Insights into the molecular adaptation of Staphylococcus aureus to copper Schiff´s base at proteome level

Abstract

The molecular adaptation of S.aureus to copper Schiff’s base (CSB) was studied by comparative proteome analysis. Proteome analysis was performed by employing nano LC-MALDI mass spectrometry with isobaric tags (iTRAQ) and 2D-DIGE techniques. In CSB adapted <em>MSSA476</em> (ErCu), considerable decrease in Cu ion concentration and copZ protein is observed as compared to non-CSB adapted MSSA476 strain by ICP-MS and nano LC-MALDI experiments respectively. CopZ protein is a copper transport protein, which aids the export of Cu⁺ across the membrane. Decreased Cu⁺ and CopZ suggest reduced Cu⁺ influx in to the cell. It has been already established that repression of Putative septation protein SpoVG leads to repressed biofilm formation in S. aureus. Approximately two fold increase in SpoVG in CSB adapted MSSA476 as observed by 2D-DIGE experiment suggests enhanced ability for biofilm formation in the adapted strain. Enhanced biofilm formation is further confirmed by biofilm assay experiment. Contrary to popular belief, it has been demonstrated previously that repression of accessory gene regulatory (agr) system is necessary to form a biofilm. Repression of agr system is confirmed by reduced agrA protein observed by nano LC-MALDI experiment in ErCu. Because of the agr repression, reduced virulence in ErCu strain was demonstrated and was further confirmed by virulence analysis experiments on C.elegans. SpoVG is known to be positively regulated by sigmaB factor; on the other hand sigmaB has a negative effect on agr sytem. Therefore, sigmaB can be proposed as a common factor, which is involved in the interrelation of SpoVG expression and agr system expression in ErCu. However, the proteomics analysis was not able to conclusively determine the expression of sigmaB incase of ErCu. Nevertheless, upregulation of SpoVG and down-regulation of agr system in ErCu were proved beyond doubt, which facilitates enhanced biofilm formation in ErCu. The extracellular matrix of S.aureus biofilm is known to immobilize toxic metals by ionic interaction of opposite charges (DNA), by precipitation, biosorption etc. It has been established that enhanced biofilm formation is the likely cause of oxidative stress tolerance towards excess copper concentration in this strain. It can be proposed that CSB adapted <em>MSSA476</em> has developed a mechanism to increase biofilm formation which prevents excess Cu⁺ influx in to the cell and therefore prevents excess oxidative stress exerted by excess Cu⁺ within the cell. It is demonstrated that <em>MSSA476</em> required only few generations to achieve tolerance to inhibitory concentrations of CSB due to enhanced biofilm formation properties. This emphasizes once again the importance of dealing with biofilms as a prime target for S.aureus therapies.