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BackgroundCandida parapsilosis is a frequent cause of candidemia worldwide. Its incidence is associated with the use of medical implants, such as central venous catheters or parenteral nutrition. This species has reduced susceptibility to echinocandins and is susceptible to polyenes and azoles. Multiple outbreaks caused by fluconazole non-susceptible strains have been reported recently. A similar trend has been observed among the C. parapsilosis isolates received in the last two years at the Spanish Mycology Reference Laboratory.MethodsYeast were identified by molecular biology and antifungal susceptibility testing was performed using EUCAST protocol. ERG11 gene was sequenced to identify resistance mechanisms, and typification was carried out by microsatellite analysis.ResultsWe examined the susceptibility profile of the C. parapsilosis isolates available at our Reference Laboratory since 2000 (around 1,300 strains). During the last two years, the number of isolates with acquired resistance to fluconazole and voriconazole has increased in at least eight different Spanish hospitals. Typification of the isolates revealed that some prevalent clones had spread through several hospitals of the same geographical region. One of these clones was found in hospitals from the region of Catalonia, another in hospitals from Madrid and Burgos, and two other different genotypes from Santander.ConclusionsOur data suggests that the epidemiological situation caused by the COVID-19 pandemic might have induced a selection of fluconazole-resistant C. parapsilosis isolates that were already present at the hospitals. Further measures must be taken to avoid the establishment of clinical outbreaks that could threaten the life of infected patients.

The aim of this review is to give an overall idea of Cryptococcus biology paying special attention to its capacity to adapt through its morphogenetic program to the hostile host environment. This morphogenetic program consists of a significant increase in capsule size and the formation of Titan cells. Research on Titan cells had been hampered by the need of obtaining these cells from the lungs of infected mice. The production of Titan cells in vitro has supposed a major step in understanding the role of this morphotype in the virulence of Cryptococcus. In this regard, Cryptococcus has acquired the capacity of inducing a heterogeneous population during infection that allows it to evade the host immune system attack, proliferate, and disseminate to the CNS where it produces meningoencephalitis which is fatal if not treated properly.

Human studies have shown associations between cryptococcal meningitis and reduced IgM memory B cell levels, and studies in IgM- and/or B cell-deficient mice have demonstrated increased Cryptococcus neoformans dissemination from lungs to brain. Since immunoglobulins are part of the immune milieu that C. neoformans confronts in a human host, and its ability to form titan cells is an important virulence mechanism, we determined the effect of human immunoglobulins on C. neoformans titan cell formation in vitro. (i) Fluorescence microscopy showed normal human IgG and IgM bind C. neoformans. (ii) C. neoformans grown in titan cell-inducing medium with IgM, not IgG, inhibited titan-like cell formation. (iii) Absorption of IgM with laminarin or curdlan (branched and linear 1-3-beta-d-glucans, respectively) decreased this effect. (iv) Transmission electron microscopy revealed that cells grown with IgM had small capsules and unique features not seen with cells grown with IgG. (v) Comparative transcriptional analysis of cell wall, capsule, and stress response genes showed that C. neoformans grown with IgM, not IgG or phosphate-buffered saline (PBS), had decreased expression of chitin synthetase, CHS1, CHS2, and CHS8, and genes encoding cell wall carbohydrate synthetases α-1-3-glucan (AGS1) and β-1,3-glucan (FKS1). IgM also decreased expression of RIM101 and HOG1, genes encoding central regulators of C. neoformans stress response pathways and cell morphogenesis. Our data show human IgM affects C. neoformans morphology in vitro and suggest that the hypothesis that human immunoglobulins may affect C. neoformans virulence in vivo warrants further investigation.

The encapsulated fungus Cryptococcus neoformans (Cn) causes cryptococcal meningitis (CM). There are ~180,000 deaths per year worldwide attributed to CM, which is the most common cause of meningitis in adults with HIV in sub-Saharan Africa. HIV infection with advanced immunodeficiency is the most important predisposing risk factor for CM, highlighting the critical role that T cell mediated immunity plays in disease prevention. Numerous studies in the past decade demonstrate that antibody immunity also plays a role in resistance to CM, although its role has taken more time to establish. In mice, B cells reduce early dissemination from lungs to brain, and naive mouse IgM can enhance fungal containment in the lungs. In concert with these findings, human studies show that patients with CM have lower IgM memory B cell levels and/or different serological profiles than controls. In this article, we review recent data on the role that B cells and/or antibody-based immunity play in host defense against Cn and natural resistance to CM.

