RESUMEN
Immunosenescence refers to the age-related progressive decline of immune function contributing to the increased susceptibility to infectious diseases in older people. Neurocryptococcosis, an infectious disease of central nervous system (CNS) caused by Cryptococcus neoformans (C. Neoformans) and C. gattii, has been observed with increased frequency in aged people, as result of the reactivation of a latent infection or community acquisition. These opportunistic microorganisms belonging to kingdom of fungi are capable of surviving and replicating within macrophages. Typically, cryptococcus is expelled by vomocytosis, a non-lytic expulsive mechanism also promoted by interferon (IFN)-I, or by cell lysis. However, whereas in a first phase cryptococcal vomocytosis leads to a latent asymptomatic infection confined to the lung, an enhancement in vomocytosis, promoted by IFN-I overproduction, can be deleterious, leading the fungus to reach the blood stream and invade the CNS. Cryptococcus may not be easy to diagnose in older individuals and, if not timely treated, could be potentially lethal. Therefore, this review aims to elucidate the putative causes of the increased incidence of cryptococcal CNS infection in older people discussing in depth the mechanisms of immunosenscence potentially able to predispose to neurocryptococcosis, laying the foundations for future research. A deepest understanding of this relationship could provide new ways to improve the prevention and recognition of neurocryptococcosis in aged frail people, in order to quickly manage pharmacological interventions and to adopt further preventive measures able to reduce the main risk factors.
Asunto(s)
Criptococosis , Inmunidad Innata , Inmunosenescencia , Humanos , Inmunosenescencia/inmunología , Criptococosis/inmunología , Cryptococcus neoformans/inmunología , Animales , Cryptococcus gattii/inmunología , Anciano , Susceptibilidad a Enfermedades/inmunologíaRESUMEN
Cryptococcus neoformans has emerged as a frontrunner among deadly fungal pathogens and is particularly life-threatening for many HIV-infected individuals with compromised immunity. Multiple virulence factors contribute to the growth and survival of C. neoformans within the human host, the two most prominent of which are the polysaccharide capsule and melanin. As both of these features are associated with the cell wall, we were interested to explore possible cooperative or competitive interactions between these two virulence factors. Whereas capsule thickness had no effect on the rate at which cells became melanized, build-up of the melanin pigment layer resulted in a concomitant loss of polysaccharide material, leaving melanized cells with significantly thinner capsules than their nonmelanized counterparts. When melanin was provided exogenously to cells in a transwell culture system we observed a similar inhibition of capsule growth and maintenance. Our results show that melanin sequesters calcium thereby limiting its availability to form divalent bridges between polysaccharide subunits required for outer capsule assembly. The decreased ability of melanized cells to incorporate exported polysaccharide into the growing capsule correlated with the amount of shed polysaccharide, which could have profound negative impacts on the host immune response.
Asunto(s)
Calcio , Pared Celular , Cryptococcus neoformans , Melaninas , Cryptococcus neoformans/metabolismo , Cryptococcus neoformans/crecimiento & desarrollo , Melaninas/metabolismo , Calcio/metabolismo , Pared Celular/metabolismo , Cápsulas Fúngicas/metabolismo , Humanos , Polisacáridos/metabolismo , Polisacáridos Fúngicos/metabolismoRESUMEN
An important host defence mechanism against pathogens is intracellular killing, which is achieved through phagocytosis, a cellular process for engulfing and neutralizing extracellular particles. Phagocytosis results in the formation of matured phagolysosomes, which are specialized compartments that provide a hostile environment and are considered the end point of the degradative pathway. However, all fungal pathogens studied to date have developed strategies to manipulate phagosomal function directly and also indirectly by redirecting phagosomes from the degradative pathway to a non-degradative pathway with the expulsion and even transfer of pathogens between cells. Here, using the major human fungal pathogens Aspergillus fumigatus, Candida albicans, Cryptococcus neoformans and Histoplasma capsulatum as examples, we discuss the processes involved in host phagosome-fungal pathogen interactions, with a focus on fungal evasion strategies. We also discuss recent approaches to targeting intraphagosomal pathogens, including the redirection of phagosomes towards degradative pathways for fungal pathogen eradication.
