The Cyclin Cln1 Controls Polyploid Titan Cell Formation following a Stress-Induced G2 Arrest in Cryptococcus.

The pathogenic yeast Cryptococcus neoformans produces polyploid titan cells in response to the host lung environment that are critical for host adaptation and subsequent disease. We analyzed the in vivo and in vitro cell cycles to identify key aspects of the C. neoformans cell cycle that are important for the formation of titan cells. We identified unbudded 2C cells, referred to as a G2 arrest, produced both in vivo and in vitro in response to various stresses. Deletion of the nonessential cyclin Cln1 resulted in overproduction of titan cells in vivo and transient morphology defects upon release from stationary phase in vitro. Using a copper-repressible promoter PCTR4-CLN1 strain and a two-step in vitro titan cell formation assay, our in vitro studies revealed Cln1 functions after the G2 arrest. These studies highlight unique cell cycle alterations in C. neoformans that ultimately promote genomic diversity and virulence in this important fungal pathogen. IMPORTANCE Dysregulation of the cell cycle underlies many human genetic diseases and cancers, yet numerous organisms, including microbes, also manipulate the cell cycle to generate both morphologic and genetic diversity as a natural mechanism to enhance their chances for survival. The eukaryotic pathogen Cryptococcus neoformans generates morphologically distinct polyploid titan cells critical for host adaptation and subsequent disease. We analyzed the C. neoformans in vivo and in vitro cell cycles to identify changes required to generate the polyploid titan cells. C. neoformans paused cell cycle progression in response to various environmental stresses after DNA replication and before morphological changes associated with cell division, referred to as a G2 arrest. Release from this G2 arrest was coordinated by the cyclin Cln1. Reduced CLN1 expression after the G2 arrest was associated with polyploid titan cell production. These results demonstrate a mechanism to generate genomic diversity in eukaryotic cells through manipulation of the cell cycle that has broad disease implications.

ATI-2307 Exhibits Equivalent Antifungal Activity in Cryptococcus neoformans Clinical Isolates With High and Low Fluconazole IC50.

Half maximal inhibitory concentrations (IC50) to the experimental drug ATI-2307 and complete inhibition (IC90) of the common clinically used antifungal drug amphotericin B were determined by microbroth dilution assay for a collection of 69 clinical isolates of Cryptococcus neoformans from Uganda that had high fluconazole IC50 values. The majority of the clinical isolates tested had fluconazole IC50 at or above 8 µg/mL, but were susceptible to both amphotericin B (IC90 ≤1 µg/mL) and ATI-2307 (IC50 ≤0.0312 µg/mL). No correlation between increased fluconazole minimum inhibitory concentration (MIC) and ATI-2307 or amphotericin B MIC was observed, suggesting that the cellular changes impacting fluconazole susceptibility did not impact the effectiveness of ATI-2307. Our results suggest that ATI-2307 is a promising new antifungal drug for use in the context of high fluconazole or other antifungal drug MICs and/or in combination drug therapy regimens.

The Adrenal Clock Prevents Aberrant Light-Induced Alterations in Circadian Glucocorticoid Rhythms.

The glucocorticoid (GC) rhythm is entrained to light-dark (LD) cycles via a molecular clock in the suprachiasmatic nucleus (SCN) and is maintained by an adrenal clock synchronized by SCN-dependent signals. Targeted deletion of the core clock gene Bmal1 can disrupt adrenal clock function. The requirement of the adrenal clock to stabilize the circadian GC rhythm during exposure to aberrant LD cycles was determined using novel aldosterone synthase (AS)Cre/+::Bmal1Fl/Fl mice in which Bmal1 deletion occurred during postnatal adrenal transdifferentiation. To examine whether adrenal Bmal1 deletion results in loss of the adrenal clock, mice were crossed with mPER2::Luciferase (mPER2Luc/+) mice. Adrenals from ASCre/+::Bmal1+/+::PER2Luc/+ [control (CTRL)] mice show mPER2Luc rhythms ex vivo, whereas slices from ASCre/+::Bmal1Fl/Fl::PER2Luc/+ [knockout (KO)] mice show dampened rhythms. To monitor corticosterone rhythmicity, mice were implanted with subcutaneous microdialysis probes and sampled at 60-minute intervals for up to 3 days under 12:12-hour [τ (T) 24] LD or 3.5:3.5-hour (T7) LD cycles. Corticosterone rhythms were entrained to T24 LD in CTRL and KO mice. Under T7 LD, circadian corticosterone rhythms persisted in most CTRL mice but not KO mice. Hyperadrenocorticism also was observed in KO mice under T7 LD, reflected by increased corticosterone peak amplitude, total daily corticosterone, and responses to ACTH. Analysis of dysregulated adrenal genes in KO mice exposed to aberrant light identified candidates involved in cholesterol metabolism and trafficking, including steroidogenic acute regulatory protein, which could increase steroidogenesis. Our results show that the adrenal clock functions to buffer steroidogenic responses to aberrant light and stabilize circadian GC rhythmicity.

