Purification and characterization of a collagenolytic enzyme from a pathogen of the great barrier reef sponge, Rhopaloeides odorabile

Abstract

Background: In recent years there has been a global increase in reports of disease affecting marine sponges. While disease outbreaks have the potential to seriously impact on the survival of sponge populations, the ecology of the marine environment and the health of associated invertebrates, our understanding of sponge disease is extremely limited.

Methodology/principal findings: A collagenolytic enzyme suspected to enhance pathogenicity of bacterial strain NW4327 against the sponge Rhopaloeides odorabile was purified using combinations of size exclusion and anion exchange chromatography. After achieving a 77-fold increase in specific activity, continued purification decreased the yield to 21-fold with 7.2% recovery (specific activity 2575 collagen degrading units mg(-1)protein) possibly due to removal of co-factors. SDS-PAGE of the partially pure enzyme showed two proteins weighing approximately 116 and 45 kDa with the heavier band being similar to reported molecular weights of collagenases from Clostridium and marine Vibrios. The enzyme degraded tissue fibres of several sponge genera suggesting that NW4327 could be deleterious to other sponge species. Activity towards casein and bird feather keratin indicates that the partially purified collagenase is either a non-selective protease able to digest collagen or is contaminated with non-specific proteases. Enzyme activity was highest at pH 5 (the internal pH of R. odorabile) and 30 degrees C (the average ambient seawater temperature). Activity under partially anaerobic conditions also supports the role of this enzyme in the degradation of the spongin tissue. Cultivation of NW4327 in the presence of collagen increased production of collagenase by 30%. Enhanced enzyme activity when NW4327 was cultivated in media formulated in sterile natural seawater indicates the presence of other factors that influence enzyme synthesis.

Conclusions/significance: Several aspects of the sponge disease etiology were revealed, particularly the strong correlation with the internal tissue chemistry and environmental temperature. This research provides a platform for further investigations into the virulence mechanisms of sponge pathogens.

Figure 1. SDS PAGE analysis of fractions obtained at 20 to 30 minutes during anion exchange chromatography.

Lane numbers (upper row of top panel) indicate the time in minutes (Min.) and lower row in top panel indicate Abs₅₇₀ values obtained after assaying 500 µL of the fractions by the sensitive ninhydrin assay (Abs.). Values on the right panel indicate molecular weights of the standard protein markers.

Figure 2. HPLC chromatogram obtained with analytical molecular size exclusion chromatography using Superdex 200 HR.

The chromatograms depicted show the absorbance of the eluted material at 254 nm (lower trace) and at 280 nm (upper trace). Note that the absorbance values at 280 nm have been offset by 7.5 mAU to distinguish it from the values at 254 nm.