Dysregulating PHO Signaling via the CDK Machinery Differentially Impacts Energy Metabolism, Calcineurin Signaling, and Virulence in Cryptococcus neoformans.

Fungal pathogens uniquely regulate phosphate homeostasis via the cyclin-dependent kinase (CDK) signaling machinery of the phosphate acquisition (PHO) pathway (Pho85 kinase-Pho80 cyclin-CDK inhibitor Pho81), providing drug-targeting opportunities. Here, we investigate the impact of a PHO pathway activation-defective Cryptococcus neoformans mutant (pho81Δ) and a constitutively activated PHO pathway mutant (pho80Δ) on fungal virulence. Irrespective of phosphate availability, the PHO pathway was derepressed in pho80Δ with all phosphate acquisition pathways upregulated and much of the excess phosphate stored as polyphosphate (polyP). Elevated phosphate in pho80Δ coincided with elevated metal ions, metal stress sensitivity, and a muted calcineurin response, all of which were ameliorated by phosphate depletion. In contrast, metal ion homeostasis was largely unaffected in the pho81Δ mutant, and Pi, polyP, ATP, and energy metabolism were reduced, even under phosphate-replete conditions. A similar decline in polyP and ATP suggests that polyP supplies phosphate for energy production even when phosphate is available. Using calcineurin reporter strains in the wild-type, pho80Δ, and pho81Δ background, we also demonstrate that phosphate deprivation stimulates calcineurin activation, most likely by increasing the bioavailability of calcium. Finally, we show that blocking, as opposed to permanently activating, the PHO pathway reduced fungal virulence in mouse infection models to a greater extent and that this is most likely attributable to depleted phosphate stores and ATP, and compromised cellular bioenergetics, irrespective of phosphate availability. IMPORTANCE Invasive fungal diseases cause more than 1.5 million deaths per year, with an estimated 181,000 of these deaths attributable to Cryptococcal meningitis. Despite the high mortality, treatment options are limited. In contrast to humans, fungal cells maintain phosphate homeostasis via a CDK complex, providing drug-targeting opportunities. To investigate which CDK components are the best targets for potential antifungal therapy, we used strains with a constitutively active (pho80Δ) and an activation-defective (pho81Δ) PHO pathway, to investigate the impact of dysregulated phosphate homeostasis on cellular function and virulence. Our studies suggest that inhibiting the function of Pho81, which has no human homologue, would have the most detrimental impact on fungal growth in the host due to depletion of phosphate stores and ATP, irrespective of phosphate availability in the host.

Toxicity of Orthodontic Brackets Examined by Single Cell Tracking.

Subtle toxic effects may be masked in traditional assays that average or summate the response of thousands of cells. We overcome this by using the recent method of single cell tracking in time-lapse recordings. This follows the fate and behavior of individual cells and their progeny and provides unambiguous results for multiple simultaneous biological responses. Further, single cell tracking permits correlation between progeny relationships and cell behavior that is not otherwise possible, including disruption by toxins and toxicants of similarity between paired sister cells. Notably, single cell tracking seems not to have been previously used to study biomaterials toxicity. The culture medium was pre-conditioned by 79 days incubation with orthodontic brackets from seven separate commercial sources. Metal levels were determined by Inductively Coupled Plasma Mass Spectrometry. Metal levels varied amongst conditioned media, with elevated Cr, Mn, Ni, and Cu and often Mo, Pb, Zn, Pd, and Ag were occasionally found. The effect on human dermal fibroblasts was determined by single cell tracking. All bracket-conditioned media reduced cell division (p < 0.05), while some reduced cell migration (p < 0.05). Most bracket-conditioned media increased the rate of asynchronous sister cell division (p < 0.05), a seemingly novel measure for toxicity. No clear effect on cell morphology was seen. We conclude that orthodontic brackets have cytotoxic effects, and that single cell tracking is effective for the study of subtle biomaterials cytotoxicity.

Large scale expression and purification of secreted mouse hephaestin.

Hephaestin is a large membrane-anchored multicopper ferroxidase involved in mammalian iron metabolism. Newly absorbed dietary iron is exported across the enterocyte basolateral membrane by the ferrous iron transporter ferroportin, but hephaestin increases the efficiency of this process by oxidizing the transported iron to its ferric form and promoting its release from ferroportin. Deletion or mutation of the hephaestin gene leads to systemic anemia with iron accumulation in the intestinal epithelium. The crystal structure of human ceruloplasmin, another multicopper ferroxidase with 50% sequence identity to hephaestin, has provided a framework for comparative analysis and modelling. However, detailed structural information for hephaestin is still absent, leaving questions relating to metal coordination and binding sites unanswered. To obtain structural information for hephaestin, a reliable protocol for large-scale purification is required. Here, we present an expression and purification protocol of soluble mouse hephaestin, yielding milligram amounts of enzymatically active, purified protein using the baculovirus/insect cell system.

Retention of different-sized particles and derived gut fill estimate in tammar wallabies (Macropus eugenii): physiological and methodological considerations.

The capacity of the digestive tract is an important parameter in understanding digestive adaptations, particularly in herbivores. Measures of this capacity ('gut fill') are commonly performed in killed animals, which has ethical and logistical implications. Alternatively, dry matter gut contents (DMC) can be estimated in live animals from food intake, digesta retention and digestibility, based on physical principles (Holleman and White, Can. J. Zool. 67, 488-494, 1989). Although this method has been used to some extent, it still awaits thorough validation. Here we estimated DMC in seven tammar wallabies during 5-day feeding trials and compared the results to those gained from dissections immediately after the trials. Calculated DMC exceeded that actually measured by 29 ± 22%. A closer inspection of the data suggested that this was partly due to the fact that DMC as measured by dissection is susceptible to short-term influences such as daily variation in food intake, whereas the calculated DMC represents an integrative measure over the whole period of the feeding trial. Correlations between both the measured digesta retention times, and the calculated DMC, with the measured wet contents mass suggest that it is particularly the DMC determined via dissection that needs to be measured with care. For a comparison of gut capacities, the calculated DMC therefore can be considered adequate, but should for a more widespread use be validated in further studies including more species and experimental regimes controlling food intake variation. Additionally, we tested whether very small (100-500 µm) and small (500-1000 µm) particles were retained differently in the tammar wallabies. There was no indication of such a difference. Whether the macropod forestomach selectively passes a certain particle fraction (that represents microbes) with the generally faster-passing fluids remains to be investigated with even smaller markers, e.g. labelled bacteria.