Ruchi Bhabhra

Doxycycline-regulated gene expression in the opportunistic fungal pathogen Aspergillus fumigatus

BackgroundAlthough Aspergillus fumigatus is an important human fungal pathogen there are few expression systems available to study the contribution of specific genes to the growth and virulence of this opportunistic mould. Regulatable promoter systems based upon prokaryotic regulatory elements in the E. coli tetracycline-resistance operon have been successfully used to manipulate gene expression in several organisms, including mice, flies, plants, and yeast. However, the system has not yet been adapted for Aspergillus spp.ResultsHere we describe the construction of plasmid vectors that can be used to regulate gene expression in A. fumigatus using a simple co-transfection approach. Vectors were generated in which the tetracycline transactivator (tTA) or the reverse tetracycline transactivator (rtTA2s-M2) are controlled by the A. nidulans gpdA promoter. Dominant selectable cassettes were introduced into each plasmid, allowing for selection following gene transfer into A. fumigatus by incorporating phleomycin or hygromycin into the medium. To model an essential gene under tetracycline regulation, the E. coli hygromycin resistance gene, hph, was placed under the control of seven copies of the TetR binding site (tetO7) in a plasmid vector and co-transfected into A. fumigatus protoplasts together with one of the two transactivator plasmids. Since the hph gene is essential to A. fumigatus in the presence of hygromycin, resistance to hygromycin was used as a marker of hph reporter gene expression. Transformants were identified in which the expression of tTA conferred hygromycin resistance by activating expression of the tetO7-hph reporter gene, and the addition of doxycycline to the medium suppressed hygromycin resistance in a dose-dependent manner. Similarly, transformants were identified in which expression of rtTA2s-M2 conferred hygromycin resistance only in the presence of doxycycline. The levels of doxycycline required to regulate expression of the tetO7-hph reporter gene were within non-toxic ranges for this organism, and low-iron medium was shown to reduce the amount of doxycycline required to accomplish regulation.ConclusionsThe vectors described in this report provide a new set of options to experimentally manipulate the level of specific gene products in A. fumigatus

Disruption of the Aspergillus fumigatus Gene Encoding Nucleolar Protein CgrA Impairs Thermotolerant Growth and Reduces Virulence

ABSTRACT Aspergillus fumigatus CgrA is the ortholog of a yeast nucleolar protein that functions in ribosome synthesis. To determine how CgrA contributes to the virulence of A. fumigatus, a ΔcgrA mutant was constructed by targeted gene disruption, and the mutant was reconstituted to wild type by homologous introduction of a functional cgrA gene. The ΔcgrA mutant had the same growth rate as the wild type at room temperature. However, when the cultures were incubated at 37°C, a condition that increased the growth rate of the wild-type and reconstituted strains approximately threefold, the ΔcgrA mutant was unable to increase its growth rate. The absence of cgrA function caused a delay in both the onset and rate of germination at 37°C but had little effect on germination at room temperature. The ΔcgrA mutant was significantly less virulent than the wild-type or reconstituted strain in immunosuppressed mice and was associated with smaller fungal colonies in lung tissue. However, this difference was less pronounced in a Drosophila infection model at 25°C, which correlated with the comparable growth rates of the two strains at this temperature. To determine the intracellular localization of CgrA, the protein was tagged at the C terminus with green fluorescent protein, and costaining with propidium iodide revealed a predominantly nucleolar localization of the fusion protein in living hyphae. Together, these findings establish the intracellular localization of CgrA in A. fumigatus and demonstrate that cgrA is required for thermotolerant growth and wild-type virulence of the organism.

