Biochemical and functional characterisation of novel antimicrobial egg molecules – OVO-Mining

The chicken egg is a model of particular interest since it contains all the components that are essential for embryonic development in a closed chamber exposed to a putative aggressive milieu. The hen must anticipate the needs for embryonic growth and protection and therefore supplies in the egg, in addition to the nutrients essential for embryogenesis, many molecules that participate in the defence of the embryo. Antimicrobial activities of some egg white proteins, such as lysozyme and ovotransferrin have been known for a long time but more recently some additional activities including antiviral, antioxidant, anti-adhesive, immunomodulating or anti-inflammatory have been reported. Thus, for humans, the egg is useful because of its high nutritive value but is also a putative source of numerous active molecules that might be of major interest for different industrial areas such as food industry, cosmetics or human health. Until recently, fewer than 100 proteins were identified in the egg white, yolk or eggshell matrix. However, the development of high-throughput methods in combination with the newly available chicken genomic sequence has recently revealed the presence of more than 1000 minor egg proteins which had not yet been characterized. Our objective is to characterize biochemically and functionally some novel molecules involved in chemical defences of egg. Identification of new proteins and peptides will be carried out using high-throughput approaches: a transcriptomic approach using temporal and spatial sequences of egg formation will explore over-expression of genes coding for yolk and vitelline membrane proteins (Task1); application to hen magnum of a recently developed high-throughput antimicrobial screening will revealed numerous novel biocidal egg white proteins (Task2). Candidate proteins and peptides will be purified using affinity chromatographies for the purification of proteases and antiproteases or of proteins with affinity for bacterial membrane to alleviate the usual contamination by major egg proteins (Task 3). The antibacterial spectra of egg fractions or of purified proteins or peptides predicted as biocidal by annotations of sequences or by direct screening of their antimicrobial activity will be characterized against a large range of bacterial pathogens using the collection of the International Centre of Microbial Resources dedicated to pathogen bacteria. In addition, micro-titre plate assays will be settled to test very limited quantities of purified molecules (Task 4). Interaction between these molecules and LPS from Salmonella will be explored by developing LPS mutants and by real time interaction analysis (Biacore). The last task (5) aims to structurally (3D) characterized some of the most relevant egg molecules to allow identification of particular domains which can contribute to structure'based drug design.