Isolation and characterization of alkaline protease-deficient mutants of Pseudomonas aeruginosa in vitro and in a mouse eye model

Howe, Timothy R.; Iglewski, Barbara H.

doi:10.1128/iai.43.3.1058-1063.1984

Cited by 126 publications

(51 citation statements)

References 29 publications

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“…Alkaline protease activity within the supernatant fraction of isolates grown overnight in LB broth was determined by adding 0.5 mL of supernatant to 1.5 mL of assay buffer (20 mM Tris-HCl, 1 mM CaCl 2 buffer, pH 8.0) containing 50 mg of hide remazol blue powder (Sigma). Tubes were incubated at 37°C for 1 h with constant rotation, reactions were stopped on ice, centrifuged at 4000 g for 5 min and absorbance of the supernatant was measured at 590 nm [16]. Assays were performed in triplicate in three different occasions.…”

Section: Alkaline Protease Assaymentioning

confidence: 99%

Analysis of quorum sensing-dependent virulence factor production and its relationship with antimicrobial susceptibility in Pseudomonas aeruginosa respiratory isolates

Karatuna

Yağcı

2010

Clinical Microbiology and Infection

121

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Pseudomonas aeruginosa is an opportunistic pathogen causing severe respiratory infections. The pathogenesis of these infections is multifactorial and the production of many virulence factors is regulated by quorum sensing (QS), a cell-to-cell communication mechanism. The two well defined QS systems in P. aeruginosa, the las and rhl systems, rely on N-acyl homoserine lactone signal molecules, also termed autoinducers. We assessed the activity of QS-dependent virulence factors (including elastase, alkaline protease, pyocyanin and biofilm production) in respiratory isolates of P. aeruginosa and their relationship with antimicrobial susceptibility. We identified sixteen isolates displaying impaired phenotypic activity; among them, eleven isolates were also defective in autoinducer production, and therefore considered QS-deficient. Six of the QS-deficient isolates failed to amplify one or more of the four QS regulatory genes (lasI, lasR, rhlI, rhlR) with PCR: one isolate was negative for rhlR, two isolates were negative for rhlI and rhlR and three isolates were negative for all four genes. The isolates that were negative for virulence factor production were generally less susceptible to the antimicrobials and statistically significant correlations were observed between the lack of elastase production and resistance to piperacillin and ceftazidime; between failure in alkaline protease production and resistance to tobramycin, piperacillin, piperacillin-tazobactam, cefepime, imipenem and ciprofloxacin; and between failure in pyocyanin production and resistance to amikacin, tobramycin, ceftazidime, ciprofloxacin and ofloxacin. The results obtained indicate that, despite the pivotal role of QS in the pathogenesis of P. aeruginosa respiratory infections, QS-deficient strains are still capable of causing infections and tend to be less susceptible to antimicrobials.

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Section: Alkaline Protease Assaymentioning

confidence: 99%

Analysis of quorum sensing-dependent virulence factor production and its relationship with antimicrobial susceptibility in Pseudomonas aeruginosa respiratory isolates

Karatuna

Yağcı

2010

Clinical Microbiology and Infection

121

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“…p-galactosidase was assayed by the o-nitrophenyl-p-D-galactopyranoside assay as described by Miller (1972). Alkaline protease was determined by the sensitive assay using Hide Powder Azure (Sigma Chemical Co.) (Howe and Iglewski, 1984). The inhibition of protease activity by the inhibitor was measured by the difference in protease activity without and with the inhibitor preparation.…”

Section: Enzyme Assaysmentioning

confidence: 99%

“…This virulence is related to the secretion of several extracellular proteins, some of which are virulence factors (Liu, 1974;Wretlind etai, 1973). Among them is an alkaline protease that has been shown to be required for the establishment of oorneal infections {Howe and Iglewski, 1984).…”

Section: Introductionmentioning

confidence: 99%

The secretion genes of Pseudomonas aeruginosa alkaline protease are functionally related to those of Erwinia chrysanthemi proteases and Escherichia coliα‐haemolysin