Cryptococcus neoformans is a human pathogenic yeast that causes hundreds of thousands of deaths worldwide among susceptible individuals, in particular, HIV+ patients. This yeast has developed several adaptation mechanisms that allow replication within the host. During decades, this yeast has been well known for a very peculiar and unique structure that contributes to virulence, a complex polysaccharide capsule that surrounds the cell wall. In contrast to other fungal pathogens, such as Candida albicans or Aspergillus fumigatus, the role of morphological transitions has not been studied in the virulence of Cryptococcus neoformans since this yeast does not form hyphae during infection. However, in the last years, different groups have described the ability of this fungus to change its size during infection. In particular, Cryptococcus can form "titan cells," which are blastoconidia of an abnormal large size. Since their discovery, there is increasing evidence that these cells contribute, not only to long-term persistence in the host, but they can also actively participate in the development of the disease. Recently, several groups have simultaneously described different media that induce the appearance of titan cells in laboratory conditions. Using these conditions, new inducing factors and signaling pathways involved in this transition have been described. In this article, we will review the main phenotypic features of these cells, factors, and transduction pathways that induce cell growth, and how titan cells contribute to the disease caused by this pathogen.

Made available in DSpace on 2018-12-11T17:20:36Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-05-01 Agència de Gestió d’Ajuts Universitaris i de Recerca Secretaría de Estado de Investigación, Desarrollo e Innovación Dirección General de Investigación Científica y Técnica Cryptococcus neoformans is an encapsulated pathogenic yeast that can change the size of the cells during infection. In particular, this process can occur by enlarging the size of the capsule without modifying the size of the cell body, or by increasing the diameter of the cell body, which is normally accompanied by an increase of the capsule too. This last process leads to the formation of cells of an abnormal enlarged size denominated titan cells. Previous works characterized titan cell formation during pulmonary infection but research on this topic has been hampered due to the difficulty to obtain them in vitro. In this work, we describe in vitro conditions (low nutrient, serum supplemented medium at neutral pH) that promote the transition from regular to titan-like cells. Moreover, addition of azide and static incubation of the cultures in a CO2enriched atmosphere favored cellular enlargement. This transition occurred at low cell densities, suggesting that the process was regulated by quorum sensing molecules and it was independent of the cryptococcal serotype/species. Transition to titan-like cell was impaired by pharmacological inhibition of PKC signaling pathway. Analysis of the gene expression profile during the transition to titan-like cells showed overexpression of enzymes involved in carbohydrate metabolism, as well as proteins from the coatomer complex, and related to iron metabolism. Indeed, we observed that iron limitation also induced the formation of titan cells. Our gene expression analysis also revealed other elements involved in titan cell formation, such as calnexin, whose absence resulted in appearance of abnormal large cells even in regular rich media. In summary, our work provides a new alternative method to investigate titan cell formation devoid the bioethical problems that involve animal experimentation. Mycology Reference Laboratory National Centre for Microbiology Instituto de Salud Carlos III, Majadahonda Universidade Estadual Paulista (UNESP) Faculdade de Ciências Farmacêuticas Câmpus Araraquara Departamento de Análises Clínicas Laboratório de Micologia Clínica Genomics Unit Core Scientific Services Instituto de Salud Carlos III, Majadahonda Institut Pasteur Unité Biologie des ARN des Pathogènes Fongiques Département de Mycologie Institut de Biotecnologia i Biomedicina and Departament de Bioquímica i Biologia Molecular Universitat Autònoma de Barcelona Albert Einstein College of Medicine Universidade Estadual Paulista (UNESP) Faculdade de Ciências Farmacêuticas Câmpus Araraquara Departamento de Análises Clínicas Laboratório de Micologia Clínica Agència de Gestió d’Ajuts Universitaris i de Recerca: 2014SGR-4 Secretaría de Estado de Investigación, Desarrollo e Innovación: BFU2014-54591-C2-1-P Dirección General de Investigación Científica y Técnica: BFU2017-82574-P Secretaría de Estado de Investigación, Desarrollo e Innovación: SAF2014-54336-R Dirección General de Investigación Científica y Técnica: SAF2017-86192-R

