PROJECT SUMMARY/ABSTRACT Gram-negative bacteria constitute the majority of antibiotic resistant organisms identified as urgent threats to human health by the Center for Disease Control and Prevention. A major reason why Gram-negative bacteria are resistant to modern antibiotics is the impermeable nature of their outer membrane lipopolysaccharide (LPS) layer. This impermeable nature is due to the strong lateral interaction between neighboring LPS molecules that includes lipid A, the core saccharide, and the O-antigen. Nanotherapeutics capable of overcoming this barrier to successfully deliver antibiotics to Gram-negative bacteria will be of immense importance for human health. It is our intent to develop such nanotherapeutics by using the knowledge attained from delineating the mechanism by which a Gram-negative bacteriophage fuses its external bacterial phospholipid (BPL) membrane with the LPS of its host for initiating infection. Embedded into the Cystovirus BPL are multimeric protein complexes, referred to as spikes, that recognize the Gram-negative host and perform fusion. These proteins are analogous to, but different from, the better studied eukaryotic proteins responsible for membrane fusion (e.g. HIV env, West Nile Virus glycoprotein E, and Herpesviridae gB protein). The intent of this proposal is to investigate the mechanism by which spikes from three different members of the Cystovirus family recognize their hosts and drive membrane fusion. The goals of this proposal are to determine the structure of the spikes for establishing a structural scaffold for biochemical and biophysical studies (Aim 1), determine the mechanism of cellular recognition (Aim 2), and determine the biochemical determinants responsible for LPS- BPL fusion (Aim 3). The long-term goal of this project is to use the spikes for delivery of antibiotics to specific strains of pathogenic Gram- negative bacteria.

PROJECT NARRATIVE The proposed research is relevant to public health as it aims to overcome a major mechanism of antibiotic resistance by Gram-negative bacteria -the exclusion of antibiotics by the lipopolysaccharide (LPS) outer membrane. Our proposed studies seek to understand how a bacteriophage fuses its bacterial phospholipid membrane with the LPS of its Gram-negative host. This research will have the potential to overcome the antibiotic exclusionary properties of the LPS by generating phospholipid derived vesicles capable of targeting specific strains of Gram-negative bacteria for delivery of antibiotics.