Asunto(s)
Interacciones Huésped-Patógeno , Fagocitosis , Fagosomas , Humanos , Fagosomas/microbiología , Fagosomas/metabolismo , Fagosomas/inmunología , Interacciones Huésped-Patógeno/inmunología , Animales , Hongos/inmunología , Hongos/fisiología , Hongos/patogenicidad , Candida albicans/inmunología , Candida albicans/fisiología , Histoplasma/inmunología , Histoplasma/fisiología , Aspergillus fumigatus/inmunología , Aspergillus fumigatus/fisiología , Cryptococcus neoformans/inmunología , Cryptococcus neoformans/fisiología , Evasión Inmune , Micosis/inmunología , Micosis/microbiologíaRESUMEN
Cryptococcosis is an important fungal infection for both humans and cats, but molecular epidemiological studies on strains isolated from cats are limited. We conducted multi-locus sequence typing analysis and antifungal susceptibility testing of 14 Cryptococcus spp. strains from domestic cats in Japan and one strain isolated from a cat in Singapore. All 14 strains from domestic cats in Japan were identified as Cryptococcus neoformans molecular type VNI. The sequence types (STs) included eight cases of ST5, five cases of ST31, and one novel ST. VNI ST5 is the most frequently isolated strain in Japanese patients as well, while there are no records of VNI ST31 being isolated from Japanese patients. The Singaporean cat strain was identified as C. gattii VGIIb (C. deuterogattii), ST7. We compared these results with strains previously reported to have been isolated from cats. This comparison suggested that molecular types of Cryptococcus spp. isolated from cats may differ depending on the country. In the antifungal susceptibility testing of C. neoformans, one strain each exceeded the epidemiological cutoff value (ECV) for amphotericin B and 5-fluorocytosine, while two strains exceeded the ECV for fluconazole. This study reveals the molecular epidemiology of Cryptococcus spp. isolated from cats with cryptococcosis in Japan. It suggests that investigating Cryptococcus spp. carried by cats, which share close living environments with humans, may contribute to the health of both cats and human populations.
Cryptococcosis is an important fungal disease in both humans and cats. We genotyped strains isolated from cats with cryptococcosis in Japan. Our findings revealed that the most common genotype infecting both cats and humans in Japan is identical.
Asunto(s)
Antifúngicos , Enfermedades de los Gatos , Criptococosis , Cryptococcus neoformans , Pruebas de Sensibilidad Microbiana , Epidemiología Molecular , Tipificación de Secuencias Multilocus , Animales , Gatos , Criptococosis/microbiología , Criptococosis/epidemiología , Criptococosis/veterinaria , Japón/epidemiología , Enfermedades de los Gatos/microbiología , Enfermedades de los Gatos/epidemiología , Antifúngicos/farmacología , Cryptococcus neoformans/genética , Cryptococcus neoformans/aislamiento & purificación , Cryptococcus neoformans/clasificación , Cryptococcus neoformans/efectos de los fármacos , Técnicas de Tipificación Micológica , Cryptococcus gattii/genética , Cryptococcus gattii/aislamiento & purificación , Cryptococcus gattii/clasificación , Cryptococcus gattii/efectos de los fármacos , Genotipo , Cryptococcus/genética , Cryptococcus/clasificación , Cryptococcus/aislamiento & purificación , Cryptococcus/efectos de los fármacos , Singapur/epidemiologíaRESUMEN
The basidiomycete fungus Cryptococcus neoformans is a useful model for investigating mechanisms of fungal pathogenesis in mammalian hosts. This pathogen is the causative agent of cryptococcal meningitis in immunocompromised patients and is in the critical priority group of the World Health Organization fungal priority pathogens list. In this study, we employed a mutant lacking the OPI3 gene encoding a methylene-fatty-acyl-phospholipid synthase to characterize the role of phosphatidylcholine (PC) and lipid homeostasis in the virulence of C. neoformans. We first confirmed that OPI3 was required for growth in nutrient limiting conditions, a phenotype that could be rescued with exogenous choline and PC. Additionally, we established that loss of Opi3 and the lack of PC lead to an accumulation of neutral lipids in lipid droplets and alterations in major lipid classes. The growth defect of the opi3Δ mutant was also rescued by sorbitol and polyethylene glycol (PEG), a result consistent with protection of ER function from the stress caused by lipid imbalance. We then examined the impact of Opi3 on virulence and found that the dependence of PC synthesis on Opi3 caused reduced capsule size and this was accompanied by an increase in shed capsule polysaccharide and changes in cell wall composition. Further tests of virulence demonstrated that survival in alveolar macrophages and the ability to cause disease in mice were not impacted by loss of Opi3 despite the choline auxotrophy of the mutant in vitro. Overall, this work establishes the contribution of lipid balance to virulence factor elaboration by C. neoformans and suggests that host choline is sufficient to support proliferation during disease.
