Identificación de los mecanismos moleculares involucrados en la morfogénesis de Cryptococcus neoformans y estudio de su función durante la infección

Una de las principales características de la mayoría de los hongos es la capacidad de cambiar de morfología. En el caso de hongos patógenos, estos cambios ocurren durante la infección y juegan un papel importante en el desarrollo de la enfermedad. Las transiciones morfológicas pueden ocurrir por formación de hifas y pseudohifas, o por un crecimiento masivo de la blastoconidia. Cryptococcus neoformans es una levadura patógena cuya principal característica es una cápsula de polisacárido. Durante la infección, esta levadura cambia de tamaño sin modificar la forma de la célula. En concreto, este hongo puede incrementar su tamaño mediante dos mecanismos diferentes: aumentando el tamaño de la cápsula, o incrementando el diámetro tanto de la cápsula como del cuerpo celular, dando lugar a células gigantes (diámetro mayor de 30 micras). El principal objetivo de este trabajo es investigar los mecanismos que regulan la morfogénesis en C. neoformans, e intentar esclarecer los factores del huésped que inducen estos cambios. En la primera parte de este trabajo, se evaluó la síntesis de la cápsula utilizando una cepa que expresa el gen CAP59 (necesario para la síntesis capsular), fusionado al promotor regulable GAL7. La síntesis de la cápsula se produjo de manera dispersa y no polarizada. Además, se estudió si el incremento capsular era dependiente de la actividad mitocondrial utilizando para ello inhibidores de la fosforilación oxidativa. Observamos que este fenómeno no ocurría en presencia de inhibidores de los diferentes complejos de la cadena transportadora de electrones como antimicina A, ácido salicilhidroxámico o rotenona. Los resultados obtenidos han mostrado nuevos aspectos sobre la dinámica de aparición y crecimiento de la cápsula y abren nuevas vías de investigación necesarias para entender mejor la biología de la cápsula..

Role of IL-17 in Morphogenesis and Dissemination of Cryptococcus neoformans during Murine Infection

Cryptococcus neoformans is a pathogenic yeast that can form Titan cells in the lungs, which are fungal cells of abnormally large size. The factors that regulate Titan cell formation in vivo are still unknown, although an increased proportion of these fungal cells of infected mice correlates with induction of Th2-type responses. Here, we focused on the role played by the cytokine IL-17 in the formation of cryptococcal Titan cells using Il17a-/- knockout mice. We found that after 9 days of infection, there was a lower proportion of Titan cells in Il17a-/- mice compared to the fungal cells found in wild-type animals. Dissemination to the brain occurred earlier in Il17a-/- mice, which correlated with the lower proportion of Titan cells in the lungs. Furthermore, knockout-infected mice increased brain size more than WT mice. We also determined the profile of cytokines accumulated in the brain, and we found significant differences between both mouse strains. We found that in Il17a-/-, there was a modest increase in the concentrations of the Th1 cytokine TNF-α. To validate if the increase in this cytokine had any role in cryptococcal morphogenesis, we injected wild-type mice with TNF-α t and observed that fungal cell size was significantly reduced in mice treated with this cytokine. Our results suggest a compensatory production of cytokines in Il17a-/- mice that influences both cryptococcal morphology and dissemination.This work was supported by Grant SAF2017-86912-R and PID 2020-114546RB-100 from the Spanish Ministry for Science and Innovation. Roselletti E. is funded by an international collaboration with the company Lesaffre International Sarl. Garcia-Rodas R. is funded by a “Juan de la Cierva” contract from the Spanish Ministry for Economics, Industry, and Competitivity (Reference: IJCI-2015-25683). Trevijano-Contador N. is funded by an “Ayudas de Atracción de Talento Investigador” contract of the Community of Madrid (Reference: 2019-T2/BMD-14926).S

Cell Wall Integrity Pathway Involved in Morphogenesis, Virulence and Antifungal Susceptibility in Cryptococcus neoformans