Cryptococcus neoformans is a pathogenic yeast that can form titan cells in the lungs, which are fungal cells of abnormal enlarged size. Little is known about the factors that trigger titan cells. In particular, it is not known how the host environment influences this transition. In this work, we describe the formation of titan cells in two mouse strains, CD1 and C57BL/6J. We found that the proportion of C. neoformans titan cells was significantly higher in C57BL/6J mice than in CD1. This higher proportion of titan cells was associated with a higher dissemination of the yeasts to the brain. Histology sections demonstrated eosinophilia in infected animals, although it was significantly lower in the CD1 mice which presented infiltration of lymphocytes. Both mouse strains presented infiltration of granulocytes, but the amount of eosinophils was higher in C57BL/6J. CD1 mice showed a significant accumulation of IFN-γ, TNF-α and IL17, while C57BL/BL mice had an increase in the anti-inflammatory cytokine IL-4. IgM antibodies to the polysaccharide capsule and total IgE were more abundant in the sera from C57BL/6J, confirming that these animals present a Th2-type response. We conclude that titan cell formation in C. neoformans depends, not only on microbe factors, but also on the host environment.

Cryptococcus neoformans is a human pathogenic yeast that causes hundreds of thousands of deaths worldwide among susceptible individuals, in particular, HIV+ patients. This yeast has developed several adaptation mechanisms that allow replication within the host. During decades, this yeast has been well known for a very peculiar and unique structure that contributes to virulence, a complex polysaccharide capsule that surrounds the cell wall. In contrast to other fungal pathogens, such as Candida albicans or Aspergillus fumigatus, the role of morphological transitions has not been studied in the virulence of Cryptococcus neoformans since this yeast does not form hyphae during infection. However, in the last years, different groups have described the ability of this fungus to change its size during infection. In particular, Cryptococcus can form "titan cells," which are blastoconidia of an abnormal large size. Since their discovery, there is increasing evidence that these cells contribute, not only to long-term persistence in the host, but they can also actively participate in the development of the disease. Recently, several groups have simultaneously described different media that induce the appearance of titan cells in laboratory conditions. Using these conditions, new inducing factors and signaling pathways involved in this transition have been described. In this article, we will review the main phenotypic features of these cells, factors, and transduction pathways that induce cell growth, and how titan cells contribute to the disease caused by this pathogen.

Cryptococcus neoformansis an encapsulated pathogenic yeast that can change the size of the cells during infection. In particular, this process can occur by enlarging the size of the capsule without modifying the size of the cell body, or by increasing the diameter of the cell body, which is normally accompanied by an increase of the capsule too. This last process leads to the formation of cells of an abnormal enlarged size denominated titan cells. Previous works characterized titan cell formation during pulmonary infection but research on this topic has been hampered due to the difficulty to obtain themin vitro. In this work, we describein vitroconditions (low nutrient, serum supplemented medium at neutral pH) that promote the transition from regular to titan-like cells. Moreover, addition of azide and static incubation of the cultures in a CO2enriched atmosphere favored cellular enlargement. This transition occurred at low cell densities, suggesting that the process was regulated by quorum sensing molecules and was independent of the cryptococcal serotype/species. Transition to titan-like cell formation was impaired by pharmacological inhibition of PKC and TOR signaling pathways. Mutants affected in capsule synthesis did not form titan-like cells. Analysis of the gene expression profile in titan-like cells indicated that they overexpressed membrane proteins and transporters, being the gene encoding the Cig1 mannoprotein involved in iron uptake from heme groups the gene most differentially expressed compared to cells of regular size. We also investigated the gene expression profile of titan-like cells isolated from mice, and observed that during infection these cells mainly overexpressed genes related to metabolism and respiration. In summary, our work provides a new alternative method to investigate titan cell formation devoid the bioethical problems that involve animal experimentation.AUTHOR SUMMARYCryptococcus neoformansis a fungal pathogen that has a significant incidence in HIV+ patients in particular, in Subsaharian Africa, Asia and South America. This yeast poses an excellent model to investigate fungal virulence because it develops many strategies to adapt to the host and evade the immune response. One of the adaptation mechanisms involves the formation of Titan Cells, which are yeast of an abnormal large size. However, research on these cells has been limited to in vivo studies (mainly in mice) because they were not reproducibly found in vitro. In this work, we describe several conditions that induce the appearance of cells that mimic titan cells, and that we denominated as titan-like cells. The main factor that induced titan-like cells was the addition of serum to nutrient limited media. This has allowed to easily performing new approaches to characterize several signaling pathways involved in their development. We found that the formation of these cells is regulated by quorum sensing molecules, and that pathways such as PKC and TOR kinases regulate the process of cellular enlargement. We have also to perform transcriptomic analysis, which led to the identification of new genes that could be involved in the process. This work will open different research lines that will contribute to the elucidation of the role of these cells during infection and on the development of cryptococcal disease.