Asunto(s)
Criptococosis , Cryptococcus neoformans , Modelos Animales de Enfermedad , Cryptococcus neoformans/patogenicidad , Cryptococcus neoformans/genética , Cryptococcus neoformans/metabolismo , Cryptococcus neoformans/crecimiento & desarrollo , Animales , Virulencia , Criptococosis/microbiología , Ratones , Metabolismo de los Lípidos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fosfatidilcolinas/metabolismo , Factores de Virulencia/genética , Factores de Virulencia/metabolismo , Cápsulas Fúngicas/metabolismo , Cápsulas Fúngicas/genética , Pared Celular/metabolismo , Colina/metabolismo , Femenino , Gotas Lipídicas/metabolismoRESUMEN
OBJECTIVE: Cryptococcosis predominantly presents as a meningoencephalitis in Thailand. Early and expeditious diagnosis is essential for reducing both mortality and morbidity associated with cryptococcal meningitis. We aim to define and establish the diagnostic performances between the benchmark commercially available diagnostic kit (CrAg® LFA) and the large-scale prototype of an inexpensive in-house immunochromatographic test (ICT) based on monoclonal antibody (MAb) 18B7. METHODS: We have developed the large-scale prototype for the rapid detection of cryptococcal polysaccharide antigens by utilizing a single antibody sandwich ICT format employing MAb 18B7, which is highly specific to Cryptococcus neoformans glucuronoxylomannan (GXM) antigens. An in-house MAb18B7 ICT was manufactured in accordance with industry standards under the control of the International Organization for Standardization (ISO) 13485. RESULTS: The diagnostic sensitivity, specificity, and accuracy for the in-house MAb 18B7 ICT were 99.10%, 97.61%, and 97.83%, respectively. The agreement kappa (κ) coefficient was 0.968 based on the retrospective evaluation of 580 specimens from patients living in northern Thailand with clinically suspected cryptococcosis. CONCLUSION: The data suggest that this in-house MAb 18B7 ICT will be highly beneficial for addressing the issue of cryptococcal infection in Thailand. Moreover, it is anticipated that this inexpensive ICT can play a pivotal role in various global strategies aimed at eradicating cryptococcal meningitis among individuals living with HIV by 2030.
Asunto(s)
Anticuerpos Monoclonales , Antígenos Fúngicos , Cromatografía de Afinidad , Criptococosis , Cryptococcus neoformans , Sensibilidad y Especificidad , Humanos , Tailandia , Anticuerpos Monoclonales/inmunología , Cromatografía de Afinidad/métodos , Criptococosis/diagnóstico , Cryptococcus neoformans/inmunología , Cryptococcus neoformans/aislamiento & purificación , Antígenos Fúngicos/análisis , Antígenos Fúngicos/inmunología , Estudios Retrospectivos , Anticuerpos Antifúngicos/sangre , Polisacáridos/análisis , Polisacáridos/inmunología , Masculino , Femenino , Adulto , Pruebas Diagnósticas de Rutina/métodos , Persona de Mediana Edad , Anciano , Adulto JovenRESUMEN
Cryptococcosis is a systemic zoonotic disease that is challenging to diagnose based on clinical findings in cats and dogs due to the nonspecific nature of its clinical presentation. This case report aims to document the first confirmed case of disseminated cryptococcosis caused by Cryptococcus neoformans in a dog in Turkey and to highlight the potential link between natural disasters such as earthquakes and the emergence of zoonotic diseases in domestic animals. A two-and-a-half-year-old spayed female Cocker Spaniel presented with increased respiratory sounds, skin lesions, facial swelling and enlarged lymph nodes. These symptoms appear to be a complication of Demodex infestation due to the stress experienced by the dog following exposure to a severe earthquake. Diagnostic procedures including cytologic examination, fungal culture and DNA sequence analysis, which confirmed the infection was caused by C. neoformans. Due to the delay in the correct diagnosis of the disease, which, contrary to common data, started as an allergic reaction on the skin and was later diagnosed as a Demodex infestation, the dog died of severe respiratory failure during the treatment with itraconazole. The case highlights the critical role of veterinary emergency and critical care in the diagnosis and management of zoonotic diseases post-natural disasters. It also highlights the need for increased awareness and preparedness among veterinary professionals to address animal health challenges following such events.
