Histoplasma capsulatum , a pathogenic dimorphic fungus that causes pulmonary and systemic disease in the normal and compromised host, poses a particular threat to patients who are immunologically defective, particularly those with the Acquired Immunodeficiency Syndrome (AIDS). In the AIDS patient progressive disseminated histoplasmosis is a significant cause of morbidity and mortality. Histoplasma capsulatum causes disease by parasitizing and surviving within non-activated macrophages. Little is known about the mechanisms by which the fungus survives and thrives within this environment. By understanding the genetic bases of how H. capsulatum subverts the normal killing mechanism of the macrophage and creates an hospitable intracellular niche, it may be possible to design more effective therapies and vaccines that can be used to treat the compromised patient and those at high risk to develop disease. Histoplasma capsulatum invades the human host through the lungs. The ability to survive within alveolar macrophages is therefore the first obstacle the organism must overcome to cause infection and a prime target for therapeutic intervention. Macrophages are highly phagocytic cells that kill microbes they have ingested by production of reactive oxygen and nitrogen metabolites, synthesis of bactericidal peptides, and formation of lysosomal acid hydrolases. Intracellular pathogens have developed diverse strategies to survive within macrophages, and in recent years we have uncovered a few of the tricks that used to avoid macrophage killing. However, the mechanisms by which H. capsulatum achieves the goal of intracellular survival is less well delineated. It neither escape from phagosomes nor inhibit phagosome-lysosome fusion; rather, these organisms survive within phagolysosomes, and appear to alter the intraphagosomal environment by moderating the pH within this organelle. We do not understand the mechanism by which this occurs, nor do we know whether the phagocytic event that mediates the internalization of these yeast is equivalent to the paradigms that have been established primarily using opsonized erythrocytes. There are 4 goals in our application. We plan to: (1) isolate and identify genes that are turned on and the products that are expressed during the process of attachment to- and survival within- macrophage; (2) clone and sequence the amplified mRNAs and perform comparative analysis of the peptide sequences they code for and select those that have low homology frequency with human sequences; (3) clone and characterize selected cDNA sequences from 2 cDNA libraries made from mRNA purified during attachment to macrophage infection and then perform gene knock-out experiments to identify the role these genes play in attachment and survival; and (4) locate the position of the cloned genes in our H. capsulatum yeast artificial chromosome (YAC) library.
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