Atypical fatal sarcocystosis associated with Sarcocystis neurona in a White-nosed coati (Nasua narica molaris).

The protozoan parasite Sarcocystis neurona is an important cause of disease in horses (equine protozoal myeloencephalitis, EPM) and marine mammals. Isolated reports of clinical EPM-like disease have been documented in a zebra, raccoon, domestic cat, domestic dog, ferret, skunk, mink, lynx, red panda and fisher. The predominant disease is encephalomyelitis associated with schizonts in neural tissues. Here, we report highly disseminated sarcocystosis, in many tissues of a captive White-nosed coati (Nasua narica molaris). The 14year old, neutered male coati was euthanized due to progressive weakness, lethargy, and inappetence. Schizonts, including free and intracellular merozoites were detected in many cell types, and differed morphologically from S. neurona schizonts in horses. Only a few sarcocysts were seen in skeletal muscle and the myocardium. Immunohistochemically, the protozoa reacted positively to S. neurona but not to Toxoplasma gondii antibodies. Severe inflammtory disease detected in the stomach, intestine, adrenal and thyroid glands, ciliary body of eye, and urinary bladder associated with schizonts in the coati has not been reported earlier in any host with EPM. Although, a few schizonts were found in the brain, encephalitis was minimal and not the cause of clinical signs. Multilocus PCR-DNA sequencing using DNA derived from the coati lung tissue identified an S. neurona infection using the 18S, 28S and ITS-1 markers, and a novel genotype using primer pairs against antigenic surface proteins (SnSAG3, SnSAG4, SnSAG1-5-6) and microsatellite markers (MS, SN7, SN9). Although the genotype was similar to the widely distributed Type VI strain, it possessed a novel allele at SnSAG5, and a different MS combination of repeats at SN7 and SN9. Whether this severe parasitism was related to the host or the parasite needs further investigation.

Construction of a Recyclable Genetic Marker and Serial Gene Deletions in the Human Pathogenic Mucorales Mucor circinelloides.

Mucor circinelloides is a human pathogen, biofuel producer, and model system that belongs to a basal fungal lineage; however, the genetics of this fungus are limited. In contrast to ascomycetes and basidiomycetes, basal fungal lineages have been understudied. This may be caused by a lack of attention given to these fungi, as well as limited tools for genetic analysis. Nonetheless, the importance of these fungi as pathogens and model systems has increased. M. circinelloides is one of a few genetically tractable organisms in the basal fungi, but it is far from a robust genetic system when compared to model fungi in the subkingdom Dikarya. One problem is the organism is resistant to drugs utilized to select for dominant markers in other fungal transformation systems. Thus, we developed a blaster recyclable marker system by using the pyrG gene (encoding an orotidine-5'-phosphate decarboxylase, ortholog of URA3 in Saccharomyces cerevisiae). A 237-bp fragment downstream of the pyrG gene was tandemly incorporated into the upstream region of the gene, resulting in construction of a pyrG-dpl237 marker. To test the functionality of the pyrG-dpl237 marker, we disrupted the carRP gene that is involved in carotenoid synthesis in pyrG- mutant background. The resulting carRP::pyrG-dpl237 mutants exhibit a white colony phenotype due to lack of carotene, whereas wild type displays yellowish colonies. The pyrG marker was then successfully excised, generating carRP-dpl237 on 5-FOA medium. The mutants became auxotrophic and required uridine for growth. We then disrupted the calcineurin B regulatory subunit cnbR gene in the carRP::dpl237 strain, generating mutants with the alleles carRP::dpl237 and cnbR::pyrG These results demonstrate that the recyclable marker system is fully functional, and therefore the pyrG-dpl237 marker can be used for sequential gene deletions in M. circinelloides.

Systematic functional analysis of kinases in the fungal pathogen Cryptococcus neoformans.

Cryptococcus neoformans is the leading cause of death by fungal meningoencephalitis; however, treatment options remain limited. Here we report the construction of 264 signature-tagged gene-deletion strains for 129 putative kinases, and examine their phenotypic traits under 30 distinct in vitro growth conditions and in two different hosts (insect larvae and mice). Clustering analysis of in vitro phenotypic traits indicates that several of these kinases have roles in known signalling pathways, and identifies hitherto uncharacterized signalling cascades. Virulence assays in the insect and mouse models provide evidence of pathogenicity-related roles for 63 kinases involved in the following biological categories: growth and cell cycle, nutrient metabolism, stress response and adaptation, cell signalling, cell polarity and morphology, vacuole trafficking, transfer RNA (tRNA) modification and other functions. Our study provides insights into the pathobiological signalling circuitry of C. neoformans and identifies potential anticryptococcal or antifungal drug targets.

Systematic functional profiling of transcription factor networks in Cryptococcus neoformans.

Cryptococcus neoformans causes life-threatening meningoencephalitis in humans, but its overall biological and pathogenic regulatory circuits remain elusive, particularly due to the presence of an evolutionarily divergent set of transcription factors (TFs). Here, we report the construction of a high-quality library of 322 signature-tagged gene-deletion strains for 155 putative TF genes previously predicted using the DNA-binding domain TF database, and examine their in vitro and in vivo phenotypic traits under 32 distinct growth conditions. At least one phenotypic trait is exhibited by 145 out of 155 TF mutants (93%) and ∼85% of them (132/155) are functionally characterized for the first time in this study. The genotypic and phenotypic data for each TF are available in the C. neoformans TF phenome database (http://tf.cryptococcus.org). In conclusion, our phenome-based functional analysis of the C. neoformans TF mutant library provides key insights into transcriptional networks of basidiomycetous fungi and human fungal pathogens.