Asunto(s)
Criptococosis , Enfermedades de los Perros , Zoonosis , Animales , Perros , Femenino , Criptococosis/diagnóstico , Criptococosis/veterinaria , Criptococosis/tratamiento farmacológico , Criptococosis/microbiología , Enfermedades de los Perros/microbiología , Enfermedades de los Perros/diagnóstico , Enfermedades de los Perros/parasitología , Enfermedades de los Perros/tratamiento farmacológico , Zoonosis/microbiología , Zoonosis/diagnóstico , Zoonosis/parasitología , Turquía , Cryptococcus neoformans/aislamiento & purificación , Desastres , Humanos , Resultado Fatal , Antifúngicos/uso terapéuticoRESUMEN
Cryptococcus neoformans (Cn) is an opportunistic fungal microorganism that causes life-threatening meningoencephalitis. During the infection, the microbial population is heterogeneously composed of cells with varying generational ages, with older cells accumulating during chronic infections. This is attributed to their enhanced resistance to phagocytic killing and tolerance of antifungals like fluconazole (FLC). In this study, we investigated the role of ergosterol synthesis, ATP-binding cassette (ABC) transporters, and mitochondrial metabolism in the regulation of age-dependent FLC tolerance. We find that old Cn cells increase the production of ergosterol and exhibit upregulation of ABC transporters. Old cells also show transcriptional and phenotypic characteristics consistent with increased metabolic activity, leading to increased ATP production. This is accompanied by increased production of reactive oxygen species, which results in mitochondrial fragmentation. This study demonstrates that the metabolic changes occurring in the mitochondria of old cells drive the increase in ergosterol synthesis and the upregulation of ABC transporters, leading to FLC tolerance. IMPORTANCE: Infections caused by Cryptococcus neoformans cause more than 180,000 deaths annually. Estimated 1-year mortality for patients receiving care ranges from 20% in developed countries to 70% in developing countries, suggesting that current treatments are inadequate. Some fungal cells can persist and replicate despite the usage of current antifungal regimens, leading to death or treatment failure. Aging in fungi is associated with enhanced tolerance against antifungals and resistance to killing by host cells. This study shows that age-dependent increase in mitochondrial reactive oxygen species drive changes in the regulation of membrane transporters and ergosterol synthesis, ultimately leading to the heightened tolerance against fluconazole in old C. neoformans cells. Understanding the underlying molecular mechanisms of this age-associated antifungal tolerance will enable more targeted antifungal therapies for cryptococcal infections.
Asunto(s)
Antifúngicos , Cryptococcus neoformans , Farmacorresistencia Fúngica , Fluconazol , Mitocondrias , Especies Reactivas de Oxígeno , Cryptococcus neoformans/efectos de los fármacos , Fluconazol/farmacología , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Antifúngicos/farmacología , Especies Reactivas de Oxígeno/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Ergosterol/metabolismo , Criptococosis/microbiología , Criptococosis/inmunología , Pruebas de Sensibilidad Microbiana , Humanos , Regulación Fúngica de la Expresión GénicaRESUMEN
PURPOSE: The opportunistic pathogens causing Cryptococcal meningitis are Cryptococcus neoformans and Cryptococcus gattii species complexes. At present, clinical detection methods for this condition include culture, ink staining, and cryptococcal antigen detection. In addition, enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and real-time quantitative PCR (qPCR) can be applied for the detection of Cryptococcus. Nevertheless, these methods cannot achieve point-of-care detection (POCT); thus, there is a pressing need to establish a fast, sensitive, and effective detection method. METHODS: Recombinase polymerase amplification (RPA) and clustered regularly spaced short palindromic repeat (CRISPR) techniques are effective tools for achieving rapid POCT. In this study, RPA was combined with CRISPR-Cas12a to establish a fast, sensitive, and specific detection method for cryptococcal meningitis. RESULTS: This study included RPA-Cas12a fluorescence detection and RPA-Cas12a immunochromatographic detection, which can be performed within 50 min. Moreover, the detection limit was as low as 102 copies/µL. Interestingly, the developed method demonstrated satisfactory specificity and no cross-reactivity with other fungi and bacteria. 36 clinical samples were tested, and the consistency between the test results and those obtained using the commonly used clinical culture method was 100 %. CONCLUSION: In this study, a rapid detection method for Cryptococcus neoformans and Cryptococcus gattii species complexes was developed based on CRISPR-Cas12a technology, characterized by its high sensitivity and specificity, ease of use, and cost-effectiveness, making it suitable for on-site detection.
Asunto(s)
Sistemas CRISPR-Cas , Cryptococcus gattii , Cryptococcus neoformans , Sensibilidad y Especificidad , Cryptococcus gattii/genética , Cryptococcus gattii/aislamiento & purificación , Cryptococcus neoformans/genética , Cryptococcus neoformans/aislamiento & purificación , Humanos , Meningitis Criptocócica/diagnóstico , Meningitis Criptocócica/microbiología , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Proteínas Bacterianas , Endodesoxirribonucleasas , Proteínas Asociadas a CRISPRRESUMEN
Cryptococcal infection of central nervous system commonly involves meningitis or meningoencephalitis, but rarely mimics inflammatory myelitis. We present short segment myelitis as a dominant manifestation caused by Cryptococcus neoformans in a patient with nephrotic syndrome under immunosuppressive therapy. This case report highlights Cryptococcus neoformans as a potential etiological factor for short segment myelitis in immunocompromised hosts.