Cryptococcus neoformans is an environmental encapsulated yeast that behaves as an opportunistic pathogen in immunocompromised individuals. The capsule is the main virulence factor of this pathogen. This structure is highly dynamic, and it can change its size and structure according to the environmental conditions. During infection, C. neoformans significantly enlarges the size of the capsule by the addition of new polysaccharide. It is believed that capsule growth is an energy-cost process, but this aspect has never been addressed. In this work, we have evaluated the role of mitochondrial activity on capsule growth using specific inhibitors of the electron respiratory chain. We observed that capsule growth was impaired in the presence of inhibitors of the respiratory chain as salicylhydroxamic acid or antimycin A. Furthermore, capsule growth correlated with an increase of the mitochondrial membrane potential and higher production of reactive oxygen species. Our results confirm that capsule growth depends on mitochondrial activity, and open new insights to understand the regulation of this process. OZ is funded by grant SAF2014-54336-R from the SpanishMinistry for Economics and Competitivity. NT-C is supported bya FPI fellowship (reference BES-2012-051837). SR is supported bya grant from Ciências sem fronteiras (202436/2015-2). EA had agrant from Erasmus+program (Erasmus code COVILHA01).SL-F participated in this work in the frame of a Master in‘Parasitology and Microbiology: Research and Development ’from the Pharmacy and Biology Faculties from the Complutense University of Madrid. Sí

Laboratorio de Micologia Clinica e Nucleo de Proteomica Faculdade de Ciencias Farmaceuticas Universidade Estadual Paulista Julio Mesquita Filho

Parents invest in their offspring by preparing them for defense against pathogens and parasites that only the parents have encountered, a phenomenon known as trans-generational immune priming. We investigated the underlying mechanism using the established lepidopteran model host Galleria mellonella. When larvae were fed with non-pathogenic bacteria, or the entomopathogenic species Pseudomonas entomophila and Serratia entomophila, the activity of lysozyme and phenoloxidase increased in the hemolymph, and immunity-related genes encoding antibacterial proteins such as gloverin were induced. Remarkably, the ingestion of bacteria by female larvae resulted in the differential expression of immunity-related genes in the eggs subsequently laid by the same females, providing evidence for trans-generational immune priming in G. mellonella. To determine the fate of these ingested microbes, the larval diet was supplemented with bacteria carrying a fluorescent label. We observed these bacteria crossing the midgut epithelium, their entrapment within nodules in the hemocoel, their accumulation within the ovary, and ultimately their deposition in the eggs. Therefore, we propose that trans-generational immune priming in Lepidoptera can be mediated by the maternal transfer of bacteria or bacterial fragments to the developing eggs.

