Translational Regulation Promotes Oxidative Stress Resistance in the Human Fungal Pathogen Cryptococcus neoformans
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Abstract Cryptococcus neoformans is one of the few environmental fungi that can survive within a mammalian host and cause disease. Although many of the factors responsible for establishing virulence have been recognized, how they are expressed in response to certain host derived cellular stresses is rarely addressed. Here we characterize the temporal translational response of C. neoformans to oxidative stress. We find that translation is largely inhibited through the phosphorylation of the critical initiation factor elF2α by a sole kinase. Preventing elF2α mediated translational suppression resulted in growth sensitivity to hydrogen peroxide (H 2 O 2 ). Our work suggests that translational repression in response to H 2 O 2 partly facilitates oxidative stress adaptation by accelerating the decay of abundant non-stress related transcripts while facilitating the proper expression of critical oxidative stress response factors. Carbon starvation, which seems to induce translational suppression that is independent elF2α, partly restored transcript decay and the expression of the critical oxidative stress response transcript Thioredoxin Reductase 1 (T RR1 ). Our results illustrate translational suppression as a key determinant of select mRNA decay, gene expression, and subsequent survival in response to oxidative stress. Importance Fungal survival in a mammalian host requires the coordinated expression and downregulation of a large cohort of genes in response to cellular stresses. Initial infection with C. neoformans occurs at the lungs, where it interacts with host macrophages. Surviving macrophage derived cellular stresses, such as the production of reactive oxygen and nitrogen species, is believed to promote dissemination into the central nervous system. Therefore, investigating how an oxidative stress resistant phenotype is brought about in C. neoformans furthers our understanding of not only fungal pathogenesis but also unveils mechanisms of stress induced gene reprogramming. We discovered that H 2 O 2 derived oxidative stress resulted in severe translational suppression and that this suppression was necessary for the accelerated decay and expression of tested transcripts. Surprisingly, compounding oxidative stress with carbon starvation resulted in a decrease in peroxide mediated killing, revealing unexpected synergy between stress responses.Keywords:
Translational regulation
Cryptococcus neoformans is an opportunistic fungal pathogen and the causative agent of the disease cryptococcosis. Cryptococcosis is initiated as a pulmonary infection and in conditions of immune deficiency disseminates to the blood stream and central nervous system, resulting in life-threatening meningoencephalitis. A number of studies have focused on the development of a vaccine against Cryptococcus, primarily utilizing protein-conjugated components of the Cryptococcus polysaccharide capsule as antigen. However, there is currently no vaccine against Cryptococcus in the clinic. Previous studies have shown that the glycosphingolipid, glucosylceramide (GlcCer), is a virulence factor in C. neoformans and antibodies against this lipid inhibit fungal growth and cell division. In the present study, we have investigated the possibility of using GlcCer as a therapeutic agent against C. neoformans infections in mouse models of cryptococcosis. GlcCer purified from a non-pathogenic fungus, Candida utilis, was administered intraperitoneally, prior to infecting mice with a lethal dose of C. neoformans. GlcCer administration prevented the dissemination of C. neoformans from the lungs to the brain and led to 60% mouse survival. GlcCer administration did not cause hepatic injury and elicited an anti-GlcCer antibody response, which was observed independent of the route of administration and the strains of mouse. Taken together, our results suggest that fungal GlcCer can protect mice against lethal doses of C. neoformans infection and can provide a viable vaccination strategy against Cryptococcus.
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Cryptococcus gattii
Medical microbiology
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Abstract The thioredoxin system is highly conserved system found in all living cells and comprises NADPH, thioredoxin, and thioredoxin reductase. This system plays a critical role in preserving a reduced intracellular environment, and its involvement in regulating a wide range of cellular functions makes it especially vital to cellular homeostasis. Its critical role is not limited to healthy cells, it is also involved in cancer development, and is overexpressed in many cancers. This makes the thioredoxin system a promising target for cancer drug development. As such, over the last decade, many inhibitors have been developed that target the thioredoxin system, most of which are small molecules targeting the thioredoxin reductase C‐terminal redox center. A few inhibitors of thioredoxin have also been developed. We believe that more efforts should be invested in developing protein/peptide‐based inhibitors against both thioredoxin reductase and/or thioredoxin.
