Insertional mutagenesis enables cleistothecial formation in a non-mating strain of Histoplasma capsulatum.
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.
Infectious Disease Division, University of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati, OH 45267-0560, USA.
Meggan C
MC
Laskowski
Alan G
AG
Smulian
D004269:DNA, Bacterial / Q000235:genetics; D048690:Fruiting Bodies, Fungal / Q000235:genetics* / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D020869:Gene Expression Profiling; D020868:Gene Silencing; D005800:Genes, Fungal; D006658:Histoplasma / Q000235:genetics* / Q000378:metabolism; D008855:Microscopy, Electron, Scanning; D016254:Mutagenesis, Insertional / Q000379:methods; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis; D010675:Pheromones / Q000235:genetics* / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism; D012331:RNA, Fungal / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012231:Rhizobium / Q000235:genetics; D014170:Transformation, Genetic
D004269:DNA, Bacterial; D005656:Fungal Proteins; D010675:Pheromones; D012331:RNA, Fungal; D012333:RNA, Messenger; C036483:T-DNA; D017853:Phosphotransferases (Alcohol Group Acceptor); C063242:hygromycin-B kinase; D011493:Protein Kinase C