The Functions of the Proteins Encoded by the Cid and Lrg Operons in S. Aureus

Abstract

Bacterial programmed cell death (PCD) is an emerging concept with implications in biofilm formation and acquiring of antibiotic resistance. The cid and lrg operons have been shown to be important to PCD in S. aureus and this work focuses on preparing and functionally characterizing proteins encoded by these two operons. The cid operon encodes the CidA, CidB, and CidC proteins, with CidA hypothesized to be a holin-like membrane protein and CidC a membrane-bound pyruvate oxidase. The lrg operon encodes the two membrane proteins LrgA and LrgB, with LrgA hypothesized to be an anti-holin membrane protein. The exact roles of CidB and LrgB remain undetermined to date. Pure preparations of CidA or LrgA were reconstituted into synthetic lipid vesicles that mimic the cellular membrane of S.aureus. A newly developed liposome leakage assay confirms that CidA induces the formation of nanometer membrane pores, while LrgA induces the formation of much smaller membrane pores. Pure recombinant CidC was shown to bind flavin adenine dinucleotide and to exhibit pyruvate oxidase activity in the presence of thiamine pyrophosphate. Ongoing studies include further liposome leakage assays to better define the pores induced by CidA and LrgA, as well as any influence exterted by the CidB and LrgB proteins. The interaction between Cid/Lrg membrane proteins is also investigated by isothermal titration calorimetry. This research will elucidate the in vivo functions of the Cid/Lrg proteins and shed light on bacterial programmed cell death.