Drug Development
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Abstract Vaccines are one of the greatest medical accomplishments to date, yet no fungal vaccines are currently available in humans mainly because opportunistic mycoses generally occur during immunodeficiencies necessary for vaccine protection. In previous studies, a live, attenuated Cryptococcus neoformans Δ sgl1 mutant accumulating sterylglucosides was found to be avirulent and protected mice from a subsequent lethal infection even in absence of CD4 + T cells, a condition most associated with cryptococcosis (e.g., HIV). Here, we tested three strategies of vaccination against cryptococcosis. First, in our preventative model, protection was achieved even after a 3-fold increase of the vaccination window. Second, because live C. neoformans Δ sgl1 -vaccinated mice challenged more than once with WT strain had a significant decrease in lung fungal burden, we tested C. neoformans Δ sgl1 as an immunotherapeutic. We found that therapeutic administrations of HK C. neoformans Δ sgl1 subsequent to WT challenge significantly improve the lung fungal burden. Similarly, therapeutic administration of HK C. neoformans Δ sgl1 post WT challenge resulted in 100% or 70% survival depending on the time of vaccine administration, suggesting that HK Δ sgl1 is a robust immunotherapeutic option. Third, we investigated a novel model of vaccination in preventing reactivation from lung granuloma using C. neoformans Δ gcs1 . Remarkably, we show that administration of HK Δ sgl1 prevents mice from reactivating Δ gcs1 upon inducing immunosuppression with corticosteroids or by depleting CD4 + T cells. Our results suggest that HK Δ sgl1 represents a clinically relevant, efficacious vaccine that confers robust host protection in three models of vaccination against cryptococcosis even during CD4-deficiency. Importance Cryptococcosis results in ∼180,000 global deaths per year in immunocompromised individuals. Current antifungal treatment options are potentially toxic, lacking in areas of need, and exhibit limited efficacy. In addition to these lackluster therapeutic options, no fungal vaccines are currently available for clinical use. Due to the increasing rate of immunocompromised individuals, there is a dire need for the development of improved antifungal therapeutics. Presently, we have demonstrated the high efficacy of a clinically relevant heat-killed mutant strain of Cryptococcus neoformans in inducing advantageous host protection in three models of vaccination against cryptococcosis during immunodeficiencies most associated with this disease.
Immunosuppression
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Background: Cryptococcosis is an infectious disease with worldwide distribution caused by Cryptococcus species. Cryptococcus neoformans and Cryptococcus gattii are the common species in human cryptococcal infection. Cryptococcus neoformans is a major cause of illness in people diagnosed with Human Immunodeficiency Virus (HIV), with an estimated 220,000 cases of cryptococcal meningitis occurring worldwide each year. This is a retrospective study on the frequency of cryptococcosis in different age groups and gender; as well as to identify the types of Cryptococcus species isolated in this population in University Malaya Medical Center from year 2013 to September 2019. Methods and materials: Data on cryptococcosis diagnosed in University Malaya Medical Center from year 2013 to September 2019 was extracted from the laboratory information system at the center. 59 newly diagnosed cryptococcosis cases were included in this study. Cryptococcus species were isolated from blood, cerebrospinal fluid, pus, body fluid, tissue and skin. Methods of identification of Cryptococcus species were via API 20C AUX, Vitek 2 and MALDI-TOF. Results: In this population of study, the frequency of cryptococcosis is 83.06% and 16.94% in males and females respectively. The frequency of Cryptococcus neoformans is 81.36%, with 83.33% comprising males (age 10–87) and 16.67% comprising females (age 33–81). The frequency of Cryptococcus neoformans var. grubii is 3.39% (2 males aged 33 and 53), Cryptococcus albidus is 3.39% (2 females aged 65 and 70), Cryptococcus uniguttulatus is 3.39% (two males aged 35 and 87) and Cryptococcus sp. is 3.39% (2 males aged 25 and 36). Cryptococcus gattii (34 year old male), Cryptococcus laurentii (71 year old male) and Cryptococcus humicola (59 years old male) are 1.70% in frequency each. Conclusion: In conclusion, the frequency of cryptococosis in this period of study is high in the male population with Cryptococcus neoformans as the commonest isolated species. Early diagnosis followed by treatment of cryptococcosis offers good to excellent prognosis for the patients. However, the prognosis varies in the immunocompromised. Targeted screening programmes should be carried out to detect early cryptococcal disease, especially in HIV-infected persons to reduce morbidity and mortality, and improve the overall quality of life.