Phase-Dependent Shifting of the Adrenal Clock by Acute Stress-Induced ACTH.

The adrenal cortex has a molecular clock that generates circadian rhythms in glucocorticoid production, yet it is unclear how the clock responds to acute stress. We hypothesized that stress-induced ACTH provides a signal that phase shifts the adrenal clock. To assess whether acute stress phase shifts the adrenal clock in vivo in a phase-dependent manner, mPER2:LUC mice on a 12:12-h light:dark cycle underwent restraint stress for 15 min or no stress at zeitgeber time (ZT) 2 (early subjective day) or at ZT16 (early subjective night). Adrenal explants from mice stressed at ZT2 showed mPER2:LUC rhythms that were phase-advanced by ~2 h, whereas adrenals from mice stressed at ZT16 showed rhythms that were phase-delayed by ~2 h. The biphasic response was also observed in mice injected subcutaneously either with saline or with ACTH at ZT2 or ZT16. Blockade of the ACTH response with the glucocorticoid, dexamethasone, prevented restraint stress-induced phase shifts in the mPER2:LUC rhythm both at ZT2 and at ZT16. The finding that acute stress results in a phase-dependent shift in the adrenal mPER2:LUC rhythm that can be blocked by dexamethasone indicates that stress-induced effectors, including ACTH, act to phase shift the adrenal clock rhythm.

Chronic subordination stress phase advances adrenal and anterior pituitary clock gene rhythms.

Circadian rhythms in glucocorticoids are the product of interactions between the hypothalamic-pituitary-adrenal (HPA) axis and the mammalian clock gene system. The adrenal clock can generate the glucocorticoid rhythm that in turn synchronizes other peripheral clocks to maintain homeostasis. Stress acutely activates and chronically upregulates the HPA axis, suggesting that the adrenal clock could be modulated by stress. However, there is no direct evidence that stress affects the adrenal clock rhythm. We tested the hypothesis that a model of chronic subordination stress (CSS) that has a major impact on HPA axis regulation, metabolism, and emotional behavior alters adrenal and pituitary clock gene rhythms. Clock gene rhythms were assessed using mPER2::Luciferase (PER2Luc) knockin mice in which in vitro bioluminescence rhythms reflect the Per2 clock gene expression. PER2Luc mice that experienced CSS for 2 wk showed positive energy balance reflected by increased body weight and food intake. Additionally, CSS phase advanced the adrenal (∼2 h) and the pituitary (∼1 h) PER2Luc rhythm compared with control mice. The activity rhythm was not affected. The adrenal clock phase shift was associated with increased feed conversion efficiency, suggesting that the metabolic phenotype in CSS mice may be related to altered adrenal clock rhythmicity. Interestingly, a single subordination experience followed by 8 h sensory housing also phase advanced the adrenal, but not the pituitary, PER2Luc rhythm. Overall, these data demonstrate a stress-induced phase shift in a peripheral clock gene rhythm and differential stress sensitivity of two peripheral clocks within the HPA axis, suggesting a link between clock desynchrony and individual vulnerability to stress.

Phase-dependent resetting of the adrenal clock by ACTH in vitro.

