The importance of iron to the outcome of host-parasite interactions was first observed in Yersinia pestis, the causative agent of bubonic and pneumonic plague. Vertebrates present pathogens with a highly iron-deficient environment by the chelation of iron to host protein ligands. This nonspecific host defense system reduces the available iron to levels insufficient for bacterial growth. In addition to a cell bound ferric ion transport system, Y. pestis has the ability to use hemin as sole source of iron and can store hemin when grown at 26 degrees C; on characteristic of the pigmentation phenotype (Pgm+). Pgm+ cells also possess the following traits: 1) synthesis of several unique membrane and cytoplasmic peptides; 2) synthesis of large molecular weight components which bind iron; 3) sensitivity to the bacteriocin pesticin; 4) growth at 37 degrees C in a defined medium containing only sub-micromolar quantities of chelated iron; and 5) virulence in the mouse model. Pgm- mutants are avirulent without an exogenous iron source.
The specific aims of this proposal are to 1) define the mammalian iron compounds used as nutritional iron sources by Y. pestis as well as characterize the systems involved in their uptake; 2) isolate and identify genes involved in the expression of the various iron transport systems; 3) isolate and identify genes involved in hemin storage and determine their role in iron acquisition and stoous iron uptake systems to the virulence of Y. pestis. Growth response studies will identify the mammalian iron sources utilized by both Pgm+ and Pgm- cells of Y. pestis. Tn10 mutagenesis will then be used to insertionally inactivate genes involved in the utilization of various iron sources and expression of specific Pgm+ traits. Genes encoding these traits will be cloned and used to verify Tn10 mutant phenotypes, induce more specific mutations where necessary, and correlate gene products with specific functions. The effect of mutations on virulence will be determined by lethality studies in the mouse model. These results will provide general insights into iron acquisition methods and the importance of iron accumulation to microbial pathogenesis which will be relevant to other highly virulent facultative intracellular parasites.