Cryptococcus gattii
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Thioredoxin is a redox protein found overexpressed in some human tumors. Thioredoxin is secreted by tumor cells and enhances the sensitivity of the cancer cells to other growth factors. Redox activity is essential for stimulation of cell growth by thioredoxin. Cells transfected with thioredoxin cDNA show increased tumor growth and decreased apoptosis in vivo and decreased sensitivity to apoptosis induced by a variety of agents both in vitro and in vivo. Cells transfected with a redox-inactive mutant thioredoxin show inhibited tumor growth in vivo. Dietary selenium has been shown to prevent some forms of human cancer. Selenocysteine is an essential component of thioredoxin reductase, the flavoenzyme that is responsible for the reduction of thioredoxin. Selenium added to the culture medium increases thioredoxin reductase activity due to an increase in thioredoxin reductase protein but mostly due to an increase in the specific activity of the enzyme. Some diaryl chalcogenide (selenium and tellurium) compounds have been studied as inhibitors of thioredoxin reductase. The most active were diaryl tellurium compounds, which were noncompetitive inhibitors of thioredoxin reductase with Ki values of 2-10 microM. Several of the compounds inhibited cancer cell colony formation in vitro with IC50s as low as 2 microM.
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The thioredoxin system consisting of NADP(H), thioredoxin reductase and thioredoxin provides reducing equivalents to a large and diverse array of cellular processes. Despite a great deal of information on the kinetics of individual thioredoxin-dependent reactions, the kinetic regulation of this system as an integrated whole is not known. We address this by using kinetic modeling to identify and describe kinetic behavioral motifs found within the system.Analysis of a realistic computational model of the Escherichia coli thioredoxin system revealed several modes of kinetic regulation in the system. In keeping with published findings, the model showed that thioredoxin-dependent reactions were adaptable (i.e. changes to the thioredoxin system affected the kinetic profiles of these reactions). Further and in contrast to other systems-level descriptions, analysis of the model showed that apparently unrelated thioredoxin oxidation reactions can affect each other via their combined effects on the thioredoxin redox cycle. However, the scale of these effects depended on the kinetics of the individual thioredoxin oxidation reactions with some reactions more sensitive to changes in the thioredoxin cycle and others, such as the Tpx-dependent reduction of hydrogen peroxide, less sensitive to these changes. The coupling of the thioredoxin and Tpx redox cycles also allowed for ultrasensitive changes in the thioredoxin concentration in response to changes in the thioredoxin reductase concentration. We were able to describe the kinetic mechanisms underlying these behaviors precisely with analytical solutions and core models.Using kinetic modeling we have revealed the logic that underlies the functional organization and kinetic behavior of the thioredoxin system. The thioredoxin redox cycle and associated reactions allows for a system that is adaptable, interconnected and able to display differential sensitivities to changes in this redox cycle. This work provides a theoretical, systems-biological basis for an experimental analysis of the thioredoxin system and its associated reactions.
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