV are considered to play an important role in pathogenesis of this organism. Although the sequence analysis of P. aeruginosa genome predicts the presence of several genes encoding other potential proteases in the genome, little has been known about the proteases involving in pathogenesis. Recently, Porphyromonas gingivalis gingipains and Serratia marcescens serralysin have been shown to activate protease-activated receptor 2 (PAR-2), thereby modulating host inflammatory and immune responses. Accordingly, we hypothesized that unknown protease(s) from P. aeruginosa would also modulate such responses through PARs. In this study, we found that P. aeruginosa produces a novel l arge e xo p rotease (LepA) distinct from known proteases such as AprA, LasA, LasB and protease IV. Sequence analysis of LepA showed a molecular feature of the proteins transported by the two-partner secretion pathway. Our results indicated that LepA activates NF-κB-driven promoter through human PAR-1, -2 or -4 and cleaves the peptides corresponding to the tethered ligand region of human PAR-1, -2 and -4 at a specific site with exposure of their tethered ligands. Considered together, these results suggest that LepA would require PARs to modulate various host responses against bacterial infection.     

Secretion of Elastinolytic Enzymes and Their Propeptides by Pseudomonas aeruginosa

Elastase of Pseudomonas aeruginosa is synthesized as a preproenzyme. The signal sequence is cleaved off during transport across the inner membrane and, in the periplasm, proelastase is further processed. We demonstrate that the propeptide and the mature elastase are both secreted but that the propeptide is degraded extracellularly. In addition, reduction of the extracellular proteolytic activity led to the accumulation of unprocessed forms of LasA and LasD in the extracellular medium, which shows that these enzymes are secreted in association with their propeptides. Furthermore, a hitherto undefined protein with homology to a Streptomyces griseus aminopeptidase accumulated under these conditions.     

Syndecan-1 shedding is enhanced by LasA, a secreted virulence factor of Pseudomonas aeruginosa

Microbial pathogens frequently take advantage of host systems for their pathogenesis. Shedding of cell surface molecules as soluble extracellular domains (ectodomains) is one of the host responses activated during tissue injury. In this study, we examined whether pathogenic bacteria can modulate shedding of syndecan-1, the predominant syndecan of host epithelia. Our studies found that overnight culture supernatants of Pseudomonas aeruginosa and Staphylococcus aureus enhanced the shedding of syndecan-1 ectodomains, whereas culture supernatants of several other Gram-negative and Gram-positive bacteria had only low levels of activity. Because supernatants from all tested strains of P. aeruginosa (n = 9) enhanced syndecan-1 shedding by more than 4-fold above control levels, we focused our attention on this Gram-negative bacterium. Culture supernatants of P. aeruginosa increased shedding of syndecan-1 in both a concentration- and time-dependent manner, and augmented shedding by various host cells. A 20-kDa shedding enhancer was partially purified from the supernatant through ammonium sulfate precipitation and gel chromatography, and identified by N-terminal sequencing as LasA, a known P. aeruginosa virulence factor. LasA was subsequently determined to be a syndecan-1 shedding enhancer from the findings that (i) immunodepletion of LasA from the partially purified sample resulted in abrogation of its activity to enhance shedding and (ii) purified LasA increased shedding in a concentration-dependent manner. Our results also indicated that LasA enhances syndecan-1 shedding by activation of the host cell's shedding mechanism and not by direct interaction with syndecan-1 ectodomains. Enhanced syndecan-1 shedding may be a means by which pathogenic bacteria take advantage of a host mechanism to promote their pathogenesis.     

Role of bacterial proteases in pseudomonal and serratial keratitis

Pseudomonas aeruginosa and Serratia marcescens can cause refractory keratitis resulting in corneal perforation and blindness. These bacteria produce various kinds of proteases. In addition to pseudomonal elastase (LasB) and alkaline protease, LasA protease and protease IV have recently been found to be more important virulence factors of P. aeruginosa . S. marcescens produces a cysteine protease in addition to metalloproteases. These bacterial proteases have a number of biological activities, such as degradation of tissue constituents and host defense-oriented proteins, as well as activation of zymogens (Hageman factor, prekallikrein and pro-matrix metalloproteinases) through limited proteolysis. In this article, the properties of these bacterial proteases are reviewed and the pathogenic roles of these proteases in pseudomonal keratitis are discussed.     

