Insertional mutagenesis enables cleistothecial formation in a non-mating strain of Histoplasma capsulatum

Abstract

Background: Histoplasma capsulatum is a pathogenic ascomycete fungus that rapidly loses mating ability in culture. Loss of mating ability, as well as the organism's low rate of targeted gene replacement, limits techniques available for genetic studies in H. capsulatum. Understanding molecular mechanisms regulating mating in this organism may allow us to reverse or prevent loss of mating in H. capsulatum strains, introducing a variety of classical genetics techniques to the field. We generated a strain, UC1, by insertional mutagenesis of the laboratory strain G217B, and found that UC1 acquired the ability to form mating structures called cleistothecia. The aim of this study was to determine the mechanism by which UC1 gained the ability to form cleistothecia. We also present initial studies demonstrating that UC1 can be used as a tool to determine molecular correlates of mating in H. capsulatum.

Results: The strain UC1 was found to have increased RNA levels of the mating locus transcription factor (MAT1-1-1), and the putative alpha pheromone (PPG1) compared to G217B. Agrobacterium-mediated transformation and integration of T-DNA from the vector pCB301-GFP-HYG were found to be partially responsible for the increased RNA levels of these genes; however, the site of integration appeared to play the largest role in the strain's ability to form cleistothecia. Silencing HMK1, a putative FUS3/KSS1 homolog, had no effect on cleistothecial production by UC1. Protein kinase C (PKC1) RNA and protein levels were increased in UC1 compared to G217B, and pheromone production was found to be linked with Pkc1 activity in H. capsulatum.

Conclusions: The site of the T-DNA integration event appears to play the largest role in UC1's ability to form cleistothecia. We show that the UC1 strain can be used as a tool to study cleistothecia production in H. capsulatum by manipulating the strain, or by identifying differences between UC1 and G217B. Using these approaches, we were able to link Pkc1 activity with pheromone production in H. capsulatum; however, further studies are required to determine molecular mechanisms behind this. These studies may reveal regulatory mechanisms that can be manipulated to restore mating ability in H. capsulatum laboratory strains.

Figure 1

Cleistothecia formed by mating crosses. A: Cleistothecia formed by UH3 and UC1, DIC image, 400×. B: Cleistothecia formed by UH3 and UC26, DIC image, 400×. C: Dissected cleistothecia from UH3 and UC26 pairing, DIC image, 400×. D: Alpha projection of Z-stack taken of cleistothecia formed by UH3 and UC1, confocal image of autofluorescence, 600×. The coiled surface hyphae are identified by short arrows while the net of short, branched hyphae are identified by long arrows.

Figure 2

SEM images of cleistothecia formed by UH3 and UC1. A: Dissected cleistothecia, 200×. B: View A, 1000×. C: View B, 2500×. D: Whole cleistothecia, 100×. E: View D, 500×. F: Microconidia, 2000×. In panels A and D, cleistothecia are identified by symbol *, while coiled surface hyphae are identified by short arrows while the net of short, branched hyphae are identified by long arrows where appropriate.

Figure 3

Molecular differences between G217B, UC1, and UC26. A-C: MAT1-1-1, PPG1, and HMK1 RNA levels in G217B, UC1, and UC26 mycelial samples as measured by qRT-PCR. D, E: STE2 and STE3 RNA levels in G217B and UC1 mycelial samples, measured by qRT-PCRr. F, G: BEM1 RNA levels in G217B, UC1, and UC26 yeast (F) and mycelial (G) samples, measured by qRT-PCR. Values represent the average and standard error of quadruplicate samples except 3A: UC1, n = 6; UC26, n = 4; 3D: UC1 n = 3; 3F: G217B & UC1, n = 3; 3G: n = 3. * = p ≤ 0.05 ** = p ≤ 0.01 *** = p ≤ 0.001 # = below level of detection.

Figure 4

Effects of T-DNA insertion from two different vectors on RNA levels of MAT1-1-1, PPG1 and BEM1. Comparison of G217B, UC1, and UC26 with strains with pCB301-HYG-GFP integrated at alternate sites (Alt), ALT strains with hph excised (Alt cre), or strains with pCB301-Blast integrated into the genome (G217B Blast). RNA levels of MAT1-1-1 (A), PPG1 (B), and BEM1 (C) in mycelial samples were compared by qRT-PCR. Alt samples represent the average of values obtained from triplicate samples of 4 different strains. Alt cre and G217 Blast samples represent the average of values obtained from triplicate samples of two different strains. n = 3 except 4A: UC1, n = 6; UC26, n = 4; 4B: n = 4 for G217B, UC1, and UC26. ** = p ≤ 0.01 # = below level of detection.

Figure 5

Overexpression of MAT1-1-1 and BEM1 in G217B. A: Detection of c-myc tagged recombinant fusion protein using anti-c-myc antisera on a Western blot of homogenates of H. capsulatum strains overexpressing Bem1 (lane 2), Mat1-1-1 (lane 5) or a control strain (lane 1). Detection of HSP60 as a loading control is shown on a duplicate blot in lane 3 and lane 4. B: RNA levels of MAT1-1-1 in mycelial phase G217B (n = 4), UC1 (n = 6), and UC26 (n = 4) compared to levels in strains overexpressing MAT1-1-1 and BEM1 in the G217B background (n = 3) # = below level of detection. C: RNA levels of PPG1 in mycelial phase G217B (n = 4), UC1 (n = 7), and UC26 (n = 4) compared to levels in strains overexpressing MAT1-1-1 and BEM1 in the G217B background (n = 3). *** = p ≤ 0.001.

Figure 6

Effects of silencing HMK1 on cleistothecia formation. A: HMK1 RNA levels found in yeast phase of the silenced strain (UC1-HMK1-RNAi) compared to those found in the empty vector control strain by qRT-PCR. Values represent averages and standard error of triplicate samples. B: Number of cleistothecia counted from three pairings of UC1 + UH3, or UC1 with HMK1 silenced + UH3.

Figure 7

Microarray analysis of UC26 and G217B gene expression. Functional annotation of genes up-down regulated greater than 3-fold in UC26 compared to G217B was performed using BLAST2GO Biological processes assigned to upregulated (Panel A) or downregulated genes (Panel B) are shown. Genes with altered gene expression to which molecular function was assigned, are shown in Panel C and D.

Figure 8

PKC1 RNA and protein levels in G217B, UC1 and UC26. A: PKC1 RNA levels in mycelial phase G217B, UC1, and UC26, by qRT-PCR. B: PKC1 RNA levels in strains with pCB301-HYG-GFP integrated into alternate sites of the genome, compared with PKC1 RNA levels in G217B and UC1. C: Pkc1 activity found in activated cell lysates of G217B, UC1, and UC26. All values represent averages and standard error of triplicate samples. * = p ≤ 0.05.

Figure 9

Effects of PKC inhibitor on pheromone production. Effects of PKC inhibitor, chelerythrine chloride (25 μM), on PPG1 RNA levels in mycelial samples of UC1 and UC26 after 1 hour exposure, compared to UC1 and UC26 exposed to HMM alone. Values represent averages and standard error of triplicate samples.