Due to its location, the fungal cell wall is the compartment that allows the interaction with the environment and/or the host, playing an important role during infection as well as in different biological functions such as cell morphology, cell permeability and protection against stress. All these processes involve the activation of signaling pathways within the cell. The cell wall integrity (CWI) pathway is the main route responsible for maintaining the functionality and proper structure of the cell wall. This pathway is highly conserved in the fungal kingdom and has been extensively characterized in Saccharomyces cerevisiae. However, there are still many unknown aspects of this pathway in the pathogenic fungi, such as Cryptococcus neoformans. This yeast is of particular interest because it is found in the environment, but can also behave as pathogen in multiple organisms, including vertebrates and invertebrates, so it has to adapt to multiple factors to survive in multiple niches. In this review, we summarize the components of the CWI pathway in C. neoformans as well as its involvement in different aspects such as virulence factors, morphological changes, and its role as target for antifungal therapies among others.This work was supported by Grant PID2020-114546RB-100 from the Spanish Ministry for Science and Innovation. De Oliveira H.C. was supported by Inova Fiocruz/Fundação Oswaldo Cruz. Rossi SA is funded by postdoctoral fellowship from Fundacão de Amparo á pesquisa do Estado de São Paulo (reference FAPESP-BEPE 2020/09919-0). García-Barbazán Iwas supported by a FPI fellowship (reference PRE2018-083436). Trevijano-Contador N is funded by a “Ayudas de Atracción de Talento Investigador” Contract of Community of Madrid (reference 2019-T2/BMD-14926).S

Role of Cln1 during melanization of Cryptococcus neoformans

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 CompetitivityS

Capsule growth in Cryptococcus neoformans is coordinated with cell cycle progression

UNLABELLED: 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.O.Z. is funded by grants SAF2008-03761 and SAF2011-25140 from the Spanish Ministry for Economics and Competitivity. R.G.-R. is supported by an FPI fellowship (reference BES-2009-015913) from the Spanish Ministry of Science and Innovation. N.T.-C. is supported by an FPI fellowship (reference BES-2012-051837) from the Spanish Ministry for Economics and Competitivity. A.C. is supported by NIH grants HL059842-3, A1033774, A1052733, and AI033142. R.J.B.C. is supported by T32 AI07506 (NIH/NIAID).S

Cryptococcus neoformans can form titan-like cells in vitro in response to multiple signals

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 CO2 enriched 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.OZ is funded by grant SAF2014-54336-R and SAF2017-86192-R1 from the Spanish Ministry for Economics, Industry and Competitivity. JA is funded by grants BFU2014-54591-C2-1-P and BFU2017-82574-P (Spanish Ministry for Economics, Industry and Competitivity) and an “Ajut 2014SGR-4” (Generalitat de Catalunya). NT-C was supported by a FPI fellowship (reference BES-2012-051837). SAR was supported by a fellowship from Coordenação de aperfeiçoamento de pessoal de nivel superior, CAPES, program Ciências Sem Fronteiras (202436/2015-2). HCdO is funded by postdoctoral fellowship from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP-BEPE 2016/20631-3). RG-R is funded by a "Juan de la Cierva" Contract from the Spanish Ministry for Economics, Industry and Competitivity (reference: IJCI-2015-25683). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S

Global Emergence of Resistance to Fluconazole and Voriconazole in Candida parapsilosis in Tertiary Hospitals in Spain During the COVID-19 Pandemic