Asunto(s)
Criptococosis , Cryptococcus neoformans , Mielitis , Humanos , Mielitis/microbiología , Mielitis/tratamiento farmacológico , Mielitis/diagnóstico , Criptococosis/tratamiento farmacológico , Criptococosis/microbiología , Criptococosis/diagnóstico , Cryptococcus neoformans/aislamiento & purificación , Masculino , Huésped Inmunocomprometido , Persona de Mediana Edad , Síndrome Nefrótico/complicaciones , Inmunosupresores/uso terapéutico , Imagen por Resonancia MagnéticaRESUMEN
Accurate and reliable detection of fungal pathogens presents an important hurdle to manage infections, especially considering that fungal pathogens, including the globally important human pathogen, Cryptococcus neoformans, have adapted diverse mechanisms to survive the hostile host environment and moderate virulence determinant production during coinfections. These pathogen adaptations present an opportunity for improvements (e.g., technological and computational) to better understand the interplay between a host and a pathogen during disease to uncover new strategies to overcome infection. In this study, we performed comparative proteomic profiling of an in vitro coinfection model across a range of fungal and bacterial burden loads in macrophages. Comparing data-dependent acquisition and data-independent acquisition enabled with parallel accumulation serial fragmentation technology, we quantified changes in dual-perspective proteome remodeling. We report enhanced and novel detection of pathogen proteins with data-independent acquisition-parallel accumulation serial fragmentation (DIA-PASEF), especially for fungal proteins during single and dual infection of macrophages. Further characterization of a fungal protein detected only with DIA-PASEF uncovered a novel determinant of fungal virulence, including altered capsule and melanin production, thermotolerance, and macrophage infectivity, supporting proteomics advances for the discovery of a novel putative druggable target to suppress C. neoformans pathogenicity.
Asunto(s)
Cryptococcus neoformans , Proteínas Fúngicas , Macrófagos , Proteómica , Cryptococcus neoformans/patogenicidad , Proteómica/métodos , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Virulencia , Macrófagos/microbiología , Macrófagos/metabolismo , Criptococosis/microbiología , Humanos , Proteoma/análisis , Proteoma/metabolismo , Melaninas/metabolismo , Melaninas/biosíntesis , Animales , Interacciones Huésped-Patógeno , Factores de Virulencia/metabolismo , RatonesRESUMEN
Cryptococcosis is a fungal infection that is becoming increasingly prevalent worldwide, particularly among individuals with compromised immune systems, such as HIV patients. Amphotericin B (AmB) is the first-line treatment mainly combined with flucytosine. The scarcity and the prohibitive cost of this regimen urge the use of fluconazole as an alternative, leading to increased rates of treatment failure and relapses. Therefore, there is a critical need for efficient and cost-effective therapy to enhance the efficacy of AmB. In this study, we evaluated the efficacy of the HIV protease inhibitors (PIs) to synergize the activity of AmB in the treatment of cryptococcosis. Five PIs (ritonavir, atazanavir, saquinavir, lopinavir, and nelfinavir) were found to synergistically potentiate the killing activity of AmB against Cryptococcus strains with Æ©FICI ranging between 0.09 and 0.5 against 20 clinical isolates. This synergistic activity was further confirmed in a time-kill assay, where different AmB/PIs combinations exhibited fungicidal activity within 24 hrs. Additionally, PIs in combination with AmB exhibited an extended post-antifungal effect on treated cryptococcal cells for approximately 10 hrs compared to 4 hours with AmB alone. This promising activity against cryptococcal cells did not exhibit increased cytotoxicity towards treated kidney cells, ruling out the risk of drug combination-induced nephrotoxicity. Finally, we evaluated the efficacy of AmB/PIs combinations in the Caenorhabditis elegans model of cryptococcosis, where these combinations significantly reduced the fungal burden of the treated nematodes by approximately 2.44 Log10 CFU (92.4%) compared to the untreated worms and 1.40 Log10 ((39.4%) compared to AmB alone. The cost-effectiveness and accessibility of PIs in resource-limited geographical areas compared to other antifungal agents, such as flucytosine, make them an appealing choice for combination therapy.
