Neisseria gonorrhoeae (GC) infects a variety of mucosal epithelial surfaces, including the eye, causing hyperacute conjunctivitis. Untreated, these infections can cause visual impairment and blindness. GC infect only humans due to a tropism for human forms of host cell receptors and a requirement for human transferring (Tf). Tf is a host iron-binding glycoprotein that shuttles iron from sites of absorption to cells of the body. To acquire essential iron in vivo GC produce specific Tf-binding proteins (Tbps) in their outer membrane that allow the piracy of iron from host Tf. GC mutants devoid of Tbps are unable to initiate urethral infection in male volunteers. The goal of the proposed study is to improve current understanding of how pathogenic microbes alter the physiology of cells of the ocular surface, to attain sufficient iron for growth. GC likely play an active role in enhancing their supply of iron on the mucosal surface, by manipulating host conjunctival epithelial cells to downregulate transferrin receptors (TfR), and altering their TfR cycling and TfR distribution patterns. Since animal models for GC eye infections are lacking, conjunctival cell lines and where appropriate, donor tissues will be used to assess uninfected and infected conjunctival cells for downregulated Tf-receptor (TfR) by semi-quantitative RT-PCR. Using a functional assay with radiolabeled Tf and live cells, the levels of host cell TfR will be measured and the TfR cycling rate will be determined in the presence and absence of infection. The other key host iron homeostasis proteins, ferritin, iron regulatory protein-1 (IRP1) and IRP2 levels and biological activity will also be monitored during the course of GC infection. Wild type and isogenic GC mutants lacking specific virulence determinants will be used for the assays. In addition, a specially designed DNA microarray, the 'iron chip', will be used for simultaneous comprehensive analysis of GC alteration of host gene regulation of some 200 genes known to play a role in cellular iron homeostasis. These studies will aid in delineating the specific bacterial products and host signaling factors that may significantly contribute to the processes of iron withholding by host epithelial cells. These studies may lead to novel treatment strategies, since withholding iron can arrest bacterial growth. ? ?
