Cloning and characterization of elastase genes from Pseudomonas aeruginosa

Schád, Péter; Bever, Robert; Nicas, Thalia I.; Leduc, Frédéric; Hanne, Larry F.; Iglewski, Barbara H.

doi:10.1128/jb.169.6.2691-2696.1987

Cited by 63 publications

(57 citation statements)

References 40 publications

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“…lasB-mediated elastolytic activity was determined by the elastin-Congo red (ECR) assay as described previously (35). Uninoculated TSB was included as a negative control.…”

Section: Methodsmentioning

confidence: 99%

Phenotypic Characterization of Clonal and Nonclonal Pseudomonas aeruginosa Strains Isolated from Lungs of Adults with Cystic Fibrosis

et al. 2007

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The emergence of virulent Pseudomonas aeruginosa clones is a threat to cystic fibrosis (CF) patients globally. Characterization of clonal P. aeruginosa strains is critical for an understanding of its clinical impact and developing strategies to meet this problem. Two clonal strains (AES-1 and AES-2) are circulating within CF centers in eastern Australia. In this study, phenotypic characteristics of 43 (14 AES-1, 5 AES-2, and 24 nonclonal) P. aeruginosa isolates were compared to gain insight into the properties of clonal strains. All 43 isolates produced bands of the predicted size in PCRs for vfr, rhlI, rhlR, lasA, lasB, aprA, rhlAB, and exoS genes; 42 were positive for lasI and lasR, and none had exoU. Thirty-seven (86%) isolates were positive in total protease assays; on zymography, 24 (56%) produced elastase/staphylolysin and 22 (51%) produced alkaline protease. Clonal isolates were more likely than nonclonal isolates to be positive for total proteases (P ‫؍‬ 0.02), to show elastase and alkaline protease activity by zymography (P ‫؍‬ 0.04 and P ‫؍‬ 0.01, respectively), and to show elastase activity by the elastin-Congo red assay (P ‫؍‬ 0.04). There were no other associations with genotype. Overall, increasing patient age was associated with decreasing elastase activity (P ‫؍‬ 0.03). Thirty-two (74%) isolates had at least one N-acylhomoserine lactone (AHL) by thin-layer chromatography. rhl-associated AHL detection was associated with the production and level of total protease and elastase activity (all P < 0.01). Thirty-three (77%) isolates were positive for ExoS by Western blot analysis, 35 (81%) produced rhamnolipids, and 34 (79%) showed chitinase activity. Findings suggest that protease activity during chronic infection may contribute to the transmissibility or virulence of these clonal strains.Pseudomonas aeruginosa lung infection is a major determinant of morbidity and mortality in cystic fibrosis (CF) patients (16). The pathogenesis of P. aeruginosa infection depends on multiple cell-associated and extracellular virulence factors including proteases (elastase [LasB], alkaline protease [AprA], and staphylolysin [LasA]), hemolysins (rhamnolipids), and toxins such as exoenzyme S (ExoS) and exotoxin A (13). Proteases contribute to pathogenesis in the lung through the induction of tissue necrosis and inflammation, destruction of surface receptors on neutrophils resulting in the inhibition of chemotaxis, phagocytosis, and the oxidative burst and degradation of surfactant proteins (27, 32).Many P. aeruginosa virulence factors, including the proteases, are regulated by cell-to-cell communication systems that rely on diffusible N-acylhomoserine lactones (AHLs) to monitor population size in a process known as "quorum sensing" (QS) (7, 31). P. aeruginosa has two AHL-regulated circuits: las, consisting of the transcriptional activator LasR and the AHL synthase LasI, which directs the synthesis of N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL), and rhl, consisting of RhlR and RhlI, which directs the synthesis of N-...

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“…lasB-mediated elastolytic activity was determined by the elastin-Congo red (ECR) assay as described previously (35). Uninoculated TSB was included as a negative control.…”

Section: Methodsmentioning

confidence: 99%

Phenotypic Characterization of Clonal and Nonclonal Pseudomonas aeruginosa Strains Isolated from Lungs of Adults with Cystic Fibrosis

et al. 2007

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“…P. aeruginosa elastase was kindly supplied by Dr. Barbara Iglewski (Rochester, New York) and also purchased from Nagase and Company, Ltd., Tokyo, Japan. These preparations were homogeneous as demonstrated by SDS-PAGE and functionally active as demonstrated by digestion of Hide powder azure according to previously published methods (29). There was <1 pg/ mL of LPS in these preparations as demonstrated by limulus amebocyte lysate assay (Associates of Cape Cod., Inc.).…”

