RESUMO
The environmental yeast Cryptococcus neoformans is the most common cause of deadly fungal meningitis in primarily immunocompromised populations. A number of factors contribute to cryptococcal pathogenesis. Among them, inositol utilization has been shown to promote C. neoformans development in nature and invasion of central nervous system during dissemination. The mechanisms of the inositol regulation of fungal virulence remain incompletely understood. In this study, we analyzed inositol-induced capsule growth and the contribution of a unique inositol catabolic pathway in fungal development and virulence. We found that genes involved in the inositol catabolic pathway are highly induced by inositol, and they are also highly expressed in the cerebrospinal fluid of patients with meningoencephalitis. This pathway in C. neoformans contains three genes encoding myo-inositol oxygenases that convert myo-inositol into d-glucuronic acid, a substrate of the pentose phosphate cycle and a component of the polysaccharide capsule. Our mutagenesis analysis demonstrates that inositol catabolism is required for C. neoformans virulence and deletion mutants of myo-inositol oxygenases result in altered capsule growth as well as the polysaccharide structure, including O-acetylation. Our study indicates that the ability to utilize the abundant inositol in the brain may contribute to fungal pathogenesis in this neurotropic fungal pathogen. IMPORTANCE The human pathogen Cryptococcus neoformans is the leading cause of fungal meningitis in primarily immunocompromised populations. Understanding how this environmental organism adapts to the human host to cause deadly infection will guide our development of novel disease control strategies. Our recent studies revealed that inositol utilization by the fungus promotes C. neoformans development in nature and invasion of the central nervous system during infection. The mechanisms of the inositol regulation in fungal virulence remain incompletely understood. In this study, we found that C. neoformans has three genes encoding myo-inositol oxygenase, a key enzyme in the inositol catabolic pathway. Expression of these genes is highly induced by inositol, and they are highly expressed in the cerebrospinal fluid of patients with meningoencephalitis. Our mutagenesis analysis indeed demonstrates that inositol catabolism is required for C. neoformans virulence by altering the growth and structure of polysaccharide capsule, a major virulence factor. Considering the abundance of free inositol and inositol-related metabolites in the brain, our study reveals an important mechanism of host inositol-mediated fungal pathogenesis for this neurotropic fungal pathogen.
Assuntos
Cryptococcus neoformans/metabolismo , Cryptococcus neoformans/patogenicidade , Cápsulas Fúngicas/química , Inositol/metabolismo , Meningite Criptocócica/microbiologia , Animais , Encéfalo/metabolismo , Encéfalo/microbiologia , Cryptococcus neoformans/química , Cryptococcus neoformans/genética , Feminino , Cápsulas Fúngicas/genética , Cápsulas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Humanos , Masculino , Meningite Criptocócica/metabolismo , Camundongos , Oxigenases/genética , Oxigenases/metabolismo , Coelhos , VirulênciaRESUMO
Background: Cryptococcal meningitis has high mortality. Flucytosine is a key treatment but is expensive and rarely available. The anticancer agent tamoxifen has synergistic anti-cryptococcal activity with amphotericin in vitro. It is off-patent, cheap, and widely available. We performed a trial to determine its therapeutic potential. Methods: Open label randomized controlled trial. Participants received standard care - amphotericin combined with fluconazole for the first 2 weeks - or standard care plus tamoxifen 300 mg/day. The primary end point was Early Fungicidal Activity (EFA) - the rate of yeast clearance from cerebrospinal fluid (CSF). Trial registration https://clinicaltrials.gov/ct2/show/NCT03112031. Results: Fifty patients were enrolled (median age 34 years, 35 male). Tamoxifen had no effect on EFA (-0.48log10 colony-forming units/mL/CSF control arm versus -0.49 tamoxifen arm, difference -0.005log10CFU/ml/day, 95% CI: -0.16, 0.15, p=0.95). Tamoxifen caused QTc prolongation. Conclusions: High-dose tamoxifen does not increase the clearance rate of Cryptococcus from CSF. Novel, affordable therapies are needed. Funding: The trial was funded through the Wellcome Trust Asia Programme Vietnam Core Grant 106680 and a Wellcome Trust Intermediate Fellowship to JND grant number WT097147MA.
