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Bacterial proteases and their inhibition - the missing link in normalizing the hostile environment in chronic wounds

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Introduction

Excessive protease levels impair new granulation tissue formation. Which role bacterial proteases play is unclear. Bacteria colonize wounds early forming complex, multi species communities within the wound. The biofilm phenotype shields from the host immune system and antimicrobials. Pseudomonas aeruginosa is associated with poor healing rates. Pseudomonas aeruginosa produces several “virulence factors/toxins” some of those are highly potent proteases. The expression of different Pseudomonas aeruginosa proteases was analyzed in planktonic and biofilm cultures and how protease activities can be reduced by polymers in wound dressings.

Methods

Pseudomonas aeruginosa (PAO1, DSM 22644) was grown in LBo medium diluted 1:100 from overnight cultures. After 8h, 24h and 48h culture supernatants were analyzed (planktonic cultures). Biofilm cultures were established dialysis membranes placed on top of LBo agar petri dishes. After 8h, 24h and 48h the uniform bacterial layer was scraped off washed 4x in 10 volumes of ddH2O (for each condition cleared supernatants were pooled). The biofilm phenotype was confirmed by the detection of extracellular DNA in the cleared supernatants. Protease activity was measured according to Koritsas (Anal Biochem. 227:22-6 (1995)). Gelatin zymography was performed according to Eming et al., (Biomaterials 29:2932-40 (2008)).

Results

Biofilm phenotype: high molecular weight DNA (> 48 kb) in the wash fluids of Pseudomonas aeruginosa biofilm cultures after 8h (faint), 24h and 48h. This indicates the formation of an extracellular matrix suggestive of a biofilm phenotype.

Protease expression: global extracellular protease activities increased in a time- and culture condition-dependent manner. The expression pattern of the different Pseudomonas aeruginosa proteases was determined by gelatin zymography.

Protease inhibition by polyacrylatesuperabsorber: inhibition of P. aeruginosa protease activity was blocked by two different polyacrylatesuperabsorbers (SAP). There were significant differences between the two SAP qualities.

Conclusions

Bacteria, as shown for Pseudomonas aeruginosa here, can contribute to the excessive protease levels in chronic wounds. Polyacrylate superabsorbers significantly inhibit protease activities, partly explaining the positive results in heavily colonized wounds clinically. 

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