Gram-positive organisms, including the pathogens Staphylococcus aureus, Streptococcus pneumoniae and Enterococcus faecalis, have dynamic cell envelopes that mediate interactions with the environment and serve as the first line of defense against toxic molecules. Major components of the cell envelope include peptidoglycan, which is a well-established target for antibiotics, teichoic acids, capsular polysaccharides, surface proteins, and phospholipids. These components can undergo modification to promote pathogenesis, decrease susceptibility to antibiotics and host immune defenses, and enhance survival in hostile environments. This chapter will cover the structure, biosynthesis and important functions of major cell envelope components in Gram-positive bacteria. Possible targets for new antimicrobials will be noted.

Identifying targets of antibacterial compounds remains a challenging step in antibiotic development. We have developed a two-pronged functional genomics approach to predict mechanism of action that uses mutant fitness data from antibiotic-treated transposon libraries containing both upregulation and inactivation mutants. We treated a Staphylococcus aureus transposon library containing 690,000 unique insertions with 32 antibiotics. Upregulation signatures, identified from directional biases in insertions, revealed known molecular targets and resistance mechanisms for the majority of these. Because single gene upregulation does not always confer resistance, we used a complementary machine learning approach to predict mechanism from inactivation mutant fitness profiles. This approach suggested the cell wall precursor Lipid II as the molecular target of the lysocins, a mechanism we have confirmed. We conclude that docking to membrane-anchored Lipid II precedes the selective bacteriolysis that distinguishes these lytic natural products, showing the utility of our approach for nominating antibiotic mechanism of action.

Staphylococcus aureus is a leading cause of life-threatening infections worldwide. The MIC of an antibiotic against S. aureus, as well as other microbes, is determined by the affinity of the antibiotic for its target in addition to a complex interplay of many other cellular factors. Identifying nontarget factors impacting resistance to multiple antibiotics could inform the design of new compounds and lead to more-effective antimicrobial strategies. We examined large collections of transposon insertion mutants in S. aureus using transposon sequencing (Tn-Seq) to detect transposon mutants with reduced fitness in the presence of six clinically important antibiotics—ciprofloxacin, daptomycin, gentamicin, linezolid, oxacillin, and vancomycin. This approach allowed us to assess the relative fitness of many mutants simultaneously within these libraries. We identified pathways/genes previously known to be involved in resistance to individual antibiotics, including graRS and vraFG (graRS/vraFG), mprF, and fmtA, validating the approach, and found several to be important across multiple classes of antibiotics. We also identified two new, previously uncharacterized genes, SAOUHSC_01025 and SAOUHSC_01050, encoding polytopic membrane proteins, as important in limiting the effectiveness of multiple antibiotics. Machine learning identified similarities in the fitness profiles of graXRS/vraFG, SAOUHSC_01025, and SAOUHSC_01050 mutants upon antibiotic treatment, connecting these genes of unknown function to modulation of crucial cell envelope properties. Therapeutic strategies that combine a known antibiotic with a compound that targets these or other intrinsic resistance factors may be of value for enhancing the activity of existing antibiotics for treating otherwise-resistant S. aureus strains.