Assuntos
Anfotericina B/uso terapêutico , Cryptococcus neoformans/efeitos dos fármacos , Fluconazol/uso terapêutico , Meningite Criptocócica/tratamento farmacológico , Tamoxifeno/uso terapêutico , Adulto , Antifúngicos/uso terapêutico , Quimioterapia Combinada , Feminino , Humanos , Síndrome do QT Longo/induzido quimicamente , Masculino , Meningite Criptocócica/líquido cefalorraquidiano , Meningite Criptocócica/metabolismo , Tamoxifeno/efeitos adversosRESUMO
Cryptococcal meningitis (CM) is the leading cause of mortality among patients infected with human immunodeficiency virus (HIV). Although treatment strategies for CM are continually being developed, the mortality rate is still high. Therefore, we need to explore more therapeutic strategies that are aimed at hindering its pathogenic mechanism. In the field of CM, several studies have observed rapid iron accumulation and lipid peroxidation within the brain, all of which are hallmarks of ferroptosis, which is a type of programmed cell death that is characterized by iron dependence and lipid peroxidation. In recent years, many studies have confirmed the involvement of ferroptosis in many diseases, including infectious diseases such as Mycobacterium tuberculosis infection and coronavirus disease-2019 (COVID-19). Furthermore, ferroptosis is considered as immunogenic and pro-inflammatory as the ferroptotic cells release damage-associated molecular pattern molecules (DAMPs) and alarmin, both of which regulate immunity and pro-inflammatory activity. Hence, we hypothesize that there might be a relationship between this unique cell death modality and CM. Herein, we review the evidence of ferroptosis in CM and consider the hypothesis that ferroptotic cell death may be involved in the cell death of CM.
Assuntos
COVID-19/metabolismo , Ferroptose , Ferro/metabolismo , Peroxidação de Lipídeos , Meningite Criptocócica/metabolismo , Tuberculose/metabolismo , COVID-19/imunologia , COVID-19/patologia , Ferroptose/imunologia , Glutationa/metabolismo , Humanos , Inflamação/imunologia , Metabolismo dos Lipídeos , Meningite Criptocócica/imunologia , Meningite Criptocócica/patologia , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Tuberculose/imunologia , Tuberculose/patologiaRESUMO
HIV-1 compartmentalization in reservoir sites remains a barrier to complete HIV eradication. It is unclear whether there is variation in HIV-1 env and gag between cerebrospinal fluid (CSF) and plasma of individuals with HIV-associated cryptococcal meningitis (CM). We compared HIV-1 env characteristics and the gag cytotoxic T-lymphocyte (CTL) escape mutations from CSF and plasma samples. Employing population-based Sanger sequencing, we sequenced HIV-1 env from CSF of 25 patients and plasma of 26 patients. For gag, 15 CSF and 21 plasma samples were successfully sequenced. Of these, 18 and 9 were paired env and gag CSF/plasma samples, respectively. There was no statistically significant difference in the proportion of CCR5-using strains in the CSF and plasma, (p = 0.50). Discordant CSF/plasma virus co-receptor use was found in 2/18 pairs (11.1%). The polymorphisms in the HIV-1 V3 loop were concordant between the two compartments. From the HIV-1 gag sequences, three pairs had discordant CTL escape mutations in three different epitopes of the nine analyzed. These findings suggest little variation in the HIV-1 env between plasma and CSF and that the CCR5-using strains predominate in both compartments. HIV-1 gag CTL escape mutations also displayed little variation in CSF and plasma suggesting similar CTL selective pressure.
