Abstract As major components of the cellular envelope, lipids play myriad roles in the survival of bacterial pathogens. Pathogens can adjust the biochemical properties (such as electrostatic charge) of their membrane lipids to cope with the changing and challenging conditions of their surroundings. We study lipids in the mitis group streptococci, specifically, Streptococcus mitis, S. oralis, and S. pneumoniae, which share a very close phylogenetic relationship. S. mitis and S. oralis are opportunistic pathogens that are among the leading causes of bacteremia (bloodstream infection) and infective endocarditis (heart valve infection). S. pneumoniae is a major human pathogen responsible for most cases of community-acquired pneumonia and meningitis in children and the elderly, and is a major cause of childhood mortality worldwide. Despite the clinical significance of these organisms and the critical functions of the lipids in host-microbe interactions, the membrane biology of mitis group streptococci is understudied and poorly understood. Using liquid chromatography/mass spectrometry (LC/MS)-based lipidomics, we analyzed lipids in the cell envelopes of mitis group streptococci and identified several novel surface properties which we propose are important for coping with antibiotics, antimicrobial peptides, and unfavorable conditions inside hosts. The overarching hypothesis of this project is that the ability of mitis group streptococci to drastically alter the charged lipid compositions of their cellular envelope impacts their interactions with hosts and their responses to antimicrobial stress. We propose to analyze the structure and biosynthesis of anionic (Aim 1) and zwitterionic lipids (Aim 2) in mitis group streptococci, and their roles in host- pathogen interactions (Aim 3). The knowledge gained will lead to a better understanding of the survival and virulence of these pathogens in the host and may facilitate the development of novel therapeutics to combat antibiotic resistance.