This application seeks to define the molecular mechanism of complement C3 binding interactions with bacterial surfaces. Specifically we will evaluate how Pseudomonas aeruginosa C3 binding to an outer membrane porin (OprF) promotes phagocyte interactions and how this may be exploited to enhance opsonophagocytosis and complement-mediated killing. We also discovered that the C-terminal region of P. aeruginosa OprF has high structural identity with the OmpA domain of several other Gram- negative bacteria, one of which is known to bind C3. Therefore, this study will not only contribute to an understanding of P. aeruginosa pathogenesis but also provide insights into the biology of complement interactions in other Gram-negative bacteria. This will, in turn, lead to new and more effective strategies for the treatment or prevention of P. aeruginosa and perhaps other Gram-negative infections. This proposal will use state-of-the art molecular, biochemical, and genetic approaches to probe interactions of complement and Gram-negative porins. To date, the mechanistic basis for resistance to complement-mediated killing of P. aeruginosa is not completely understood. We recently discovered that OprF is one ligand recognized by human complement C3.
Aim 1 will evaluate conservation of OprF-C3 binding and Psl masking among P. aeruginosa clinical isolates. The goal of Aim 2 is to determine if OprF levels can be modulated to improve serum-mediated killing.
Aim 3 will focus on mapping regions of OprF that are required for C3 interactions. Finally, in Aim 4 we will evaluate conservation of C3 interaction with surface exposed porin domains in other Gram-negative pathogens. Understanding the molecular mechanism of C3 binding to bacterial surfaces offers a potential means for preventing and/or treating diseases caused by these Gram-negative pathogens.
Pseudomonas aeruginosa is versatile opportunistic pathogens that can cause devastating persistent infections. The complement system is a highly conserved innate immune pathway and the role of complement in our first line defense against pathogens is widely appreciated. However, most P. aeruginosa strains are highly resistant to the killing effects of complement. This proposed study will determine the basic molecular mechanism underlying bacterial porin and complement factor C3 interactions. The primary goal of this research is to enhance complement-mediated interactions and promote opsonophagocytosis and complement-mediated killing of P. aeruginosa and perhaps other important pathogens. This research could directly benefit an estimated 500,000 Americans diagnosed with burn-wounds and/or pulmonary infections.