Assuntos
Infecções Oportunistas Relacionadas com a AIDS/metabolismo , Infecções por HIV/complicações , Meningite Criptocócica/etiologia , Meningite Criptocócica/metabolismo , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Infecções Oportunistas Relacionadas com a AIDS/sangue , Infecções Oportunistas Relacionadas com a AIDS/líquido cefalorraquidiano , Infecções Oportunistas Relacionadas com a AIDS/diagnóstico , Adulto , Sequência de Aminoácidos , Substituição de Aminoácidos , Botsuana , Contagem de Linfócito CD4 , Estudos Transversais , Suscetibilidade a Doenças , Feminino , Infecções por HIV/virologia , Humanos , Hospedeiro Imunocomprometido , Masculino , Meningite Criptocócica/sangue , Meningite Criptocócica/líquido cefalorraquidiano , Pessoa de Meia-Idade , Mutação , RNA Viral , Carga Viral , Produtos do Gene env do Vírus da Imunodeficiência Humana/sangue , Produtos do Gene env do Vírus da Imunodeficiência Humana/líquido cefalorraquidiano , Produtos do Gene gag do Vírus da Imunodeficiência Humana/sangue , Produtos do Gene gag do Vírus da Imunodeficiência Humana/líquido cefalorraquidianoRESUMO
The incidence of infectious diseases affecting the central nervous system (CNS) has been increasing over the last several years. Among the reasons for the expansion of these diseases and the appearance of new neuropathogens are globalization, global warming, and the increased proximity between humans and wild animals due to human activities such as deforestation. Neurotropism affecting normal brain function is shared by organisms such as viruses, bacteria, fungi, and parasites. Neuroinfections caused by these agents activate immune responses, inducing neuroinflammation, excitotoxicity, and neurodegeneration. Purinergic signaling is an evolutionarily conserved signaling pathway associated with these neuropathologies. During neuroinfections, host cells release ATP as an extracellular danger signal with pro-inflammatory activities. ATP is metabolized to its derivatives by ectonucleotidases such as CD39 and CD73; ATP and its metabolites modulate neuronal and immune mechanisms through P1 and P2 purinergic receptors that are involved in pathophysiological mechanisms of neuroinfections. In this review we discuss the beneficial or deleterious effects of various components of the purinergic signaling pathway in infectious diseases that affect the CNS, including human immunodeficiency virus (HIV-1) infection, herpes simplex virus type 1 (HSV-1) infection, bacterial meningitis, sepsis, cryptococcosis, toxoplasmosis, and malaria. We also provide a description of this signaling pathway in emerging viral infections with neurological implications such as Zika and SARS-CoV-2.
Assuntos
Infecções do Sistema Nervoso Central/metabolismo , Receptores Purinérgicos P1/metabolismo , Receptores Purinérgicos P2X/metabolismo , Receptores Purinérgicos P2Y/metabolismo , Complexo AIDS Demência/metabolismo , Betacoronavirus , COVID-19 , Infecções por Coronavirus/metabolismo , Encefalite por Herpes Simples/metabolismo , Humanos , Malária/metabolismo , Meningites Bacterianas/metabolismo , Meningite Criptocócica/metabolismo , Pandemias , Pneumonia Viral/metabolismo , SARS-CoV-2 , Sepse/metabolismo , Transdução de Sinais , Toxoplasmose Cerebral/metabolismo , Infecção por Zika virus/metabolismoRESUMO
BACKGROUND: Cryptococcal meningitis is most commonly found in HIV-infected patients. In HIV-negative patients, its low incidence can lead to prolonged time to diagnosis. Detailed case reports of chronic cryptococcal meningitis are scarce, but could provide clues for earlier diagnosis in this patient category. CASE PRESENTATION: A 60-year old man presented June 2015 with intermittent headaches for several months without any fever. Initial work-up showed a leukocytosis, raised CSF opening pressure and raised leukocytes and protein in the CSF. An MRI revealed leptomeningeal contrast enhancement and cerebellar oedema. While malignancy and various infectious causes were excluded, the patient had a spontaneous clinical and radiological recovery. One year later, the patient returned with complaints of headaches. Also, cerebellar oedema and leptomeningeal contrast enhancement had recurred. Eventually in March 2017, the novel cryptococcal antigen lateral flow assay (CrAg LFA) was positive on CSF, and one colony of Cryptococcus neoformans was cultured from CSF. The patient was treated with the standard antifungal regimen which resulted in resolution of his headaches. In retrospect, the cryptococcal antigen test was already positive on a serum sample from June 2015. Interestingly, post-treatment immunological analysis revealed both a low mannose-binding lectin (MBL) concentration and low naïve CD4 counts. CONCLUSIONS: We present a patient with cryptococcal meningitis in an HIV-negative patient with low MBL and low naïve CD4 count suffering a chronic relapsing meningo-encephalitis with relatively mild symptoms for around 2 years. In patients with an unexplained meningo-encephalitis such as this case, early performance of CrAg LFA on serum and/or CSF is an inexpensive and rapid method to reduce time-to diagnosis.
