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Title: The role of bacterial proteases in the development of chronic wounds
Author: Suleman, Louise
ISNI:       0000 0004 6058 0166
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2015
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Abstract:
A large number of bacteria are able to secrete extracellular proteases that play vital roles in nutrient acquisition and biofilm formation but are also important biochemical mediators in bacterial virulence, facilitating host invasion. Microorganisms within chronic wounds have been hypothesised to form biofilms, which result in perpetuated inflammation and delayed wound closure. Whilst it is thought that exaggerated secretion of host-derived proteases within chronic wounds prevents successful wound closure, the contribution of bacterial proteases secreted from planktonic microorganisms and biofilms in chronic wound pathology has yet to be elucidated. It is therefore the primary research aim of this thesis is to assess the proteolytic activity of Pseudomonas aeruginosa and Staphylococcus aureus isolated from equine and human chronic wounds and determine whether there is a difference in activity between planktonic-conditioned medium (PCM) and biofilm-conditioned medium (BCM). The next aim will be to identify these bacterial-derived proteases using zymography and mass spectrometry, and determine whether these proteases reduce wound closure in in vitro scratch wound models. More specifically, the effect of P. aeruginosa and S. aureus PCM and BCM from equine and human clinical isolates, and also purified proteases of these preparations, on the in vitro wound closure of equine normal fibroblasts (NFs), equine chronic wound granulation tissue fibroblasts (GTFs) and human dermal fibroblasts (HDFs), shall be investigated. In these wound models, zymography will be utilised to determine the release of host-derived matrix metalloproteases (MMPs). In this thesis I have identified, for the first time, high protease activity in equine chronic wound-derived P. aeruginosa PCM and BCM, by which 52kDa and 42kDa proteases were detected. P. aeruginosa PCM and BCM were shown to significantly reduce the wound closure of NFs (P < 0.0001) and GTFs (P < 0.0001). Furthermore, the soluble products of P. aeruginosa biofilms, but not planktonic P. aeruginosa, elicited the release of metalloproteases from equine NFs and GTFs. In human studies, high protease activity specific to P. aeruginosa BCM but not PCM (P < 0.0001) was determined. P. aeruginosa BCM significantly reduced the wound closure of HDFs (P <0.0001) and cell viability (P < 0.0001), and further induced the release of metalloproteases from HDFs. P. aeruginosa in biofilm form secreted 62kDa and 52kDa proteases, however, through the partial purification of these proteases, it was determined that these proteases did not play a role in the reduction of wound closure in HDFs. The P. aeruginosa-derived partially purified proteases did however reduce HDF cell viability (P < 0.05). The results in this thesis support the theory that bacterial-derived proteases may contribute to the emphasised proteolytic environment of human chronic wounds. Furthermore, the presence of bacterial biofilms within chronic wounds may induce the release of host-derived proteases. However the role of these specific P. aeruginosa-derived bacterial proteases in chronic wound pathology remains undetermined.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.706618  DOI: Not available
Keywords: Q Science (General) ; QR Microbiology
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