The Mould-specific M46 gene is not essential for yeast-mould dimorphism in the pathogenic fungus Histoplasma capsulatum

Abstract

Histoplasma capsulatum (Hc) is the causative agent for the respiratory infection histoplasmosis. The fungus exists in the environment as a saprophytic multi-cellular mould. Spores are inhaled by mammals whereupon the organism will convert into the single-celled yeast morphotype resulting in infection. The shift to the yeast morphotype is required for pathogenesis. Most studies on dimorphism have examined yeast-phase-specific genes and few mould-phase-specific genes have been investigated. It is likely, that some mould-phase-specific genes must be downregulated for the yeast to form or upregulated for the mould to form. We isolated a strongly expressed mould-specific gene, M46, from an expression library enriched for mould upregulated genes in Hc strain G186AS. To determine if M46 is involved in dimorphism, M46 was ectopically expressed in yeast phase growing temperature, and an m46 knockout strain was created via allelic replacement. Ectopically expressing M46 in yeast, did not induce filamentous growth. Genomic disruption of M46 by allelic replacement did not alter the morphology of the mould as seen in bright field microscopy, scanning electron microscopy, and transmission electron microscopy. A growth curve study, revealed that M46 is not involved in maintaining the growth rate of cells. These findings indicate that the mould specific M46 gene is not necessary nor essential for dimorphism, maintaining the normal mould morphology, and growth rate of Histoplasma capsulatum.

Keywords: Histoplasma capsulatum; M46; dimorphic; fungus; pathogenic.

Figure 1.

Putative protein and genomic sequence of M46 (GenBank assession no. KM078708) in Hc. The sequence is numbered from the transcriptional start site. On the left, numbers are designated as nucleotide sequence, and numbers on the right are amino acid sequences. The putative TA rich sequence and transcriptional start sites are underlined. Exon 1 encodes for entire open reading frame. Exon 2 is enclosed in [] and contains the polyadenylation signal (underline and bold). M46 has a 92 bp intron (lowercase), The GT, AG, and GCTAAC mark the beginning, end, and branching signal of the intron (underlined lowercase).

Figure 2.

M46 ectopical expression plasmid. The TEF1 promoter was fused in frame to the M46 ORF and sub-cloned into the pCR2.1 (Invitrogen) multiple cloning site creating pMOE. The PaURA5-tel tet fragment from pRPU1 was ligated into the ApaI site of pMOE to generate pDCMO1.

Figure 3.

Ectopical expression of M46 in Hc yeast. Y. Yeast cells not expressing M46. Y1,Y2; Cells ectopically expressing M46. A. Cells grown on solid HMM media. Images were taken with the Canon EOS 20D camera at 63X. B. Cells grown in HMM liquid media. Images were taken with a Confocal 150 (Zeiss) microscope.

Figure 4.

Genomic replacement map of m46. A 4.8 kb fragment of M46 containing a 2.0 kb left flanking region and a 2.6 kb right flanking region was amplified via PCR (primers indicated with arrows; M46pf1, M46pr1), and cut with restriction enzymes EcoNI and BsmI to delete a 700 bp fragments that contains 69% of the ORF. This fragment was replaced with the hygromycin antibiotic resistant marker (Hph 3.1 kb). Primers designed to confirm the allelic replacement are indicated with arrows; M46pf2, M46pr2. The primers were designed to amplify ∼ 500 upstream and downstream the M46 chromosome region.

Figure 5.

Hc parental and m46 knock out strain mould grown in liquid and solid HMM media. Parental strain (left) and m46 knockout strain (right). A. Cells were grown at 25 °C in HMM liquid media for mould morphology. Images were taken with a Confocal 150 (Zeiss) at 63X. B. Cells were grown at 25 °C on HMM plates for mould morphology. Images were taken with a Canon EOS 20 D camera.

Figure 6.

Scanning electron microscopy of parental and m46 knockout strain in Hc. Parental strain (left) and m46 knockout strain (right) mould. Cells were sputtered with 15 nm of platinum. Images were taken at 50,000 X, Bar = 100 nm.

Figure 7.

Transmission electron microscopy of parental and m46 knockout strain in Hc. Parental strain (left) and m46 knockout strain (right) mould. Images were taken at 30,000 X, Bar = 0.5 um.

Figure 8.

Growth curve of m46 knockout and parental strain mould in Hc. Cells were grown as yeast for 7 days at 37^oC and shifted to 25^oC to grow as mould for 10 days. The hyphal elongation was measured every 24 hrs from day 5 to day 10, a total of 6 days. The measurements were taken by using a 10X ocular lens micrometer on a CZM4 Labomed microscope.