Immunization with aPseudomonas aeruginosaElastase Peptide Reduces Severity of Experimental Lung Infections Due toP. aeruginosaorBurkholderia cepacia

Sokol, Pamela A.; Kooi, Cora; Hodges, R. S.; Cachia, Paul J.; Woods, Donald E.

doi:10.1086/315470

Cited by 52 publications

(34 citation statements)

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“…In vitro assays demonstrate that PSCP is capable of cleaving biologically relevant substrates, including collagen (McKevitt et al, 1989), human IgA, IgG, IgM, transferrin and lactoferrin (C. Kooi & P. A. Sokol, unpublished observation). Previously, we have demonstrated a possible role for the extracellular zinc metalloprotease, PSCP, in B. cepacia virulence (Sokol et al, 2000). In this study, we demonstrate that the expression of a thermolysin-like protease by B. cepacia K56-2 contributes to virulence in an agar bead model of lung infection.…”

Section: Discussionsupporting

confidence: 56%

“…McKevitt et al (1989) demonstrated that purified PSCP induces bronchopneumonia in rat lungs characterized by polymorphonuclear leukocyte infiltration and proteinaceous exudate in the airways. Immunization with a peptide corresponding to a conserved zinc metalloprotease epitope significantly decreased the severity of experimental B. cepacia lung infections (Sokol et al, 2000). Further study of the role of extracellular proteases in the pathogenesis of B. cepacia infections has been limited by the lack of isogenic protease mutants.…”

Section: Introductionmentioning

confidence: 99%

“…Saggital slices of the left lung were mounted and stained with haemotoxylin and eosin. The percentage of the lung infiltrated with inflammatory exudate was quantified as described previously using a point counting method (Dunnil, 1962;Sokol et al, 2000). The lungs of 3-4 animals in each group were removed and homogenized (Polytron Homogenizer; Brinkman Instruments) in 3 ml PBS (10 mM sodium phosphate pH 7?2, 150 mM NaCl).…”

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confidence: 99%

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An extracellular zinc metalloprotease gene of Burkholderia cepacia

et al. 2003

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Burkholderia cepacia produces at least one extracellular zinc metalloprotease that may be involved in virulence. A B. cepacia zinc metalloprotease gene was cloned using a Burkholderia pseudomallei zinc metalloprotease gene as a probe. The predicted amino acid sequences of these B. cepacia and a B. pseudomallei extracellular zinc metalloproteases indicate that they are similar to the thermolysin-like family of metalloproteases (M4 family of metalloendopeptidases) and they are likely to be secreted via the general secretory pathway. zmpA isogenic mutants were constructed in B. cepacia genomovar III strains Pc715j and K56-2 by insertional inactivation of the zmpA genes. The zmpA mutants produced less protease than the parent strains. The B. cepacia strain K56-2 zmpA mutant was significantly less virulent than its parent strain in a chronic respiratory infection model; however, there was no difference between the virulence of B. cepacia strain Pc715j and a Pc715j zmpA mutant. The results indicate that this extracellular zinc metalloprotease may play a greater role in virulence in some strains of B. cepacia.

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Section: Discussionsupporting

confidence: 56%

Section: Introductionmentioning

confidence: 99%

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confidence: 99%

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An extracellular zinc metalloprotease gene of Burkholderia cepacia

et al. 2003

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“…Neutralizing monoclonal antibodies (MAbs) raised against ZmpA cross-react with a Pseudomonas aeruginosa LasB epitope (25,26). Immunization with a peptide ( 341 HGFTEQNG 349 ) corresponding to this conserved P. aeruginosa LasB epitope significantly decreases the severity of experimental B. cenocepacia lung infections (48). Although there is evidence from animal infection models that ZmpA plays a major role in the virulence of B. cenocepacia, the direct role of ZmpA in B. cenocepacia pathogenesis is not understood.…”

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Functional Analysis of the Burkholderia cenocepacia ZmpA Metalloprotease

2005

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Burkholderia cenocepacia ZmpA is expressed as a preproenzyme typical of thermolysin-like proteases such as Pseudomonas aeruginosa LasB and Bacillus thermoproteolyticus thermolysin. The zmpA gene was expressed using the pPRO-EXHTa His 6 tag expression system, which incorporates a six-His tag at the N-terminal end of the protein, and recombinant ZmpA was purified using Ni-nitrilotriacetic acid affinity chromatography. Upon refolding of the recombinant His 6 -pre-pro-ZmpA (62 kDa), the fusion protein was autoproteolytically cleaved into 36-kDa (mature ZmpA) and 27-kDa peptides. Site-directed mutagenesis was employed to infer the identity of the active site residues of ZmpA and to confirm that the enzyme undergoes autoproteolytic cleavage. Oligonucleotide mutagenesis was used to replace H 465 with G 465 or A 465 , E 377 with A 377 or D 377 , or H 380 with P 380 or A 380 . Mutagenesis of H 465 , E 377 , or H 380 resulted in the loss of both autocatalytic activity and proteolytic activity. ZmpA with either substitution in H 380 was not detectable in B. cenocepacia cell extracts. The activity of the recombinant ZmpA was inhibited by EDTA and 1,10 phenanthroline, indicating that it is a zinc metalloprotease. ZmpA, however, was not inhibited by phosphoramidon, a classical inhibitor of the thermolysin-like proteases. The refolded mature ZmpA enzyme was proteolytically active against various substrates including hide powder azure, type IV collagen, fibronectin, neutrophil ␣-1 proteinase inhibitor, ␣ 2 -macroglobulin, and gamma interferon, suggesting that B. cenocepacia ZmpA may cause direct tissue damage to the host or damage to host tissues through a modulation of the host's immune system.

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“…Several murine models of B. cepacia infection have been developed (8,42,44), and B. cepacia has been reported in mixed culture with other bacteria in clinical specimens from horses with pneumonia (7) and in specific-pathogen-free piglets (40) and as the sole organism involved in a case of vegetative endocarditis in a horse (47). In this report, we describe an epidemic outbreak of subclinical mastitis associated with infection by the B. cepacia complex in a large flock of dairy sheep.…”

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confidence: 99%