The adrenal cortex has a molecular clock that generates circadian rhythms in glucocorticoids, yet how the clock is synchronized to the external environment is unknown. Using mPER2::Luciferase (mPER2Luc) knockin mice, in which luciferase is rhythmically expressed under the control of the mouse Per2 clock gene, we hypothesized that ACTH transmits entrainment signals to the adrenal. Adrenal explants were administered ACTH at different phases of the mPER2Luc rhythm. Treatment with ACTH 1-39 produced a phase delay that was phase-dependent, with a maximum at circadian time (CT)18; ACTH did not alter the period or amplitude of the rhythm. Forskolin produced a parallel response, suggesting that the phase delay was cAMP-mediated. The response to ACTH was concentration-dependent and peptide-specific. Pulse administration (60 min) of ACTH 1-39 also produced phase delays restricted to late CTs. In contrast to ACTH 1-39, other ACTH fragments, including α-melanocyte-stimulating hormone, which do not activate the melanocortin 2 (MC2/ACTH) receptor, had no effect. The finding that ACTH in vitro phase delays the adrenal mPER2luc rhythm in a monophasic fashion argues for ACTH as a key resetter, but not the sole entrainer, of the adrenal clock.

Subdiaphragmatic vagotomy prevents drinking-induced reduction in plasma corticosterone in water-restricted rats.

Dehydrated rats exhibit a rapid inhibition of the hypothalamic-pituitary-adrenal axis after rehydration. Drinking activates vagal afferents that project to neurons in the nucleus tractus solitarius (NTS). We hypothesized that when dehydrated rats drink, vagal afferents stimulate NTS neurons initiating inhibition of hypothalamic-pituitary-adrenal activity. Experiments assessed NTS activity by measuring Fos expression. Rats were water restricted for 1 or 6 d, limiting access to water to 30 min/d in the morning. Drinking after single or repeated restriction increased Fos, demonstrating increased NTS activity. We next examined the contribution of the vagus by comparing hormonal responses after total subdiaphragmatic vagotomy or sham surgery. Water restriction for 6 d increased plasma arginine vasopressin (AVP), ACTH, and adrenal and plasma corticosterone in both groups. In sham rats, drinking reduced plasma AVP, ACTH, adrenal and plasma corticosterone by 7.5 min. In total subdiaphragmatic vagotomy rats, whereas drinking reduced plasma AVP, ACTH, and adrenal corticosterone, drinking did not reduce plasma corticosterone. To identify the source of vagal activity, hormonal responses to restriction-induced drinking were measured after common hepatic branch vagotomy (HBV). Although pituitary hormonal responses were not affected by HBV, the adrenal and plasma corticosterone responses to water restriction were reduced; in addition, drinking in HBV rats decreased adrenal corticosterone without changing plasma corticosterone. These data indicate that an intact vagus is necessary to reduce plasma corticosterone when water-restricted rats drink and that the common hepatic vagal branch contributes to the response. These findings implicate the vagus in augmenting rapid removal of circulating corticosterone during relief from stress.

Evidence for widespread epithelial damage and coincident production of monocyte chemotactic protein 1 in a murine model of intestinal ricin intoxication.

The development of small-animal models is necessary to understand host responses and immunity to emerging infectious diseases and potential bioterrorism agents. In this report we have characterized a murine model of intestinal ricin intoxication. Ricin administered intragastrically (i.g.) to BALB/c mice at doses ranging from 1 to 10 mg/kg of body weight induced dose-dependent morphological changes in the proximal small intestine (i.e., duodenum), including widespread villus atrophy and epithelial damage. Coincident with epithelial damage was a localized increase in monocyte chemotactic protein 1, a chemokine known to be associated with inflammation of the intestinal mucosa. Immunity to intestinal ricin intoxication was achieved by immunizing mice i.g. with ricin toxoid and correlated with elevated levels of antitoxin mucosal immunoglobulin A (IgA) and serum IgG antibodies. We expect that this model will serve as a valuable tool in identifying the inflammatory pathways and protective immune responses that are elicited in the intestinal mucosa following ricin exposure and will prove useful in the evaluation of antitoxin vaccines and therapeutics.