Asunto(s)
Anfotericina B , Antifúngicos , Criptococosis , Sinergismo Farmacológico , Inhibidores de la Proteasa del VIH , Anfotericina B/farmacología , Anfotericina B/uso terapéutico , Antifúngicos/farmacología , Antifúngicos/uso terapéutico , Inhibidores de la Proteasa del VIH/uso terapéutico , Inhibidores de la Proteasa del VIH/farmacología , Animales , Criptococosis/tratamiento farmacológico , Humanos , Caenorhabditis elegans/microbiología , Caenorhabditis elegans/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Cryptococcus neoformans/efectos de los fármacos , Quimioterapia Combinada , Ritonavir/uso terapéutico , Ritonavir/farmacología , Cryptococcus/efectos de los fármacosRESUMEN
Cryptococcus neoformans is a widely distributed opportunistic pathogenic fungus. While C. neoformans commonly infects immunocompromised individuals, it can also affect those who are immunocompetent. Transmission of C. neoformans primarily occurs through the respiratory tract, leading to the development of meningitis. The mortality rate of Cryptococcal meningitis is high, and treatment options are limited. Cryptococcus neoformans infections pose a significant public health threat and currently lack targeted and effective response strategies. This study aimed to screen T lymphocyte (cytotoxic T lymphocyte and helper T lymphocyte) and B lymphocyte epitopes derived from four C. neoformans antigens and develop two multi-epitope vaccines by combining them with various adjuvants. Molecular docking results demonstrated that the vaccines bind stably to Toll-like receptor 4 ( and induce innate immunity. The credibility of the molecular docking results was validated through subsequent molecular dynamics simulations. Furthermore, the results of immune simulation analyses underscored the multi-epitope vaccine's capability to effectively induce robust humoral and cellular immune responses within the host organism. These two vaccines have demonstrated theoretical efficacy against C. neoformans infection as indicated by computer analysis. Nevertheless, additional experimental validation is essential to substantiate the protective efficacy of the vaccines.
A multi-epitope Cryptococcus neoformans vaccine covering the most common A and D phenotypes was designed using bioinformatics methods.
Asunto(s)
Biología Computacional , Cryptococcus neoformans , Epítopos de Linfocito B , Epítopos de Linfocito T , Vacunas Fúngicas , Simulación del Acoplamiento Molecular , Cryptococcus neoformans/inmunología , Cryptococcus neoformans/química , Vacunas Fúngicas/inmunología , Epítopos de Linfocito T/inmunología , Epítopos de Linfocito B/inmunología , Humanos , Criptococosis/inmunología , Criptococosis/prevención & control , Receptor Toll-Like 4/inmunología , Antígenos Fúngicos/inmunología , Simulación de Dinámica Molecular , Adyuvantes Inmunológicos , InmunoinformáticaRESUMEN
Cryptococcus neoformans (Cn) is an opportunistic yeast that causes meningoencephalitis in immunocompromised individuals. Calorie restriction (CR) prolongs Cn replicative lifespan (RLS) and mimics low-glucose environments in which Cn resides during infection. The effects of CR-mediated stress can differ among strains and have only been studied in MATα cells. Cn replicates sexually, generating two mating types, MATα and MATa. MATα strains are more dominant in clinical and environmental isolates. We sought to compare the effects of CR stress and longevity regulation between congenic MATα and MATa. Although MATα and MATa cells extended their RLS in response to CR, they engaged different pathways. The sirtuins were upregulated in MATα cells under CR, but not in MATa cells. RLS extension was SIR2-dependent in KN99α, but not in KN99a. The TOR nutrient-sensing pathway was downregulated in MATa strains under CR, while MATα strains demonstrated no difference. Lower oxidative stress and higher ATP production were observed in KN99α cells, possibly due to higher SOD expression. SIR2 was important for mitochondrial morphology and function in both mating types. Increased ATP production during CR powered the upregulated ABC transporters, increasing efflux in MATα cells. This led to enhanced fluconazole tolerance, while MATa cells remained sensitive to fluconazole. Our investigation highlights differences in the response of the mating types to CR.
Asunto(s)
Restricción Calórica , Cryptococcus neoformans , Genes del Tipo Sexual de los Hongos , Cryptococcus neoformans/fisiología , Cryptococcus neoformans/genética , Estrés Oxidativo , Regulación Fúngica de la Expresión Génica , Adenosina Trifosfato/metabolismoRESUMEN
BACKGROUND: Cryptococcosis is an infectious disease caused by encapsulated heterobasidiomycete yeasts. As an opportunistic pathogen, cryptococcal inhalation infection is the most common. While Primary cutaneous cryptococcosis is extremely uncommon. CASE PRESENTATION: A 61-year-old woman with a history of rheumatoid arthritis on long-term prednisone developed a red plaque on her left thigh. Despite initial antibiotic treatment, the erythema worsened, leading to rupture and fever. Microbiological analysis of the lesion's secretion revealed Candida albicans, Pseudomonas aeruginosa, and methicillin-resistant Staphylococcus epidermidis. Skin biopsy showed thick-walled spores, and culture confirmed primary cutaneous infection with Cryptococcus neoformans. Histopathological stains were positive, and mass spectrometry identified serotype A of the pathogen. The patient was treated with oral fluconazole and topical nystatin, resulting in significant improvement and near-complete healing of the skin lesion within 2.5 months. CONCLUSIONS: Primary cutaneous cryptococcosis was a primary skin infection exclusively located on the skin. It has no typical clinical manifestation of cutaneous infection of Cryptococcus, and culture and histopathology remain the gold standard for diagnosing. The recommended medication for Primary cutaneous cryptococcosis is fluconazole. When patients at risk for opportunistic infections develop skin ulcers that are unresponsive to antibiotic, the possibility of primary cutaneous cryptococcosis needs to be considered.
