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

Abstract

BackgroundHistoplasma 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.ResultsThe 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.ConclusionsThe 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.

Frequency and Genetic Diversity of the MAT1 Locus of Histoplasma capsulatum Isolates in Mexico and Brazil

“…To date, there have been a few relevant studies in the United States about the use of genetic tools to determine sexual compatibility in H. capsulatum, in which the Ϫ mating type predominates (6)(7)(8). Recent findings have involved the product of the Velvet A gene (VeA), which belongs to the proteins of the Velvet family, in mating structure formation (cleistothecial) and virulence of H. capsulatum (9).…”

“…The classical studies of sexual compatibility in H. capsulatum were performed by mating fungal specimens in culture plates. However, this procedure is difficult because H. capsulatum isolates rapidly lose the ability to mate in vitro (5); therefore, molecular methods were developed to identify the mating type in this microorganism (6)(7)(8).…”

Experimental annotation of the human pathogen Histoplasma capsulatum transcribed regions using high-resolution tiling arrays

“…Consistent with this hypothesis, we find STE3 , the a-factor receptor whose expression has been observed only in mutants of G217B[17]; the ortholog of N. crassa RID , which is required for the RIP process and therefore expected to be expressed only during meiosis[18]; and the ortholog of T. reesei AXE2 , a hemicellulolytic enzyme whose expression is dependent on carbon source[19]. …”

Site-Directed and Random Insertional Mutagenesis in Medically Important Fungi

“…One mutant actually gained the ability to form cleistothecia, a mating structure that was not present in the parent strain. This phenotypic trait was not due to the hph gene; and, thus, the strain may be used as a tool to study mating in Histoplasma (Laskowski and Smulian, 2010).…”

“…Researchers have developed protocols that have improved the efficiency of transformation using T-DNA in C. neoformans (Idnurm et al, 2004), Histoplasma and Blastomyces (Brandhorst et al, 2002;Edwards et al, 2011;Gauthier et al, 2010;Laskowski and Smulian, 2010;Marion et al, 2006;Smulian et al, 2007;. In addition, T-DNA mutagenesis protocols have been developed for Coccidioides (Abuodeh et al, 2000), Trichoderma spp.…”