Functional characterization of Staphylococcus aureus lipase 2 (SAL2) as a collagen adhesin.

Extracellular lipases of many pathogens have been characterized as human virulence factors. Staphylococcus aureus produces a variety of enzymes that aid in the pathogenesis of the bacterium to invade and destroy host tissues, resulting in a wide range of clinical illnesses. The lipase is one such enzyme, and the lipases produced by S. aureus (SAL1, SAL2 and SAL3) have been associated with the virulence of the bacterium. In the present study, we cloned, expressed and purified the mature lipase domain of SAL2 (rSAL2_296-690) and characterized its interaction with human collagen type IV using biolayer interferometry (BLI), molecular docking and simulation studies. Collagen binds to rSAL2_296-690 with an affinity of 3.261 μM. The enzymatic activity of rSAL2_296-690 was analyzed in the presence of collagen and orlistat, a potent lipase inhibitor. The activity of rSAL2_296-690 in the presence of collagen or orlistat was nearly 90 fold lower than that of the native rSAL2_296-690. The optimal pH and temperature for rSAL2_296-690 activity were found at 7 and 25 °C respectively. rSAL2_296-690 found to be stable at pH 7 and exhibits thermostability in the temperature range 15-25 °C. With CaCl₂ and ZnCl₂, an increase in activity of rSAL2_296-690 was observed whereas NiSO₄, CuSO₄, MnCl₂, CoCl2 and MgCl₂ reduced the activity. No substrate specificity was found with rSAL2_296-690, as it cleaves different substrate lengths (C2, C6, C12 and C16) and triglyceride triolein. Interaction of rSAL2_296-690 with metal-incubated collagen revealed that the binding affinity of collagen in the presence of NiSO₄, CuSO₄ and CoCl₂ significantly reduced. Enzymatic and collagen binding studies provided insights into the putative collagen binding site on SAL2_296-690, which is near the active site region of the molecule. This study thus revealed that rSAL2_296-690 as a bi-functional molecule, acts not only as a lipase but also as a collagen adhesin.