Assuntos
Linfócitos T CD4-Positivos/citologia , Lectina de Ligação a Manose/metabolismo , Meningite Criptocócica/diagnóstico , Antifúngicos/uso terapêutico , Antígenos de Fungos/líquido cefalorraquidiano , Contagem de Linfócito CD4 , Linfócitos T CD4-Positivos/metabolismo , Cryptococcus neoformans/isolamento & purificação , Humanos , Imageamento por Ressonância Magnética , Masculino , Meningite Criptocócica/imunologia , Meningite Criptocócica/metabolismo , Pessoa de Meia-Idade , RecidivaRESUMO
AIMS: LncRNAs play a vital role in the pathological and physiological process. This study aimed to explore the involvement of lncRNAs in cryptococcal meningitis. METHODS: Microarray was performed in cryptococcal meningitis patients, and then, GO and KEGG pathways were analyzed. Coexpression relationship between lncRNA and mRNA was explored. The expressions of the lncRNAs and mRNAs, and their changes after treatment were detected by PCR. RESULTS: A total of 325 mRNAs (201 upregulated and 124 downregulated) and 497 lncRNAs (263 upregulated and 234 downregulated) were identified. The top three enriched GO terms for the mRNAs were arachidonic acid binding, activin receptor binding, and replication fork protection complex. The top three pathways in KEGG were asthma, one carbon pool by folate, and allograft rejection. A total of 305 coexpression relationships were found between 108 lncRNAs and 87 mRNAs. LncRNA-DPY19L1p1 was significantly increased in patients and decreased after treatment. ROC analysis revealed DPY19L1p1 was a potential diagnostic marker (AUCROC = 0.9389). Furthermore, the target genes of DPY19L1p1 in cis or trans regulation were mainly involved in immune-related pathways like the interleukin signaling pathway. CONCLUSIONS: This study analyzed the differential lncRNA profile in cryptococcal meningitis patients and revealed DPY19L1p1 could be used for treatment evaluation and disease diagnosis.
Assuntos
Meningite Criptocócica/metabolismo , RNA Longo não Codificante/metabolismo , Adulto , Biomarcadores/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Análise em Microsséries , Pessoa de Meia-Idade , RNA Mensageiro/metabolismo , Adulto JovemRESUMO
PURPOSE: Serum cytokines/chemokines play important roles in cryptococcal meningitis, but it is unclear whether cytokines/chemokines in cerebrospinal fluid (CSF) contribute to high intracranial pressure (HICP) in HIV-associated cryptococcal meningitis (HCM). METHODS: CSF cytokines/chemokines were assayed in 17 HIV-uninfected patients, 26 HIV-infected patients without CNS infection, and 39 HCM patients at admission. Principal component analysis and correlation and logistic regression analyses were used to assess the relationships between these parameters. RESULTS: The CSF Th1, Th2, and macrophage cytokines showed an obvious increase in HCM patients as compared to the HIV-uninfected patients and HIV-infected patients without CNS infection. CSF IL-6, GM-CSF, and IL-8 were positively correlated with CSF fungal burden. Serum CD4 count, CSF Th1 cytokines (TNF-α, TNF-ß, IL-12, IL-1ß, IL-12, IL-1α, TNF-α, TNF-ß, IL-12, IL-1γ, and IL-12) and Th2 cytokines (IL-4 and IL-10) contribute to HICP. CONCLUSION: Overall, the present findings indicated that both pro- and anti-inflammatory cytokines of Th1, Th2, and macrophage origin contributed to the development of HCM. Specifically, the chemokine and cytokine cascade caused by skewing of the Th1-Th2 balance and reduced CD4 count were found to be important contributors to HICP. Summary. Our research suggested that chemokine and cytokine cascade caused by skewing of the Th1-Th2 balance in HIV-infected patients played more important role than Cryptococcus numbers and size in CSF on the development of high intracranial pressure in HIV-associated cryptococcal meningitis, providing a new understanding of mechanisms of HCM.