Thermotolerance and virulence of Aspergillus fumigatus: role of the fungal nucleolus

The ability to thrive at 37 degrees C is characteristic of all human pathogens and has long been suspected to play a role in the pathogenesis of aspergillosis. As a thermotolerant fungus, Aspergillus fumigatus is capable of growth at temperatures that approach the upper limit for all eukaryotes, suggesting that the organism has evolved unique mechanisms of stress resistance that may be relevant to its ability to adapt to the stress of growth in the host. High temperature is a strain on many biological systems, particularly those involved in complex macromolecular assemblies such as ribosomes. This review will discuss the relationship between thermotolerance and virulence in pathogenic fungi, emphasizing the link to ribosome biogenesis in A. fumigatus. Future work in this area will help determine how rapid growth is accomplished at elevated temperature and may offer new avenues for the development of novel antifungals that disrupt thermotolerant ribosome assembly.

A Fungus-Specific Ras Homolog Contributes to the Hyphal Growth and Virulence of Aspergillus fumigatus

ABSTRACT The Ras family of GTPase proteins has been shown to control morphogenesis in many organisms, including several species of pathogenic fungi. In a previous study, we identified a gene encoding a fungus-specific Ras subfamily homolog, rasB, in Aspergillus fumigatus. Here we report that deletion of A. fumigatus rasB caused decreased germination and growth rates on solid media but had no effect on total biomass accumulation after 24 h of growth in liquid culture. The ΔrasB mutant had an irregular hyphal morphology characterized by increased branching. Expression of rasBΔ113-135, a mutant transgene lacking the conserved rasB internal amino acid insertion, did not complement the deletion phenotype of delayed growth and germination rates and abnormal hyphal morphology. Virulence of the rasB deletion strain was diminished; mice infected with this strain exhibited ∼65% survival compared to ∼10% with wild-type and reconstituted strains. These data support the hypothesis that rasB homologs, which are highly conserved among fungi that undergo hyphal growth, control signaling modules important to the directional growth of fungal hyphae.

The Aspergillus fumigatus metacaspases CasA and CasB facilitate growth under conditions of endoplasmic reticulum stress

We have examined the contribution of metacaspases to the growth and stress response of the opportunistic human mould pathogen, Aspergillus fumigatus, based on increasing evidence implicating the yeast metacaspase Yca1p in apoptotic‐like programmed cell death. Single metacaspase‐deficient mutants were constructed by targeted disruption of each of the two metacaspase genes in A. fumigatus, casA and casB, and a metacaspase‐deficient mutant, ΔcasA/ΔcasB, was constructed by disrupting both genes. Stationary phase cultures of wild‐type A.  fumigatus were associated with the appearance of typical markers of apoptosis, including elevated proteolytic activity against caspase substrates, phosphatidylserine exposure on the outer leaflet of the membrane, and loss of viability. By contrast, phosphatidylserine exposure was not observed in stationary phase cultures of the ΔcasA/ΔcasB mutant, although caspase activity and viability was indistinguishable from wild type. The mutant retained wild‐type virulence and showed no difference in sensitivity to a range of pro‐apoptotic stimuli that have been reported to initiate yeast apoptosis. However, the ΔcasA/ΔcasB mutant showed a growth detriment in the presence of agents that disrupt endoplasmic reticulum homeostasis. These findings demonstrate that metacaspase activity in A. fumigatus contributes to the apoptotic‐like loss of membrane phospholipid asymmetry at stationary phase, and suggest that CasA and CasB have functions that support growth under conditions of endoplasmic reticulum stress.

Doege-Potter syndrome presenting with hypoinsulinemic hypoglycemia in a patient with a malignant extrapleural solitary fibrous tumor: a case report

