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T.C. Conibear, H. Zhu, M.D. P. Willcox; Characterisation of Protease IV as a Virulence Determinant in Clinical Ocular Isolates of Pseudomonas aeruginosa . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1918.
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© ARVO (1962-2015); The Authors (2016-present)
Protease IV (prpL) has been shown to be an important virulence factor in establishing Pseudomonas keratitis. We have previously identified a group of low protease production P. aeruginosa isolates and demonstrated that ocular virulence in these isolates is not reduced unless the strains are las quorum–sensing gene deficiency. The aim of the current study was to determine the levels of protease IV production and the activity of the enzyme in these isolates.
The low protease production P. aeruginosa isolates Paer1, –2, –3, –17, –26 and 6206, and the reference strains PAO1 and 6294 were included in the study. The polymerase chain reaction (PCR) was used to identify the presence of prpL gene by specific primer sequences from published literature. For Southern hybridisation a DIG labelled prpL probe was created by PCR using labelled dUTP nucleotide, and hybridisation detected by standard laboratory protocols. Novel primers to flank and amplify the entire prpL sequence were designed, and amplicons were sequenced by ABI Prism DNA sequencer. Protease IV peptides were detected using standard Western blotting procedure. Protease IV activity and the effect of serine protease inhibitor 1–Chloro–3–tosylamido–7–amino–2–heptanone HCl (TLCK) were assayed using a chromogenic substrate and standard zymography techniques.
PCR failed to amplify prpL in strains Paer2, –17 and –26. However, homology to over 80% of the wild–type prpL gene was confirmed in these strains by Southern blot analysis. Sequence data for the complete prpL gene demonstrated two distinct lineages of the prpL gene among the strains tested. Peptides with affinity to PrpL antisera were detected by Western blotting in the culture supernatant of all strains but the amount of PrpL in strains Paer1, –2, –3, –17 and –26 was reduced. TLCK inhibited the enzymatic activity in proteins migrating to the high molecular weight region (>200kD; 7.5% SDS–polyacrylamide gel) in all strains tested.
These results suggest that despite being expressed, PrpL may not be in an active confirmation in the supernatant of some P. aeruginosa strains isolated from the eye. However isolates that produce low levels of functional protease IV enzyme are still virulent in a murine keratitis model.
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