There is an urgent need for the development of innovative strategies to counter the growing threat of multi-drug resistance. Complement (C) presents the first line of defense against microbial intruders. Complement directs the removal of invasive bacteria through two major mechanisms: 1) the clearance of opsonized bacteria by the mononuclear phagocytic system; and 2) the disruption of bacterial cell integrity by the membrane attack complex (MAC), leading to lysis. Many Gram-negative bacteria resist C attack by expressing outer membrane components that recruit fluid phase complement regulators that serve to protect host tissues. Our long-term goal is to develop novel C-based antibiotics that can be effective against pathogenic Gram-negative bacteria. Properdin, a component of the complement alternative activation pathway (AP), is the sole positive regulator of complement. It plays an essential role in combatting pyogenic infection, serving to stabilize the labile AP convertase, a key player in the cascade. In certain circumstances properdin can also initiate C activity by binding directly to a target, forming a platform for convertase assembly. High MW properdin, manufactured in the laboratory, binds readily to Streptococcus pneumonia and N. meningitides, initiating C-dependent bactericidal activity in vitro, and conferring protection against infection in animal models. High MW properdin, however, binds a variety of biological surfaces and would potentially cause dangerous second site C activity. We posit that properdin antimicrobial activity would be more specific and safe if targeted via an antibody drug conjugate. We have previously engineered scFv-properdin bifunctional constructs and demonstrated their capacity to direct C activity to a red blood cell in vitro. Importantly, properdin-initiated complement activation occurs in the presence of endogenous fluid phase and surface bound complement inhibitors, including the host C regulators that protect many Gram-negative bacteria. We propose to engineer single chain antibody/properdin conjugates that bind to the Gram-negative outer wall and mediate bacterial lysis and clearance. To that end we propose the following specific aims: 1. Produce scFV-properdin bifunctional proteins that can recognize the major Gram-negative cell wall components (lipopolysaccharide, LPS). 2. Use in vitro and in vivo assays to characterize the capacity of scFv-properdin bifunctional proteins prepared in SA#1 to mobilize a complement response against Gram-negative bacteria.
Pathogenic bacteria that are resistant to multiple antibiotics pose a great threat to human health. They are a particular problem in hospitals and nursing homes, often causing deadly infections, and they are responsible for common diseases such as gonorrhea and food poisoning. Complement, a part of the immune system, is an essential first line of defense against microbial intruders, but many bacteria can resist complement activity. Here we propose the development of a new class of antibiotics that would boost the capacity of the complement system to recognize and destroy pathogenic bacteria.
