Blow fly Lucilia sericata nuclease digests DNA associated with wound slough/eschar and with Pseudomonas aeruginosa biofilm.

In chronic wounds, it may be clinically important to remove extracellular bacterial and patient DNA as its presence may impede wound healing and promote bacterial survival in biofilm, in which extracellular DNA forms part of the biofilm architecture. As medicinal maggots, larvae of Lucilia sericata Meigen (Diptera: Calliphoridae) have been shown to efficiently debride wounds it became of interest to investigate their excretions/secretions (ES) for the presence of a deoxyribonuclease (DNAse) activity. Excretions/secretions products were shown to contain a DNAse, with magnesium, sodium and calcium metal ion dependency, and a native molecular mass following affinity purification of approximately 45 kDa. The affinity purified DNAse degraded genomic bacterial DNA per se, DNA from the slough/eschar of a venous leg ulcer, and extracellular bacterial DNA in biofilms pre-formed from a clinical isolate of Pseudomonas aeruginosa. The latter finding highlights an important attribute of the DNAse, given the frequency of P. aeruginosa infection in non-healing wounds and the fact that P. aeruginosa virulence factors can be toxic to maggots. Maggot DNAse is thus a competent enzyme derived from a rational source, with the potential to assist in clinical wound debridement by removing extracellular DNA from tissue and biofilm, and promoting tissue viability, while liberating proteinaceous slough/eschar for debridement by the suite of proteinases secreted by L. sericata.

Degradation of eschar from venous leg ulcers using a recombinant chymotrypsin from Lucilia sericata.

BACKGROUND: Larvae of the greenbottle Lucilia sericata are used to debride nonhealing wounds and stimulate the production of fresh granulation tissue. Previous publications have shown that secretions from L. sericata contain a number of proteolytic activities including a chymotrypsin that degrades a number of extracellular matrix components such as fibronectin, laminin and collagen. OBJECTIVES: To produce a recombinant L. sericata chymotrypsin (chymotrypsin I) and determine its effects on the degradation of patient wound eschar. METHODS: An active recombinant chymotrypsin I from L. sericata was cloned and expressed in Sf9 cells and its subsequent effects ex vivo on eschar from venous leg ulcers were determined by two-dimensional electrophoresis. RESULTS: The recombinant enzyme had the attributes of a chymotrypsin, possessing sequence homology with other chymotrypsins and demonstrating attributes of the native enzyme including cleavage of the chymotrypsin substrate succinyl-alanyl-alanyl-prolyl-phenylalanyl-7-amino-4-methyl coumarin, inhibition by phenylmethylsulphonyl fluoride and lack of inhibition by amidinophenylmethylsulphonyl fluoride. Importantly, the recombinant chymotrypsin cleaved the majority of proteins from slough/eschar from venous leg ulcers in a superior manner to chymotrypsins from human and bovine sources. CONCLUSIONS: The ex vivo degradation of eschar from venous leg ulcers indicates the potential value of recombinant chymotrypsin I as a novel, stand-alone debridement agent.

Maggots and wound healing: an investigation of the effects of secretions from Lucilia sericata larvae upon interactions between human dermal fibroblasts and extracellular matrix components.

BACKGROUND: Through clinical observation, Lucilia sericata (greenbottle fly) larvae are credited with exerting the following beneficial effects upon a chronic nonhealing wound: removal of necrotic tissue ('debridement'), disinfection of the wound and active promotion of granulation tissue formation. As a major cellular component of granulation tissue, fibroblasts play an extensive role in healing. The composition of extracellular matrix (ECM) located in the wound is another important factor, partaking in a dynamic feedback loop with the fibroblasts that produce it. Fibroblast-ECM interactions therefore exert considerable influence upon new tissue formation. OBJECTIVES: To investigate the effects of L. sericata larval excretory/secretory products (ES) upon the behaviour of fibroblasts, seeded upon ECM component surfaces. METHODS: ES were collected by washing freshly hatched larvae in phosphate-buffered saline. Human dermal neonatal fibroblast cells were seeded upon fibronectin- or collagen-coated surfaces, together with untreated (or 'native') ES, heat-treated ES, or no ES (ES blank). Following incubation, fibroblast adhesion was determined using an adenosine triphosphate assay and, for confirmation, a total nucleic acid content assay. Cell spreading was observed using microscopy. The effect of ES upon fibronectin structure was observed using sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Peptide sequencing of suspected fibronectin fragments was performed using an 'electrospray' type time of flight mass spectrometer and 'Peptident' database search. RESULTS: ES significantly reduced fibroblast adhesion to both fibronectin and, to a lesser extent, collagen. Cell spreading was also reduced, yet cells remained viable. For both spreading and adhesion, heat-treated ES exerted significantly less activity than native, untreated ES. However, they still exhibited significant activity when compared with the ES blank. ES appeared to modify fibroblast adhesion indirectly via proteolytic fragmentation of the fibronectin protein surface. CONCLUSIONS: L. sericata larval secretions modify fibroblast adhesion and spreading across ECM protein surfaces, while keeping cells viable. Proteolytic activity of the ES played a significant role. If transferred to the wound situation, such alteration of fibroblast-ECM interactions may enhance new tissue formation.