Background An important feature of Cryptococcus neoformans (CN) is an ability to undergo morphological changes that enhance virulence and development of cryptococcal disease (CD). CN can change its size by capsule enlargement alone or capsule and cell body enlargement, resulting in “titan cells.” Titan cells enable CN to evade host defense mechanisms. Human and mouse β-glucan antibodies bind and inhibit CN growth in vitro. Naturally occurring antibodies in human serum bind β-glucans. In this study, we determined the effect of human IgM and IgG on CN size and titan cell formation in vitro. Methods Experiments were performed with CN var. grubii H99 (serotype A) grown in liquid Sabouraud media at 30ºC. First, we established that human IgM (Sigma Aldrich) binds H99 and Laminarin (a polymer consisting primarily of β (1–3) glucan with occasional β (1–6) branching (Sigma Aldrich) by ELISA using Goat Anti-Human IgM-AP Then, we cultured CN in titan cell medium (TCM, 5% sabouraud and 5% fetal bovine serum diluted in MOPS 50 mM at pH 7.3 plus 15 µM sodium azide) at 37ºC with CO2 for 18 hours with and without human IgM or IgG (Sigma Aldrich), after which cell size was evaluated using India Ink in a Zeiss microscope. Results We found that IgM-treated cells exhibited a significant reduction in CN capsule size and titan cell formation (total cell size) compared with controls without IgM or with IgG. Median total cell size (µm) were: IgM (15.04), IgG (20) and PBS (22.24), P < 0.05 using the Kolmogorov–Smirnow test to estimate normality and one-way ANOVA to compare between groups. There were no statistical differences in cell size after incubation with human IgG or PBS. To gain insight into how IgM may mediate its effect, we demonstrated that it bound mainly to the CN cell wall with some diffuse punctuate to the capsule by immunofluorescence. Conclusion Our results reveal that natural IgM has the ability to inhibit CN titan cell formation in cultured cells. Given the importance of titan cell formation in virulence, our results suggest that direct effects of natural antibody on CN biology may contribute to human resistance to CD. This hypothesis is under investigation in our laboratory. Disclosures All authors: No reported disclosures.

One of the main features of the majority of pathogenic fungi is the ability to switch between different types of morphological forms. These changes include the transition between cells of different shapes (such as the formation of pseudohyphae and hyphae), or the massive growth of the blastoconidia and formation of titan cells. Morphological changes occur during infection, and there is extensive evidence that they play a key role in processes required for disease, such as adhesion, invasion and dissemination, immune recognition evasion, and phagocytosis avoidance. In the present review, we will provide an overview of how morphological transitions contribute to the development of fungal disease, with special emphasis in two cases: Candida albicans as an example of yeast that switches between blastoconidia and filaments, and Cryptococcus neoformans as an example of a fungus that changes the size without modifying the shape of the cell.

Made available in DSpace on 2018-12-11T17:02:03Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-02-16 Cryptococcus sp. are responsible for around 1 million cases of meningitis every year. Fluconazole (FLU) is commonly used in the treatment of cryptococcosis, mainly in immunocompromised patients and the resistance is usually reported after long periods of treatment. In this study, the morphological characterization and virulence profile of FLU-susceptible and FLU-resistant clinical and environmental isolates of C. neoformans and C. gattii were performed both in vitro and in vivo using the Galleria mellonella model. FLU-susceptible isolates from C. neoformans were significantly more virulent than the FLU-resistant isolates. FLU-susceptible C. gattii isolates showed a different virulence profile from C. neoformans isolates where only the environmental isolate, CL, was more virulent compared with the resistant isolates. Cell morphology and capsule size were analyzed and the FLU-resistant isolates did not change significantly compared with the most sensitive isolates. Growth at 37°C was also evaluated and in both species, the resistant isolates showed a reduced growth at this temperature, indicating that FLU resistance can affect their growth. Based on the results obtained is possible suggest that FLU resistance can influence the morphology of the isolates and consequently changed the virulence profiles. The most evident results were observed for C. neoformans showing that the adaptation of isolates to antifungal selective pressure influenced the loss of virulence. Faculdade de Ciências Farmacêuticas UNESP - Universidade Estadual Paulista Campus Araraquara Departamento de Análises Clíncas Centro Nacional de Microbiologia Unidad de Micologia Instituto de Salud Carlos III Dermatology Research Group Universidad de Antioquia Faculdade de Medicina Veterinária e Zootecnia USP - Universidade de São Paulo Departamento de Patologia Faculdade de Ciências Farmacêuticas UNESP - Universidade Estadual Paulista Campus Araraquara Departamento de Análises Clíncas