Pseudomonas aeruginosa protease IV enzyme assays and comparison to other Pseudomonas proteases

Pseudomonas aeruginosa secretes multiple proteases that have been implicated as virulence factors and the detection of each specific enzyme can be difficult to determine. Unlike the three Pseudomonas enzymes that have been well characterized (elastase A, elastase B, and alkaline protease), the activity of protease IV in multiple assays has yet to be described. This study defines new assays for Pseudomonas proteases and compares protease IV activity to the activities of elastase A, elastase B, and alkaline protease. Six in vitro assays were studied: zymography, elastin congo red assay, staphylolytic assay, colorimetric peptide assay, solid-phase colorimetric peptide assay, and poly-l-lysine degradation. Casein zymography distinguished protease IV from elastase B and alkaline protease, and gelatin zymography differentiated all four proteases. The elastin congo red assay detected mainly elastase B while the staphylolytic assay was specific for elastase A. Protease IV activity was assayed specifically by the colorimetric assay and two new assays, the solid-phase colorimetric assay and degradation of poly-L-lysine in the presence of EDTA. Alkaline protease could be specifically assayed by poly-L-lysine degradation in the presence of N-alpha-p-tosyl-L-lysine chloromethyl ketone. The results identified three specific assays for protease IV, a new assay specific for alkaline protease, and showed that protease IV has a distinct enzymatic specificity relative to the three other Pseudomonas proteases.     

Crystal Structure of the LasA Virulence Factor from Pseudomonas aeruginosa: Substrate Specificity and Mechanism of M23 Metallopeptidases

Pseudomonas aeruginosa is an opportunist Gram-negative bacterial pathogen responsible for a wide range of infections in immunocompromized individuals and is a leading cause of mortality in cystic fibrosis patients. A number of secreted virulence factors, including various proteolytic enzymes, contribute to the establishment and maintenance of Pseudomonas infection. One such is LasA, an M23 metallopeptidase related to autolytic glycylglycine endopeptidases such as Staphylococcus aureus lysostaphin and LytM, and to DD-endopeptidases involved in entry of bacteriophage to host bacteria. LasA is implicated in a range of processes related to Pseudomonas virulence, including stimulating ectodomain shedding of the cell surface heparan sulphate proteoglycan syndecan-1 and elastin degradation in connective tissue. Here we present crystal structures of active LasA as a complex with tartrate and in the uncomplexed form. While the overall fold resembles that of the other M23 family members, the LasA active site is less constricted and utilizes a different set of metal ligands. The active site of uncomplexed LasA contains a five-coordinate zinc ion with trigonal bipyramidal geometry and two metal-bound water molecules. Using these structures as a starting point, we propose a model for substrate binding by LasA that explains its activity against a wider range of substrates than those used by related lytic enzymes, and offer a catalytic mechanism for M23 metallopeptidases consistent with available structural and mutagenesis data. Our results highlight how LasA is a structurally distinct member of this endopeptidase family, consistent with its activity against a wider range of substrates and with its multiple roles in Pseudomonas virulence.     

Identification of critical residues in the propeptide of LasA protease of Pseudomonas aeruginosa involved in the formation of a stable mature protease

LasA protease is a 20-kDa elastolytic and staphylolytic enzyme secreted by Pseudomonas aeruginosa. LasA is synthesized as a preproenzyme that undergoes proteolysis to remove a 22-kDa amino-terminal propeptide. Like the propeptides of other bacterial proteases, the LasA propeptide may act as an intramolecular chaperone that correctly folds the mature domain into an active protease. To locate regions of functional importance within proLasA, linker-scanning insertional mutagenesis was employed using a plasmid containing lasA as the target. Among the 5 missense insertions found in the mature domain of proLasA, all abolished enzymatic activity but not secretion. In general, the propeptide domain was more tolerant to insertions. However, insertions within a 9-amino-acid region in the propeptide caused dramatic reductions in LasA enzymatic activity. All mutant proLasA proteins were still secreted, but extracellular stability was low due to clustered insertions within the propeptide. The codons of 16 residues within and surrounding the identified 9-amino-acid region were subjected to site-directed mutagenesis. Among the alanine substitutions in the propeptide that had a major effect on extracellular LasA activity, two (L92A and W95A) resulted in highly unstable proteins that were susceptible to proteolytic degradation and three (H94A, I101A, and N102A) were moderately unstable and allowed the production of a LasA protein with low enzymatic activity. These data suggest that these clustered residues in the propeptide may play an important role in promoting the correct protein conformation of the mature LasA protease domain.     

