Cloning, expression and knockout of a mold-specific gene in Histoplasma capsulatum

Xianbin Tian

Abstract

Histoplasma capsulatum is a dimorphic pathogenic fungus. It grows as a multicelular differentiated mold form in soil or in the lab at 25°C. When inhaled into the respiratory tract or when the incubation temperature shifted to 37°C, it shifts into a unicellular yeast growth form. The yeast form is the etiologic agent of one of the most common systemic fungal diseases, histoplasmosis, which may be life threatening particularly to immunocompromised individuals. The molecular biology of its dimorphism is unclear. A general hypothesis is that phase specific genes play a critical role in the process. Several yeast specific genes have been cloned and analyzed in the past ten years. However, there are no studies of mold specific genes reported in the literature. In this project we used a modified enzymatic degradation subtraction (EDS) method to construct a subtractive library of enriched mold specific/upregulated genes. Three mold-specific genes were selected from preliminary screens for analysis: MS8, MS88 and MS95 . Sequence analysis showed that MS8 was 1278 by with an ORF (open reading frame) of 612 by encoding a putative glutamine-rich protein, which was similar to a novel gene cloned from the plant pathogenic fungus Colletotrichum gloeosporioides . MS88 was 1168 by with an ORF of 657 bp, it had no match in a GenBank homology search. MS95 was 1297 by with an ORF of 891 bp, it showed high similarity to the DDR48 gene (DNA damage responsive protein) from Saccharomyces cerevisiae . Genomic analysis showed that MS8 was present in a single copy and was interrupted by a single 96 by intron. Primer extension showed that MS8 had three closely spaced transcription start sites. Time-course northern blot analysis showed that the in vivo expression of MS8 was upregulated 11 hours after the 37°C to 25°C temperature shift. Over-expression of MS8 in yeast cells growing at 37°C caused cell clumping in liquid culture and formed sticky colonies on plates, but did not influence the yeast cell morphology. The MS8 genomic knockout strain was constructed with an improved negative/positive selection method. Although the knockout did not change the yeast-to-mold transition, it influenced the morphology of mold growing on solid media. The knockout mold colonies were about half the diameter of normal colonies, produced a dark red pigment, and had short hyphal branches which were approximately 30% wider with a marked "zig-zag" shape.