Cryptococcus neoformans is an opportunistic fungal pathogen that has several well-described virulence determinants. A polysaccharide capsule and the ability to produce melanin are among the most important. Melanization occurs both in vitro, in the presence of catecholamine and indole compounds, and in vivo during the infection. Despite the importance of melanin production for cryptococcal virulence, the component and mechanisms involved in its synthesis have not been fully elucidated. In this work, we describe the role of a G1/S cyclin (Cln1) in the melanization process. Cln1 has evolved specifically with proteins present only in other basidiomycetes. We found that Cln1 is required for the cell wall stability and production of melanin in C. neoformans. Absence of melanization correlated with a defect in the expression of the LAC1 gene. The relation between cell cycle elements and melanization was confirmed by the effect of drugs that cause cell cycle arrest at a specific phase, such as rapamycin. The cln1 mutant was consistently more susceptible to oxidative damage in a medium that induces melanization. Our results strongly suggest a novel and hitherto unrecognized role for C. neoformans Cln1 in the expression of virulence traits. We thank Rajendra Uphadya (Washington University School of Medicine, St. Louis, MI, USA) for providing the sequence of oligonucleotides for 18s gene used in this article. RG-R was supported by a FPI fellowship (reference BES-2009-015913) from the Spanish Ministry of Economics and Competitivity. NT-C is supported by a FPI fellowship (reference BES-2012-051837). OZ is funded by grant SAF2011-25140 and SAF2014-54336 from the Spanish Ministry for Economics and Competitivity Sí

Cryptococcus neoformans is an encapsulated opportunistic fungal pathogen that is found in multiple niches in the environment and that can cause fatal meningoencephalitis in susceptible patients, mainly HIV+ individuals. Cryptococcus also infects environmental hosts such as nematodes, insects and plants. In particular, C. neoformans can kill the lepidopteran Galleria mellonella, which offers a useful tool to study microbial virulence and drug efficacy. Galleria mellonella immunity relies on innate responses based on melanization, accumulation of antimicrobial peptides, and cellular responses as phagocytosis or multicellular encapsulation. In this work we have investigated the immune response of G. mellonella during cryptococcal infection. We found that G. mellonella infected with C. neoformans had a high lytic activity in their hemolymph. This response was temperature- and capsule-dependent. During interaction with phagocytic cells, C. neoformans behaved as an intracellular pathogen since it could replicate within hemocytes. Non-lytic events were also observed. In contrast to Candida species, C. neoformans did not induce melanization of G. mellonella after infection. Finally, passage of C. neoformans through G. mellonella resulted in changes in capsule structure as it has been also reported during infection in mammals. Our results highlight that G. mellonella is an optimal model to investigate innate immune responses against C. neoformans. O.Z. is funded by grant SAF2011–25140 from the Spanish Ministry for Economics and Competitivity. N.T-C. is supported by a FPI fellowship (reference BES-2012–051837). C.R. has a Sara Borrell contract from the Fondo de Investigaciones Sanitarias from Instituto de Salud Carlos III (reference number CD11/00110). I.H-F and I.G-B participated in this work in the frame of the Master “Parasitology and Microbiology: Research and Development” from the Pharmacy and Biology Faculties from the Complutense University of Madrid. Liliana Scorzoni was supported by Fundação de Apoio à Pesquisa do Estado de São Paulo (FAPESP) 2013/10917–9. S.A.R was supported by Programa PDSE-CAPES – 1909–13–4. Sí

Cryptococcus neoformans is a pathogenic yeast that can form titan cells in the lungs, which are fungal cells of abnormal enlarged size. Little is known about the factors that trigger titan cells. In particular, it is not known how the host environment influences this transition. In this work, we describe the formation of titan cells in two mouse strains, CD1 and C57BL/6J. We found that the proportion of C. neoformans titan cells was significantly higher in C57BL/6J mice than in CD1. This higher proportion of titan cells was associated with a higher dissemination of the yeasts to the brain. Histology sections demonstrated eosinophilia in infected animals, although it was significantly lower in the CD1 mice which presented infiltration of lymphocytes. Both mouse strains presented infiltration of granulocytes, but the amount of eosinophils was higher in C57BL/6J. CD1 mice showed a significant accumulation of IFN-γ, TNF-α and IL17, while C57BL/BL mice had an increase in the anti-inflammatory cytokine IL-4. IgM antibodies to the polysaccharide capsule and total IgE were more abundant in the sera from C57BL/6J, confirming that these animals present a Th2-type response. We conclude that titan cell formation in C. neoformans depends, not only on microbe factors, but also on the host environment.

