Evaluation of a Multivalent Artificial Opsonin to Target and Phagocytose Gram-Negative Bacteria

Abstract

Hospital-acquired infections (HAIs) are a significant medical concern that must be tackled. HAIs cost the United States billions of dollars annually, and in 2002, were the sixth-leading cause of death [1-2]. Klebsiella pneumoniae, a gram-negative bacterial species, accounts for up to 10% of all HAIs [3-5]. Due to the prevalence of HAIs in the hospital environment, antibiotics have been traditionally used in an attempt to treat bacterial infections. Consequently, Klebsiella pneumoniae has developed resistance to a variety of antibiotics [3]. To compound the issue, this bacterial species can form biofilms to avoid opsonization and phagocytosis [6]. As a result, novel therapeutics that transcend antibiotics and antibodies must be developed. One promising approach is to enhance the body’s own immune system when fighting a bacterial infection. We have developed an artificial opsonin to recognize and phagocytose gram-negative bacteria. The artificial opsonin consists of three parts: (1) di-tuftsin, a peptide that activates and binds to the neuropilin-1 (Nrp1) receptor on macrophages to initiate an immune response, (2) YI13WF, a peptide that has affinity to lipopolysaccharides (LPS) found on gram-negative bacterial membranes, and (3) a glycine linker to couple di-tuftsin and YI13WF together. This thesis reports on the characterization of the artificial opsonin. First, the binding stoichiometry of the opsonin to several gram-negative and gram-positive bacteria was quantified. Second, the artificial opsonin’s affinity to bind to RAW 264.7 mouse macrophages was evaluated. Finally, the efficacy of the opsonin to enhance RAW 264.7 macrophages’ phagocytic capabilities of Klebsiella pneumoniae was determined.