Membrane Permeabilization by Holin like Proteins CidA/LrgA

Abstract

Holins are a large family of membrane proteins that control the activity of bacteriophage-encoded murein hydrolases by regulating their access to the peptidoglycan substrate. The two S. aureus membrane proteins CidA and LrgA exhibit holin and, respectively, anti-holin like properties. They are believed to play an important role in the formation of biofilm by controling bacterial cell lysis during bacterial programmed cell death. This resembles the behavior of apoptotic (e.g. Bax/Bak) and antiapoptotic (e.g. Bcl-2/Bcl-XL) proteins in eukaryotes. Pure preparations of recombinant CidA/LrgA were functionally reconstituted into synthetic lipid vesicles, as confirmed by circular dichroism spectroscopy and transmission electron microscopy. Fluorescent markers were entrapped into the protein reconstituted vesicles. A new method was developed to study the protein-induced leakage from vesicles since the conventional leakage assay could not be applied to CidA/LrgA. Our initial study has revealed that CidA induces leakage of small dyes. Ongoing studies include leakage of larger molecules to determine the dimension of the pore formed by CidA and characterization of the role played by LrgA in pore formation. This research will provide insight into the in vivo function of Cid/Lrg proteins and shed light on bacterial holin-like proteins.