IntroductionDoege-Potter syndrome is a paraneoplastic syndrome characterized by non-islet cell tumor hypoglycemia secondary to a solitary fibrous tumor. This tumor causes hypoglycemia by the secretion of a prohormone form of insulin-like growth factor II. We describe the diagnosis and management of Doege-Potter syndrome and the use of transarterial chemoembolization in a patient with a malignant extrapleural solitary fibrous tumor.Case presentationOur patient was a 64-year-old Caucasian woman who initially presented with urinary incontinence and was found to have a 14.5×9.0×9.0cm retroperitoneal solitary fibrous tumor compressing her bladder. Her tumor was surgically resected but recurred with multiple hepatic metastatic lesions. The hepatic metastases progressed despite systemic chemotherapy and treatment with doxorubicin transarterial chemoembolization. Her course was complicated by the development of recurrent fasting hypoglycemia, most likely secondary to Doege-Potter syndrome. Her hypoglycemia was managed with corticosteroid therapy and frequent scheduled nutrient intake overnight.ConclusionsThe rarity of hepatic solitary fibrous tumors and consequent lack of controlled trials make this report significant in that it describes the diagnostic approach to Doege-Potter syndrome, describes our experience with the use of doxorubicin transarterial chemoembolization, and presents management options for tumor-associated hypoglycemia in the case of extensive disease not amenable to surgical resection.

Impaired Ribosome Biogenesis Disrupts the Integration between Morphogenesis and Nuclear Duplication during the Germination of Aspergillus fumigatus

ABSTRACT Aspergillus fumigatus is an important opportunistic fungal pathogen that is responsible for high mortality rates in the immunosuppressed population. CgrA, the A. fumigatus ortholog of a Saccharomyces cerevisiae nucleolar protein involved in ribosome biogenesis, contributes to the virulence of this fungus by supporting rapid growth at 37°C. To determine how CgrA affects ribosome biogenesis in A. fumigatus, polysome profile and ribosomal subunit analyses were performed on both wild-type A. fumigatus and a ΔcgrA mutant. The loss of CgrA was associated with a reduction in the level of 80S monosomes as well as an imbalance in the 60S:40S subunit ratio and the appearance of half-mer ribosomes. The gene expression profile in the ΔcgrA mutant revealed increased abundance of a subset of translational machinery mRNAs relative to the wild type, suggesting a potential compensatory response to CgrA deficiency. Although ΔcgrA conidia germinated normally at 22°C, they swelled excessively when incubated at 37°C and accumulated abnormally high numbers of nuclei. This hypernucleated phenotype could be replicated pharmacologically by germinating wild-type conidia under conditions of reductive stress. These findings indicate that the germination process is particularly vulnerable to global disruption of protein synthesis and suggest that CgrA is involved in both ribosome biogenesis and polarized cell growth in A. fumigatus.

Progesterone-Mediated Inhibition of the GnRH Pulse Generator: Differential Sensitivity as a Function of Sleep Status

Context During normal, early puberty, luteinizing hormone (LH) pulse frequency is low while awake but increases during sleep. Mechanisms underlying such changes are unclear, but a small study in early pubertal girls suggested that differential wake-sleep sensitivity to progesterone negative feedback plays a role. Objective To test the hypothesis that progesterone acutely reduces waking LH pulse frequency more than sleep-associated pulse frequency in late pubertal girls. Design Randomized, placebo-controlled, double-blinded crossover study. Setting Academic clinical research unit. Participants Eleven normal, postmenarcheal girls, ages 12 to 15 years. Intervention Subjects completed two 18-hour admissions in separate menstrual cycles (cycle days 6 to 11). Frequent blood sampling for LH assessment was performed at 1800 to 1200 hours; sleep was encouraged at 2300 to 0700 hours. Either oral micronized progesterone (0.8 mg/kg/dose) or placebo was given at 0700, 1500, 2300, and 0700 hours, before and during the first admission. A second admission, performed at least 2 months later, was identical to the first except that placebo was exchanged for progesterone or vice versa (treatment crossover). Main Outcome Measures LH pulse frequency during waking and sleeping hours. Results Progesterone reduced waking LH pulse frequency by 26% (P = 0.019), with no change observed during sleep (P = 0.314). The interaction between treatment condition (progesterone vs placebo) and sleep status (wake vs sleep) was highly significant (P = 0.007). Conclusions In late pubertal girls, progesterone acutely reduced waking LH pulse frequency more than sleep-associated pulse frequency. Differential wake-sleep sensitivity to progesterone negative feedback may direct sleep-wake LH pulse frequency changes across puberty.