Iron facilitates the RetS-Gac-Rsm cascade to inversely regulate protease IV (piv) expression via the sigma factor PvdS in Pseudomonas aeruginosa

Pseudomonas aeruginosa produces several proteases, such as an elastase (LasB protease), a LasA protease, and protease IV (PIV), which are thought as significant virulence factors during infection. Regulators of LasA and LasB expression have been identified and well characterized; however, the molecular details of this regulation of protease IV (PIV) remained largely unknown. Here, we describe the interaction between protease IV and the RetS/Rsm signalling pathway, which plays a central role in controlling the production of multiple virulence factors and the switch from planktonic to biofilm lifestyle. We show that the expression of piv was reduced in ΔretS or ΔrsmA strain grown under restrictive conditions but was induced in ΔretS or ΔrsmA mutant grown under rich conditions as compared with wild-type parent. We compare the expression of piv under various conditions and found that iron facilitates RetS/Rsm system to lead this inverse regulation. Moreover, we reveal that the RetS/Rsm pathway regulates PIV production dependent on the alternative sigma factor PvdS. Collectively, this study extends the understanding of the RetS/Rsm regulatory cascade in response to environmental signals and provides insights into how P. aeruginosa adapts to the complex conditions.     

Identification of extracellular N-acylhomoserine lactone acylase from a Streptomyces sp. and its application to quorum quenching

N-acylhomoserine lactones (AHLs) play an important role in regulating virulence factors in pathogenic bacteria. Recently, the enzymatic inactivation of AHLs, which can be used as antibacterial targets, has been identified in several soil bacteria. In this study, strain M664, identified as a Streptomyces sp., was found to secrete an AHL-degrading enzyme into a culture medium. The ahlM gene for AHL degradation from Streptomyces sp. strain M664 was cloned, expressed heterologously in Streptomyces lividans, and purified. The enzyme was found to be a heterodimeric protein with subunits of approximately 60 kDa and 23 kDa. A comparison of AhlM with known AHL-acylases, Ralstonia strain XJ12B AiiD and Pseudomonas aeruginosa PAO1 PvdQ, revealed 35% and 32% identities in the deduced amino acid sequences, respectively. However, AhlM was most similar to the cyclic lipopeptide acylase from Streptomyces sp. strain FERM BP-5809, exhibiting 93% identity. A mass spectrometry analysis demonstrated that AhlM hydrolyzed the amide bond of AHL, releasing homoserine lactone. AhlM exhibited a higher deacylation activity toward AHLs with long acyl chains rather than short acyl chains. Interestingly, AhlM was also found to be capable of degrading penicillin G by deacylation, showing that AhlM has a broad substrate specificity. The addition of AhlM to the growth medium reduced the accumulation of AHLs and decreased the production of virulence factors, including elastase, total protease, and LasA, in P. aeruginosa. Accordingly, these results suggest that AHL-acylase, AhlM could be effectively applied to the control of AHL-mediated pathogenicity.     

Detection of elastase production in Escherichia coli with the elastase structural gene from several non-elastase-producing strains of Pseudomonas aeruginosa.

The elastase structural gene from Pseudomonas aeruginosa IFO 3455 has been cloned and sequenced. Using this gene as a probe, we cloned the DNA fragments (pEL3080R, pEL10, and pEL103R) of the elastase gene from non-elastase-producing strains (P. aeruginosa IFO 3080, N-10, and PA103 respectively). These three Pseudomonas strains showed no detectable levels of elastase antigenicity by Western blotting (immunoblotting) or by elastase activity. When elastase structural genes about 8 kb in length were cloned into pUC18, an Escherichia coli expression vector, we were able to detect both elastase antigenicity and elastolytic activity in two bacterial clones (E. coli pEL10 and E. coli pEL103R). However, neither elastolytic activity nor elastase antigenicity was detected in the E. coli pEL3080R clone, although elastase mRNA was observed. The partial restriction map determined with several restriction enzymes of these three structural genes corresponded to that of P. aeruginosa IFO 3455. We sequenced the three DNA segments of the elastase gene from non-elastase-producing strains and compared the sequences with those from the elastase-producing P. aeruginosa strains IFO 3455 and PAO1. In P. aeruginosa N-10 and PA103, the sequences were almost identical to those from elastase-producing strains, except for several nucleotide differences. These minor differences may reflect a microheterogeneity of the elastase gene. These results suggest that two of the non-elastase-producing strains have the normal elastase structural gene and that elastase production is repressed by regulation of this gene expression in P. aeruginosa. Possible reasons for the lack of expression in these two strains are offered in this paper. In P. aeruginosa IFO 3080, the sequence had a 1-base deletion in the coding region, which should have caused a frameshift variation in the amino acid sequence. At present, we have no explanation for the abnormal posttransciptional behavior of this strain.