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confidence: 99%

Effect of Pseudomonas Elastase on Human Mononuclear Phagocyte α1-Antitrypsin Expression

Barbey-Morel

Perlmutter

1991

Pediatr Res

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ABSTRACT. The net balance of neutrophil elastase and its inhibitor, a,-antitrypsin (al-AT), is a critical determinant of connective tissue turnover during homeostasis and in disease states. In addition to liver-derived a,-AT, which translocates from blood to tissues, this elastase-a,-AT balance is maintained by expression of al-AT at the local tissue level in resident mononuclear phagocytes. Our previous studies have shown that this elastase-al-AT balance is also tightly controlled at a cellular level in that addition of exogenous neutrophil elastase (serpine-type elastase) to cultured mononuclear phagocytes is associated with an increase in expression of the al-AT gene. Subsequent studies have demonstrated that this novel regulatory loop involves interaction between exogenous neutrophil elastase and endogenous a,-AT inducing a structural rearrangement in the al-AT molecule and exposing highly conserved conformation-specific domain of a,-AT, which can then be recognized by a specific cell surface receptor, the serpinenzyme complex receptor. In the following study, we examined the effect of a bacterial metalloelastase, Pseudomonas aeruginosa elastase, on expression of a,-AT in human mononuclear phagocytes. We show that pseudomonas elastase inactivates monocyte-derived a,-AT by limited proteolysis but, in so doing, a,-AT becomes recognized by the serpin-enzyme complex receptor and mediates an increase in de novo synthesis of a,-AT in these cells. However, the concentrations of pseudomonas elastase needed to proteolytically inactivate al-AT in monocyte culture fluid are higher than those required for inactivation of purified plasma a,-AT. Results of experiments in this report show that this can be explained, at least in part, by binding of pseudomonas elastase to another endogenous protease inhibitor, a2-macroglobulin. Thus, the results of this study further define the elaborate mechanisms by which the host mononuclear phagocyte controls the elastase-a,-AT balance and, in turn, connective tissue turnover. (Pediatr Res 29: 133-140,1991) Abbreviations a,-AT, a,-antitrypsin LPS, lipopolysaccharide SEC, serpin-enzyme complex

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“…A third possibility is that the 8-kb fragments have a normal promoter and some unknown factor(s) may play a role for regulating elastase gene expression in these non-elastase-producing Pseudomonas strains. Schad and coworkers (25) suggested that more than one gene is involved in elastase production in P. aeruginosa strains. To resolve these issues, it is necessary to clarify the promoter region for elastase.…”

Section: Discussionmentioning

confidence: 99%

“…Several elastase-related products (8,9,21), the existence of "pro" protein (2,6,9,29), and elastase activation molecules (9,26) have been described. In addition, Goldberg and Ohman (8) and Schad et al (25) first cloned the elastase-related lasA gene, which was later determined to be an elastase-modifying gene (9,26). However, the regulatory mechanism of elastase production has not yet been investigated.…”

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confidence: 99%

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

et al. 1991

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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 pEL308OR 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 ff0 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 posttranscriptional behavior of this strain.Pseudomonas aeruginosa is an opportunistic pathogen which can cause fatal infections in vulnerable hosts (19). Several components related to its virulence have been identified and characterized (22). Among these, elastase acts to inactivate a variety of biologically important proteins or phenomena (12,14,(16)(17)(18)23). In addition, there is strong evidence that elastase plays an important role in the pathogenesis of P. aeruginosa infections (11,20). Although the regulatory mechanism of elastase production is still unknown, elastase-deficient Pseudomonas strains have been isolated (18). Furthermore, some elastase-positive Pseudomonas isolates often become elastase negative after several passages in vitro.We (6, 29) and another group (2, 25) have independently cloned and sequenced the elastase structural gene using the elastase-producing P. aeruginosa strains IFO 3455 and PA01, respectively. Several elastase-related products (8, 9, 21), the existence of "pro" protein (2,6,9,29), and elastase activation molecules (9, 26) have been described. In addition, Goldberg and Ohm...

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Cloning and characterization of elastase genes from Pseudomonas aeruginosa

Cited by 63 publications

References 40 publications

Phenotypic Characterization of Clonal and Nonclonal Pseudomonas aeruginosa Strains Isolated from Lungs of Adults with Cystic Fibrosis

Phenotypic Characterization of Clonal and Nonclonal Pseudomonas aeruginosa Strains Isolated from Lungs of Adults with Cystic Fibrosis

Effect of Pseudomonas Elastase on Human Mononuclear Phagocyte α1-Antitrypsin Expression

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

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