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

<p>Abstract</p> <p>Background</p> <p><it>Histoplasma capsulatum </it>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 <it>H. capsulatum</it>. Understanding molecular mechanisms regulating mating in this organism may allow us to reverse or prevent loss of mating in <it>H. capsulatum </it>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 <it>H. capsulatum</it>.</p> <p>Results</p> <p>The strain UC1 was found to have increased RNA levels of the mating locus transcription factor (<it>MAT1-1-1</it>), and the putative alpha pheromone (<it>PPG1</it>) compared to G217B. <it>Agrobacterium</it>-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 <it>HMK1</it>, a putative <it>FUS3/KSS1 </it>homolog, had no effect on cleistothecial production by UC1. Protein kinase C <it>(PKC1) </it>RNA and protein levels were increased in UC1 compared to G217B, and pheromone production was found to be linked with Pkc1 activity in <it>H. capsulatum</it>.</p> <p>Conclusions</p> <p>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 <it>H. capsulatum </it>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 <it>H. capsulatum</it>; 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 <it>H. capsulatum </it>laboratory strains.</p

Histoplasma capsulatum proteome response to decreased iron availability

<p>Abstract</p> <p>Background</p> <p>A fundamental pathogenic feature of the fungus <it>Histoplasma capsulatum </it>is its ability to evade innate and adaptive immune defenses. Once ingested by macrophages the organism is faced with several hostile environmental conditions including iron limitation. <it>H. capsulatum </it>can establish a persistent state within the macrophage. A gap in knowledge exists because the identities and number of proteins regulated by the organism under host conditions has yet to be defined. Lack of such knowledge is an important problem because until these proteins are identified it is unlikely that they can be targeted as new and innovative treatment for histoplasmosis.</p> <p>Results</p> <p>To investigate the proteomic response by <it>H. capsulatum </it>to decreasing iron availability we have created <it>H. capsulatum </it>protein/genomic databases compatible with current mass spectrometric (MS) search engines. Databases were assembled from the <it>H. capsulatum </it>G217B strain genome using gene prediction programs and expressed sequence tag (EST) libraries. Searching these databases with MS data generated from two dimensional (2D) in-gel digestions of proteins resulted in over 50% more proteins identified compared to searching the publicly available fungal databases alone. Using 2D gel electrophoresis combined with statistical analysis we discovered 42 <it>H. capsulatum </it>proteins whose abundance was significantly modulated when iron concentrations were lowered. Altered proteins were identified by mass spectrometry and database searching to be involved in glycolysis, the tricarboxylic acid cycle, lysine metabolism, protein synthesis, and one protein sequence whose function was unknown.</p> <p>Conclusion</p> <p>We have created a bioinformatics platform for <it>H. capsulatum </it>and demonstrated the utility of a proteomic approach by identifying a shift in metabolism the organism utilizes to cope with the hostile conditions provided by the host. We have shown that enzyme transcripts regulated by other fungal pathogens in response to lowering iron availability are also regulated in <it>H. capsulatum </it>at the protein level. We also identified <it>H. capsulatum </it>proteins sensitive to iron level reductions which have yet to be connected to iron availability in other pathogens. These data also indicate the complexity of the response by <it>H. capsulatum </it>to nutritional deprivation. Finally, we demonstrate the importance of a strain specific gene/protein database for <it>H. capsulatum </it>proteomic analysis.</p