Candida parapsilosis; Antifungal resistance; OutbreaksCandida parapsilosis; Resistencia antifúngica; BrotesCandida parapsilosis; Resistència antifúngica; BrotsBackground Candida 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 it is susceptible to polyenes and azoles. Multiple outbreaks caused by fluconazole-nonsusceptible strains have been reported recently. A similar trend has been observed among the C. parapsilosis isolates received in the last 2 years at the Spanish Mycology Reference Laboratory. Methods Yeast were identified by molecular biology, and antifungal susceptibility testing was performed using the European Committee on Antimicrobial Susceptibility Testing protocol. The ERG11 gene was sequenced to identify resistance mechanisms, and strain typing was carried out by microsatellite analysis. Results We examined the susceptibility profile of 1315 C. parapsilosis isolates available at our reference laboratory between 2000 and 2021, noticing an increase in the number of isolates with acquired resistance to fluconazole, and voriconazole has increased in at least 8 different Spanish hospitals in 2020–2021. From 121 recorded clones, 3 were identified as the most prevalent in Spain (clone 10 in Catalonia and clone 96 in Castilla-Leon and Madrid, whereas clone 67 was found in 2 geographically unrelated regions, Cantabria and the Balearic Islands). Conclusions Our data suggest that concurrently with the coronavirus disease 2019 pandemic, a selection of fluconazole-resistant C. parapsilosis isolates has occurred in Spain, and the expansion of specific clones has been noted across centers. Further research is needed to determine the factors that underlie the successful expansion of these clones and their potential genetic relatedness.O.Z. was funded by grants SAF2017–86912-R and PID2020–114546RB-I00 from the Spanish Ministry for Science and Innovation. This work was also funded by the National Centre for Microbiology (Instituto de Salud Carlos III) through the Surveillance Program of Antifungal Resistance and the Center for Biomedical Research in Network of Infectious Diseases CIBERINFECTCB21/13/00105 (O.Z. and L.A.F.), CIBERINFEC-CB21/13/00009 (M.P.-A.), CIBERES-CB06/06/0037 (C.A.-T.), and CIBERES-CB06/06/0058 (J.G). L.A.-F. was supported by Fondo de Investigación Sanitaria (MPY 117/18 and MPY 305/20). We thank Dr. David Campany Herrero (Vall d’Hebron Hospital), Noelia Garrido Peño (Móstoles Hospital), David Gómez Gómez y Aitziber Illaro Uranga (Marqués de Valdecilla Hospital), María Ángeles Machín Morón (Burgos Hospital), Jose Manuel Caro Teller (Doce de Octubre Hospital), Marina Calvo (Puerta de Hierro Hospital), and Ariadna Padulles (Bellvitge Hospital) for providing the data on antifungal consumption from their hospitals. We also thank Ángel Zaballos and Pilar Jiménez from the Genomics Core Facility from Instituto de Salud Carlos III for their technical help with the microsatellite analysis technique

Impact of Resistance to Fluconazole on Virulence and Morphological Aspects of Cryptococcus neoformans and Cryptococcus gattii Isolates

Cryptococcus spp. are responsible for around one 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

Expanding the use of alternative models to investigate novel aspects of immunity to microbial pathogens

In the present issue of Virulence, an article entitled “The maternal transfer of bacteria can mediate trans-generational immune priming in insects”1 describes an elegant study that illustrates the use of the lepidopteran Galleria mellonella to investigate a specific aspect of immunity to microbes. The authors show that exposure of mothers to bacteria results in enhanced immunity in the offspring. Furthermore, they have demonstrated that bacteria ingested by female larvae are found in the eggs, suggesting that enhanced immunity of the offspring is a consequence of direct exposure of the eggs to bacteria. This is a relevant and novel study for several reasons. The authors provide a mechanism for an important aspect of insect immunity, which is that direct transfer of bacterial fragments from the mother to the eggs primes their immune response. But in addition, this study opens the scope on the use of non-conventional models and illustrates how they can be used to investigate aspects of immunity against pathogenic microorganisms.O.Z. is funded by grant SAF-2011-25140 from the Spanish Ministry for Economics and Competitivity. N.T-C. is supported by a FPI fellowship (reference BES-2012-051837).S

Expanding the use of alternative models to investigate novel aspects of immunity to microbial pathogens

In the present issue of Virulence, an article entitled “The maternal transfer of bacteria can mediate trans-generational immune priming in insects”1 describes an elegant study that illustrates the use of the lepidopteran Galleria mellonella to investigate a specific aspect of immunity to microbes. The authors show that exposure of mothers to bacteria results in enhanced immunity in the offspring. Furthermore, they have demonstrated that bacteria ingested by female larvae are found in the eggs, suggesting that enhanced immunity of the offspring is a consequence of direct exposure of the eggs to bacteria. This is a relevant and novel study for several reasons. The authors provide a mechanism for an important aspect of insect immunity, which is that direct transfer of bacterial fragments from the mother to the eggs primes their immune response. But in addition, this study opens the scope on the use of non-conventional models and illustrates how they can be used to investigate aspects of immunity against pathogenic microorganisms.O.Z. is funded by grant SAF-2011-25140 from the Spanish Ministry for Economics and Competitivity. N.T-C. is supported by a FPI fellowship (reference BES-2012-051837).S