The sites colonized by Neisseria gonorrhoeae and Neisseria meningitidis within the human host are diverse and each site represents a unique niche with respect to nutrients, environmental factors and competing microorganisms. The growth environment has a marked effect on the metabolism and cellular composition of N. gonorrhoeae and N. meningitidis, and an altered cellular composition is often reflected in the interaction of these microorganisms with the human host. The overall goal of this project is to elucidate the specific mechanisms employed by the pathogenic Neisseria for the assimilation of the essential nutrient iron. We will focus a majority of our efforts on the major iron- regulated protein, Fep. The recently cloned fep will allow us to create mutants defective in the production of Fep by miniT13 mutagenesis. We will examine the DNA sequences 5' to the fep structural gene and investigate how iron regulates the expression of Fep. We will also examine the regulation by iron of the Fep transcript. The specific location of the gonococcal Fep in the cell will be determined by osmotic shock techniques, as well as its interaction with other cellular components by cross-linking techniques. Finally, we will use our recently described N. meningitidis Tn916 mutagenesis system to isolate specific mutants defective in iron utilization. The resulting mutated DNA will be used as probes to clone the corresponding wildtype DNA. Mutants generated in these studies will be examined in vivo in our animal models. The mouse subcutaneous chamber model will be employed for studies on the growth of N. gonorrhoeae and N. meningitidis mutants. In addition, we will examine N. meningitidis mutants in the mouse bacteremia model. These studies will allow us to evaluate the role of iron utilization in the growth and subsequent pathogenicity of infection caused by N. gonorrhoeae and N. meningitidis.
