Molecular Determinants of Calcitic Biomineralization.

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Flaws in chicken eggshell fabrication are a human health and food safety issue; the nutritious table egg is an important source of protein, minerals and vitamins for 100's of millions of individuals living in poverty worldwide. In Canada it is estimated that about 10,000 annual cases of salmonella poisoning are due to contamination of cracked eggshells that find their way into the food chain. In this sense, the reproducible nature of calcitic biomineralization of the eggshell is of critical concern to human society. My research program during the past 20 years has investigated the integrated defense strategies that function at this biomineralized barrier. My studies have revealed how protein components of the eggshell matrix modulate calcium carbonate crystal nucleation and growth in order to understand regulation of eggshell mechanical properties. Moreover, we have demonstrated the dual function of antimicrobial eggshell matrix proteins (EMPs) that regulate mineralization of the shell barrier which maintains the pathogen-free status of the egg.***The goals are to investigate the initiation of eggshell mineralization and the regulatory role of proteins. The role of specific EMPs and amorphous calcium carbonate (ACC) in eggshell formation (mineralization) and resorption (demineralization) will be determined. The dual role of antimicrobial EMPs will be further investigated.****Specific objectives:****1. Our transcriptomic analysis of specific gene expression in the isthmus segment of the oviduct identified collagen X (COL10A1), fibrillin-1 (FBN1) and cysteine rich eggshell membrane protein (CREMP) as key structural constituents. Their incorporation and relative organization in the native eggshell membranes will be investigated using specific antibodies. The assembly of these proteins into stable fibres will be investigated in vitro with purified native and/or recombinant proteins.****2. My hypothesis that the mammillary cone contains a stabilized pool of ACC as a Ca reservoir that the embryo accesses during growth will be tested by investigating the status of CaCO3 and identifying associated proteins in the calcium reserve body at the mammillary cone base.****3. Candidate proteins (such as OC17, NUCB) proposed to modulate mineralization during eggshell formation will be investigated with in vitro crystallization studies. Regulatory molecules will be validated by reconstitution experiments utilizing purified recombinant proteins to modulate the ACC to calcite transformation, and to reproduce nucleation of mammillary cones.****4. The antimicrobial activity and CaCO3 nucleation / crystal growth properties of OCX32 will be investigated to assess the dual function of this important matrix protein.****These studies will provide solutions to serious food safety issues such as salmonella contamination of the table egg.**