Asunto(s)
Antifúngicos , Criptococosis , Cryptococcus neoformans , Fluconazol , Humanos , Femenino , Persona de Mediana Edad , Cryptococcus neoformans/aislamiento & purificación , Cryptococcus neoformans/efectos de los fármacos , Criptococosis/tratamiento farmacológico , Criptococosis/microbiología , Criptococosis/diagnóstico , Criptococosis/patología , Antifúngicos/uso terapéutico , Fluconazol/uso terapéutico , Dermatomicosis/tratamiento farmacológico , Dermatomicosis/microbiología , Dermatomicosis/diagnóstico , Dermatomicosis/patología , Piel/patología , Piel/microbiología , Resultado del Tratamiento , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/complicacionesRESUMEN
Cryptococcus neoformans is an opportunistic pathogenic fungus that produces melanin during infection, an important virulence factor in Cryptococcal infections that enhances the ability of the fungus to resist immune defense. This fungus can synthesize melanin from a variety of substrates, including L-DOPA (L-3,4-dihydroxyphenylalanine). Since melanin protects the fungus from various stress factors such as oxidative, nitrosative, extreme heat and cold stress; we investigated the effects of environmental conditions on melanin production and survival. In this study, we investigated the effects of different pH values (5.6, 7.0 and 8.5) and temperatures (30 °C and 37 °C) on melanization and cell survival using a microtiter plate-based melanin production assay and an oxidative stress assay, respectively. In addition, the efficacy of compounds known to inhibit laccase involved in melanin synthesis, i.e., tunicamycin, ß-mercaptoethanol, dithiothreitol, sodium azide and caspofungin on melanization was evaluated and their sensitivity to temperature and pH changes was measured. The results showed that melanin content correlated with pH and temperature changes and that pH 8.5 and 30 °C, were best for melanin production. Besides that, melanin production protects the fungal cells from oxidative stress induced by hydrogen peroxide. Thus, changes in pH and temperature drastically alter melanin production in C. neoformans and it correlates with the fungal survival. Due to the limited antifungal repertoire and the development of resistance in cryptococcal infections, the investigation of environmental conditions in the regulation of melanization and survival of C. neoformans could be useful for future research and clinical phasing.
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Cryptococcus neoformans , Melaninas , Estrés Oxidativo , Temperatura , Cryptococcus neoformans/metabolismo , Cryptococcus neoformans/efectos de los fármacos , Melaninas/metabolismo , Concentración de Iones de Hidrógeno , Peróxido de Hidrógeno/metabolismo , Lacasa/metabolismo , Tunicamicina/farmacología , Caspofungina/farmacología , Azida Sódica/farmacología , Mercaptoetanol/farmacología , Ditiotreitol/farmacología , Criptococosis/microbiología , Viabilidad Microbiana/efectos de los fármacos , Lipopéptidos/farmacología , Lipopéptidos/metabolismoRESUMEN
The fungal pathogen Cryptococcus neoformans is well adapted to its host environment. It has several defence mechanisms to evade oxidative and nitrosative agents released by phagocytic host cells during infection. Among them, melanin production is linked to both fungal virulence and defence against harmful free radicals that facilitate host innate immunity. How C. neoformans manipulates its redox environment to facilitate melanin formation and virulence is unclear. Here we show that the antioxidant glutathione is inextricably linked to redox-active processes that facilitate melanin and titan cell production, as well as survival in macrophages and virulence in a murine model of cryptococcosis. Comparative metabolomics revealed that disruption of glutathione biosynthesis leads to accumulation of reducing and acidic compounds in the extracellular environment of mutant cells. Overall, these findings highlight the importance of redox homeostasis and metabolic compensation in pathogen adaptation to the host environment and suggest new avenues for antifungal drug development.