International audience; Opportunistic pathogens like Cryptococcus neoformans are constantly exposed to changing environments, in their natural habitat as well as when encountering a human host. This requires a coordinated program to regulate gene expression that can act at the levels of mRNA synthesis and also mRNA degradation. Here, we find that deletion of the gene encoding the major cytoplasmic 5' -> 3' exonuclease Xrn1p in C neoformans has important consequences for virulence associated phenotypes such as growth at 37 degrees C, capsule and melanin. In an invertebrate model of cryptococcosis the alteration of these virulence properties corresponds to avirulence of the xrn1 Delta mutant strains. Additionally, deletion of XRN1 impairs uni- and bisexual mating. On a molecular level, the absence of XRN1 is associated with the upregulation of other major exonuclease encoding genes (i.e. XRN2 and RRP44). Using inducible alleles of RRP44 and XRN2, we show that artificial overexpression of these genes alters LAC1 gene expression and mating. Our data thus suggest the existence of a complex interdependent regulation of exonuclease encoding genes that impact upon virulence and mating in C. neoformans. (C) 2014 Elsevier Inc. All rights reserved.

Amphotericin B (AMB) is an antifungal drug that binds to ergosterol and forms pores at the cell membrane, causing the loss of ions. In addition, AMB induces the accumulation of reactive oxygen species (ROS), and although these molecules have multiple deleterious effects on fungal cells, their specific role in the action mechanism of AMB remains unknown. In this work, we studied the role of ROS in the action mechanism of AMB. We determined the intracellular induction of ROS in 44 isolates of different pathogenic yeast species ( Candida albicans , Candida parapsilosis , Candida glabrata , Candida tropicalis , Candida krusei , Cryptococcus neoformans , and Cryptococcus gattii ). We also characterized the production of ROS in AMB-resistant isolates. We found that AMB induces the formation of ROS in all the species tested. The inhibition of the mitochondrial respiratory chain by rotenone blocked the induction of ROS by AMB and provided protection from the killing action of the antifungal. Moreover, this phenomenon was absent in strains that displayed resistance to AMB. These strains showed an alteration in the respiration rate and mitochondrial membrane potential and also had higher catalase activity than that of the AMB-susceptible strains. Consistently, AMB failed to induce protein carbonylation in the resistant strains. Our data demonstrate that the production of ROS by AMB is a universal and important action mechanism that is correlated with the fungicidal effect and might explain the low rate of resistance to the molecule. Finally, these data provide an opportunity to design new strategies to improve the efficacy of this antifungal.

The fungal pathogen Cryptococcus neoformans has several virulence factors, among which the most important is a polysaccharide capsule. The size of the capsule is variable and can increase significantly during infection. In this work, we investigated the relationship between capsular enlargement and the cell cycle. Capsule growth occurred primarily during the G1 phase. Real-time visualization of capsule growth demonstrated that this process occurred before the appearance of the bud and that capsule growth arrested during budding. Benomyl, which arrests the cells in G2/M, inhibited capsule growth, while sirolimus (rapamycin) addition, which induces G1 arrest, resulted in cells with larger capsule. Furthermore, we have characterized a mutant strain that lacks a putative G1/S cyclin. This mutant showed an increased capacity to enlarge the capsule, both in vivo (using Galleria mellonella as the host model) and in vitro. In the absence of Cln1, there was a significant increase in the production of extracellular vesicles. Proteomic assays suggest that in the cln1 mutant strain, there is an upregulation of the glyoxylate acid cycle. Besides, this cyclin mutant is avirulent at 37°C, which correlates with growth defects at this temperature in rich medium. In addition, the cln1 mutant showed lower intracellular replication rates in murine macrophages. We conclude that cell cycle regulatory elements are involved in the modulation of the expression of the main virulence factor in C. neoformans., IMPORTANCE Cryptococcus neoformans is a pathogenic fungus that has significant incidence worldwide. Its main virulence factor is a polysaccharide capsule that can increase in size during infection. In this work, we demonstrate that this process occurs in a specific phase of the cell cycle, in particular, in G1. In agreement, mutants that have an abnormal longer G1 phase show larger capsule sizes. We believe that our findings are relevant because they provide a link between capsule growth, cell cycle progression, and virulence in C. neoformans that reveals new aspects about the pathogenicity of this fungus. Moreover, our findings indicate that cell cycle elements could be used as antifungal targets in C. neoformans by affecting both the growth of the cells and the expression of the main virulence factor of this pathogenic yeast.