The central theme of the Program Project is to understand the innate host defense mechanisms against inhaled pathogens by the two pulmonary collectins, surfactant proteins A and D (SP-A, SP-D). This understanding is important not only in the context of prevalent respiratory infections but also those associated with serious global biothreats - the emergence of antibiotic-resistant organisms;evolution and rapid spread of more lethal respiratory viruses;and potential use of inhaled pathogens in acts of bioterrorism. Effective pulmonary host defense requires early recognition of microorganisms. Following deposition of an organism into the respiratory tract, there is a critical window of opportunity for pathogen clearance;delayed response favors infection. The pulmonary collectins play a front-line role in the innate defense against many Gram-negative and positive bacteria and their endotoxins, mycobacteria, pathogenic fungi, and obligate intracellular pathogens including influenza A (lAV) and other potentially lethal viruses. The innate defense properties of pulmonary collectins depend upon their rapid recognition and, in some cases, neutralization of inhaled microorganisms based on pattern recognition of highly-conserved microbial surface components such as N-linked high-mannose glycans on lAV and Gram-negative lipolysaccharide (endotoxin). Other studies show that animals deficient in lung collectins show significantly increased susceptibility to microbiological challenges. Studies of recombinant truncated SP-A and SP-D in murine models of allergy and infection have offered the possibility that aerosolized forms of these proteins delivered by inhalation may have therapeutic potential in controlling respiratory infection, inflammation, and allergy in humans. In this program project, a highly collaborative and focused group of projects using complementary approaches, including x-ray crystallography, mutagenesis, spectroscopy, in vitro and in vivo analyses using animal models, aim specifically at innate responses of collectins, and proteins such as KGF that modulate their activity, to lAV and Gram-negative bacteria both to gain mechanistic understanding and assist design of potential collectin-based therapeutics with increased antimicrobial activities. PROJECT 1: Vibrational Spectroscopy: Collectin Interactions with Physiological Ligands (Mendelsohn, R) PROJECT 1 DESCRIPTION (provided by applicant): The long term objective of this project is to elucidate the molecular basis by which collectins in the lung provide the first challenge to airborne pathogens in host defense. The surfactant specific collectins SP-A and SP-D, through their interactions with lipopolysaccharides (LPS), are responsible in part for this activity. With the technological expertise in novel vibrational spectroscopic approaches developed in this lab, we propose two specific aims to characterize the interaction of both proteins with physiological LPS derivatives. With the expertise of the other investigators in the program project, we will have access to a large number of genetic variants of these proteins, permitting us to pinpoint the primary interaction sites in a variety of biologically relevant physical preparations.
Two specific aims are proposed. First, since both collectins recognize carbohydrate and other polar ligand sites, we will determine those collectin structural factors important for binding to LPS and its variants in Langmuir films (i.e. monolayers) at the air/water interface using a unique vibrational spectroscopy experiment (Infrared Reflection Absorption Spectroscopy- IRRAS) that extracts molecular structural and orientational information from the monolayer constituents. These films mimic polar regions of bacterial Gram negative outer membrane monolayers. The integrating hypothesis for this Aim is that at the level of molecular structure, the interaction between collectins and LPS depends on particular elements of protein structure and emphasizes specific regions of the collectin-carbohydrate recognition domain. Raman micro crystallography complements IRRAS and provides very specific structural information about protein side chains involved in the initial recognition event. In the second Aim, we will complement studies of protein structural changes with IR experiments that track structural changes both in the acyl chains and in the polar regions of the LPS derivatives. We will monitor collectin/LPS interaction under conditions where Langmuir films are not the only reasonable experimental paradigm. Physical states of lipid and lipid/protein complexes from which structural information will be acquired include vesicles, monolayers, supported oriented multibilayers, and micelles.
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