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Criptococosis , Cryptococcus neoformans , Glutatión , Macrófagos , Melaninas , Oxidación-Reducción , Cryptococcus neoformans/patogenicidad , Cryptococcus neoformans/metabolismo , Cryptococcus neoformans/genética , Animales , Glutatión/metabolismo , Virulencia , Ratones , Criptococosis/microbiología , Melaninas/metabolismo , Melaninas/biosíntesis , Macrófagos/microbiología , Macrófagos/metabolismo , Macrófagos/inmunología , Modelos Animales de Enfermedad , Regulación Fúngica de la Expresión Génica , FemeninoRESUMEN
BACKGROUND: Cryptococcosis is a life-threatening disease caused by Cryptococcus neoformans or C. gattii. Neutralizing autoantibodies (auto-Abs) against granulocyte-macrophage colony-stimulating factor (GM-CSF) in otherwise healthy adults with cryptococcal meningitis have been described since 2013. We searched for neutralizing auto-Abs in sera collected from Colombian patients with non-HIV-associated cryptococcosis in a retrospective national cohort from 1997 to 2016. METHODS: We reviewed clinical and laboratory records and assessed the presence of neutralizing auto-Abs against GM-CSF in 30 HIV negative adults with cryptococcosis (13 caused by C. gattii and 17 caused by C. neoformans). RESULTS: We detected neutralizing auto-Abs against GM-CSF in the sera of 10 out of 13 (77%) patients infected with C. gattii and one out of 17 (6%) patients infected with C. neoformans. CONCLUSIONS: We report eleven Colombian patients diagnosed with cryptococcosis who had auto-Abs that neutralize GM-CSF. Among these patients, ten were infected with C. gattii and only one with C. neoformans.
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Anticuerpos Neutralizantes , Autoanticuerpos , Criptococosis , Cryptococcus gattii , Cryptococcus neoformans , Factor Estimulante de Colonias de Granulocitos y Macrófagos , Humanos , Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Autoanticuerpos/sangre , Autoanticuerpos/inmunología , Masculino , Colombia , Femenino , Adulto , Cryptococcus gattii/inmunología , Persona de Mediana Edad , Cryptococcus neoformans/inmunología , Criptococosis/inmunología , Criptococosis/diagnóstico , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Estudios Retrospectivos , Seronegatividad para VIH/inmunología , Adulto Joven , AncianoRESUMEN
The increasing prevalence of invasive fungal pathogens is dramatically changing the clinical landscape of infectious diseases, posing an imminent threat to public health. Specifically, Cryptococcus neoformans, the human opportunistic pathogen, expresses elaborate virulence mechanisms and is equipped with sophisticated adaptation strategies to survive in harsh host environments. This study extensively characterizes Wos2, an Hsp90 co-chaperone homolog, featuring bilateral functioning for both cryptococcal adaptation and the resulting virulence response. In this study, we evaluated the proteome and secretome signatures associated with wos2 deletion in enriched and infection-mimicking conditions to reveal Wos2-dependent regulation of the oxidative stress response through global translational reprogramming. The wos2Δ strain demonstrates defective intracellular and extracellular antioxidant protection systems, measurable through a decreased abundance of critical antioxidant enzymes and reduced growth in the presence of peroxide stress. Additional Wos2-associated stress phenotypes were observed upon fungal challenge with heat shock, osmotic stress, and cell membrane stressors. We demonstrate the importance of Wos2 for intracellular lifestyle of C. neoformans during in vitro macrophage infection and provide evidence for reduced phagosomal replication levels associated with wos2Δ. Accordingly, wos2Δ featured significantly reduced virulence within impacting fungal burden in a murine model of cryptococcosis. Our study highlights a vulnerable point in the fungal chaperone network that offers a therapeutic opportunity to interfere with both fungal virulence and fitness.IMPORTANCEThe global impact of fungal pathogens, both emerging and emerged, is undeniable, and the alarming increase in antifungal resistance rates hampers our ability to protect the global population from deadly infections. For cryptococcal infections, a limited arsenal of antifungals and increasing rates of resistance demand alternative therapeutic strategies, including an anti-virulence approach, which disarms the pathogen of critical virulence factors, empowering the host to remove the pathogens and clear the infection. To this end, we apply state-of-the-art mass spectrometry-based proteomics to evaluate the impact of a recently defined novel co-chaperone, Wos2, toward cryptococcal virulence using in vitro and in vivo models of infection. We explore global proteome and secretome remodeling driven by the protein and uncover the novel role in modulating the fungal oxidative stress response. Complementation of proteome findings with in vitro infectivity assays demonstrated the protective role of Wos2 within the macrophage phagosome, influencing fungal replication and survival. These results underscore differential cryptococcal survivability and weakened patterns of dissemination in the absence of wos2. Overall, our study establishes Wos2 as an important contributor to fungal pathogenesis and warrants further research into critical proteins within global stress response networks as potential druggable targets to reduce fungal virulence and clear infection.