Investigating the function of the mold specific gene M46 in the pathogenic dimorphic fungus Histoplasma capsulatum
Histoplasma capsulatum (Hc) is a dimorphic fungus that is the etiologic agent for the respiratory infection histoplasmosis. The fungus is found in the environment in contaminated soils of birds and bat excreta as a multi-cellular saprophytic mold. Once the soil is disturbed, spores are released and are inhaled into the lungs. In the lungs, the fungus converts to uni-cellular parasitic yeast (Maresca & Kobayashi, 1989). The conversion from mold to yeast is a requirement for pathogenesis. Previously, several mold specific genes have been isolated by our laboratory from a subtracted cDNA library. One such gene, is M46. M46 is a single copy gene and has a predicted protein size of 8.5 kDa. Northern blot analysis of M46 in four major Hc strains, revealed that M46 is not expressed in yeast. The gene is expressed in mold for strains G186AS and Downs, but is silent in strains G184AS and G217B. These findings suggest that M46 may not be involved in dimorphism. According to NCBI Genbank, M46 does not have a homolog, and therefore the function is unknown. Several experiments were conducted to gather more information about possible functions of M46. Over expression of M46 in the yeast did not induce filamentous growth. Examination of a M46 knock out mutant constructed by allelic replacement, with confocal microscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM), did not reveal any significant structural changes. Therefore, M46 may not be involved in maintaining the mold and yeast morphology. Growth curve studies of the M46 knockout and wild type strains, revealed that M46 is not involved in maintaining the normal growth rate in mold and yeast. Parallel comparison of gene expression of the M46 knockout and wild type strain via RNA sequencing, suggest that the function of M46 may be correlated with drug resistance and iron and sugar transport.