Disease caused by Neisseria gonorrhoeae is a significant public health problem. A key to the successful management of this disease is defining the attributes which make the gonococcus a human pathogen. Like most infectious diseases, the etiology of gonorrhea proceeds by a multifactoral process applying the basic pathogenic steps of transmission, colonization, multiplication, and production of disease. The ability of the gonococcus to multiply is dependent upon obtaining growth-essential iron. Withholding iron by vertebrate hosts is a principal mechanism of non-specific immunity against infection. Thus, the expression of a high-affinity iron-acquisition system is a prerequisite for infection. Studies demonstrate that this system involves iron-responsive elements which sense conditions of iron- deprivation and induce the expression of receptors on the gonococcal cell surface which bind the host iron bearing proteins, lactoferrin or transferrin. Next, a system for removal of iron from gonococcal- associated lactoferrin or transferrin is required followed by transport of iron across the outer and cytoplasmic membranes. The transport phase of this process appears to engage the wall-characterized iron-regulated and iron-binding protein, Fbp, as a central participant. To more clearly elucidated the molecular basis of gonococcal iron-acquisition, we intend to study the aspects related to Fbp which will define its role in this process and exploit it as a probe to define other molecular participants in this process.
The specific aims of this proposal are to (1) Characterize the genetic organization of the gonococcal Fbp; (2) Demonstrate the functional role of Fbp in the transport of growth- essential iron; and (3) Identify accessory proteins linked to the process of gonococcal iron-acquisition. The proposed experiments take advantage of the recent success in genetically characterizing the gonococcal Fbp. This has provided us with insight into the structure, regulation and the potential organization of this protein as the first gene of a multi-component operon and has led to strong evidence that FBP functions like a typical periplasmic-binding protein. We propose to extend these studies by identifying and characterizing proteins which act at the level of Fbp regulation. The atomic basis of Fbp iron-binding will be defined by a combination of chemical modification, site-directed mutagenesis, and X-ray crystallographic analysis. Additional candidate proteins which operate in conjunction with the gonococcal Fbp during the process of iron-acquisition will be identified by nearest neighbor analysis using chemical cross-linkers. We will characterize regions downstream of Fbp to identify genetically linked participants in the process of iron-acquisition. The long range goal of these studies are to understand the molecular basis for gonococcal iron-acquisition and to determine the role of this process in infection caused by N. gonorrhoeae and related pathogens.
