7. PROJECT SUMMARY/ ABSTRACT Staphylococcus aureus is a major bacterial pathogen worldwide, and this is compounded by multiple drug resistance including methicillin-resistant S. aureus (MRSA) and vancomycin-intermediate S. aureus (VISA). We have discovered that when S. aureus is grown ex vivo in serum and likely in vivo in an infection, straight-chain unsaturated fatty acids (SCUFAs) are incorporated directly into cellular lipids and become major components of the total fatty acids. SCUFAs are not found in cells grown in laboratory media, in which the fatty acids are a mixture of straight-chain fatty acids (SCFAs) and branched-chain fatty acids (BCFAs). Bacterial glycerolipid fatty acid composition is a major determinant of membrane biophysical properties, which thus impacts all aspects of cell physiology including susceptibility to membrane active antimicrobials, pathogenesis, and response to environmental stress. It is shocking to realize that all aspects of S. aureus membrane structure and function studied thus far have been done with cells with the ?wrong? fatty acid composition, i.e., they lack SCUFAs as a major component. Although both BCFAs and SCUFAs increase membrane fluidity, indications are they are not exactly functionally equivalent in membrane structure or in conferring fitness on an organism. Furthermore, laboratory media-grown cells present the wrong face to the host in that incorporation of SCUFAs into glycolipids and lipoproteins increases their reactivity with host defence systems. We propose to redress these severe omissions in staphylococcal biology. We will carry out a refined and detailed investigation of how the presence of SCUFAs in the growth environment impacts the fatty acid composition of individual classes of lipid- containing molecules in the cell, i.e. phospholipids, glycolipids, lipoteichoic acid, lipoproteins, and carotenoids. The biophysical and functional aspects of the membranes with extreme differences in the proportions of BCFAs, SCFAs, and SCUFAs, including cell physiology, membrane fluidity, antimicrobial susceptibility and virulence factor expression will be determined. This will show if the alterations of the membrane lipid profile following growth in serum is a key adaptation in S. aureus in response to host-pathogen interactions. Cells grown in serum, Tryptic Soy broth (TSB) and Mueller-Hinton (MH) medium, and cells with fatty acid compositions manipulated by feeding fatty acid auxotrophs different mixtures of fatty acids, will be studied. Detailed biophysical investigations of membranes with or without SCUFAs will be carried out through studies of the temperature-dependent fluidity of total lipids and individual lipid components. Membrane integrity will be probed through studies of peptide interactions with total lipids and individual lipid classes. The carotenoid staphyloxanthin is a further unique membrane lipid component. A carotenoid-deficient mutant will be used to study the impact of this component on membrane structure and function. Because the cytoplasmic membrane is such a critical aspect of cell function these studies are important to the fundamental biology of S. aureus including antibiotic susceptibility, pathogenesis and overall physiology. It may be possible through supplementation of medium to devise culture conditions that yield a membrane composition that resembles that of cells growing in vivo. These considerations are important in antibiotic susceptibility testing, expression of virulence factors, and attempts to develop inhibitors of fatty acid biosynthesis as antistaphylococcal drugs.