Characterization of Staphylococcus aureus CidA and LrgA proteins involved in cell death and lysis

The Staphylococcus aureus cid and lrg operons have been shown to affect murein hydrolase activity, antibiotic tolerance and stationary phase lysis. Recent studies demonstrate that their biological role is to control cell death and lysis during biofilm development, causing release of genomic DNA, which ultimately contributes as a structural component of the biofilm matrix. This system has also been proposed to control bacterial programmed cell death in a manner analogous to the Bax and Bcl-2 family of proteins involved in the control of apoptosis. Although the molecular mechanism of Cid and Lrg protein function is not known, a computational analysis of their primary structures suggests they are membrane proteins. Previous studies also indicate that the CidA and LrgA proteins are members of the holin family of proteins, known to control murein hydrolase activity during bacteriophage infection. This research focuses on the biochemical and molecular functions of CidA and LrgA in cell death and lysis. Membrane fractionation and fluorescent protein fusion studies have shown that CidA and LrgA localize to the staphylococcal membrane. Furthermore, similar to Bax/Bcl-2 and holin proteins, CidA and LrgA were found to oligomerize into high molecular weight structures. Dissecting the oligomeric complexes of CidA in a two-dimensional gel analysis revealed two major protein complexes of approximately 450 kDa and 150 kDa in size. In addition, unlike Bax/Bcl-2, but similar to holin proteins, cysteines in CidA and LrgA are found to be involved in the disulfide bond-dependent dimerization. To determine the function of disulfide-dependent oligomerization of CidA, a S. aureus mutant was generated in which the wild-type copy of the cidA gene was replaced with the cysteine mutant allele. As determined by beta-galactosidase release assays, this mutant exhibited increased cell lysis during stationary phase, suggesting that oligomerization has a negative impact on this process. Overall, these studies present new information regarding the biochemical properties and functions of this novel family of proteins and provide further insight into the control of cell lysis in S. aureus..