Guzzo

Duong

Wandersman

et al. 1991

Molecular Microbiology

124

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The extracellular alkaline protease produced by Pseudomonas aeruginosa is secreted by a specific pathway, independent of the pathway used by most of the other extracellular proteins of this organism. Secretion of this protease is dependent on the presence of several genes located adjacent to the apr gene. Complementation studies have shown that PrtD, E, and F, the three secretion functions for Erwinia chrysanthemi proteases B and C (Létoffé et al., 1990), can mediate the secretion of the alkaline protease by Escherichia coli. The secretion functions involved in alpha-haemolysin secretion in E. coli (hlyB, hlyD, tolC) can also be used to complement alkaline protease secretion by E. coli, although less efficiently. These data indicate that protease secretion mechanisms in Pseudomonas and Erwinia are very similar and are homologous to that of E. coli alpha-haemolysin.

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“…Pseudomonas aeruginosa can be identified in a range of infections, particularly those with a tendency to become chronic, such as lung infections in patients with cystic fibrosis (Wagner & Iglewski, 2008), those related to venous ulcers (Dowd et al, 2008) and infections associated with in-dwelling medical devices (Finkelstein et al, 2002). The most well-documented virulence property of P. aeruginosa is its ability to produce and secrete elastase (Woods et al, 1982), alkaline protease (Howe & Iglewski, 1984), pyocyanin (Lau et al, 2004), rhamnolipids and a range of exotoxins (Smith & Iglewski, 2003). The expression of many of these factors is known to be differentially regulated through quorum-sensing systems in response to prevailing environmental conditions (Williams et al, 2000).…”

Section: Introductionmentioning

confidence: 99%

Effects of clinical isolates ofPseudomonas aeruginosaonStaphylococcus epidermidisbiofilm formation

Pihl

Paz

Schmidtchen

et al. 2010

FEMS Immunol Med Microbiol

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Pseudomonas aeruginosa is often found in chronic infections, including cystic fibrosis lung infections and those related to chronic wounds and venous ulcers. At the latter sites, P. aeruginosa can be isolated together with Staphylococcus epidermidis, and we have therefore explored the effect of clinical isolates and laboratory strains of P. aeruginosa strains on colonization by S. epidermidis in dual-species biofilms. Biofilm formation was assayed using 16S rRNA FISH and confocal laser scanning microscopy. Among the six P. aeruginosa strains tested, one particular strain, denoted 14:2, exerted a significant inhibitory effect, and even after 6 h, S. epidermidis levels in dual-species biofilms were reduced by >85% compared with those without P. aeruginosa. Interestingly, strain 14:2 was found to be negative for classical virulence determinants including pyocyanin, elastase and alkaline protease. Therefore, we suggest that less virulent phenotypes of P. aeruginosa, which may develop over time in chronic infections, could counteract colonization by S. epidermidis, ensuring persistence and dominance by P. aeruginosa in the host micro-habitat. Further studies are required to explain the inhibitory effect on S. epidermidis, although extracellular polysaccharides produced by P. aeruginosa might play a role in this phenomenon.

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Isolation and characterization of alkaline protease-deficient mutants of Pseudomonas aeruginosa in vitro and in a mouse eye model

Cited by 126 publications

References 29 publications

Analysis of quorum sensing-dependent virulence factor production and its relationship with antimicrobial susceptibility in Pseudomonas aeruginosa respiratory isolates

Analysis of quorum sensing-dependent virulence factor production and its relationship with antimicrobial susceptibility in Pseudomonas aeruginosa respiratory isolates

The secretion genes of Pseudomonas aeruginosa alkaline protease are functionally related to those of Erwinia chrysanthemi proteases and Escherichia coliα‐haemolysin

Effects of clinical isolates ofPseudomonas aeruginosaonStaphylococcus epidermidisbiofilm formation

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