Assuntos
Infecções por HIV/metabolismo , Infecções por HIV/fisiopatologia , Pressão Intracraniana/fisiologia , Meningite Criptocócica/metabolismo , Meningite Criptocócica/fisiopatologia , Equilíbrio Th1-Th2/fisiologia , Adulto , Contagem de Linfócito CD4 , Quimiocinas/metabolismo , Citocinas/metabolismo , Feminino , Infecções por HIV/genética , Humanos , Pressão Intracraniana/genética , Masculino , Meningite Criptocócica/genética , Pessoa de Meia-Idade , Equilíbrio Th1-Th2/genéticaRESUMO
There is a limited understanding of the population pharmacokinetics (PK) and pharmacodynamics (PD) of amphotericin B deoxycholate (DAmB) for cryptococcal meningitis. A PK study was conducted in n = 42 patients receiving DAmB (1 mg/kg of body weight every 24 h [q24h]). A 2-compartment PK model was developed. Patient weight influenced clearance and volume in the final structural model. Monte Carlo simulations estimated drug exposure associated with various DAmB dosages. A search was conducted for trials reporting outcomes of treatment of cryptococcal meningitis patients with DAmB monotherapy, and a meta-analysis was performed. The PK parameter means (standard deviations) were as follows: clearance, 0.03 (0.01) × weight + 0.67 (0.01) liters/h; volume, 0.82 (0.80) × weight + 1.76 (1.29) liters; first-order rate constant from central compartment to peripheral compartment, 5.36 (6.67) h-1; first-order rate constant from peripheral compartment to central compartment, 9.92 (12.27) h-1 The meta-analysis suggested that the DAmB dosage explained most of the heterogeneity in cerebrospinal fluid (CSF) sterility outcomes but not in mortality outcomes. Simulations of values corresponding to the area under concentration-time curve from h 144 to h 168 (AUC144-168) resulted in median (interquartile range) values of 5.83 mg · h/liter (4.66 to 8.55), 10.16 mg · h/liter (8.07 to 14.55), and 14.51 mg · h/liter (11.48 to 20.42) with dosages of 0.4, 0.7, and 1.0 mg/kg q24h, respectively. DAmB PK is described adequately by a linear model that incorporates weight with clearance and volume. Interpatient PK variability is modest and unlikely to be responsible for variability in clinical outcomes. There is discordance between the impact that drug exposure has on CSF sterility and its impact on mortality outcomes, which may be due to cerebral pathology not reflected in CSF fungal burden, in addition to clinical variables.
Assuntos
Anfotericina B/farmacocinética , Antifúngicos/farmacocinética , Antifúngicos/uso terapêutico , Ácido Desoxicólico/farmacocinética , Meningite Criptocócica/tratamento farmacológico , Meningite Criptocócica/metabolismo , Adulto , Idoso , Anfotericina B/líquido cefalorraquidiano , Anfotericina B/uso terapêutico , Antifúngicos/líquido cefalorraquidiano , Ácido Desoxicólico/líquido cefalorraquidiano , Ácido Desoxicólico/uso terapêutico , Combinação de Medicamentos , Feminino , Humanos , Masculino , Meningite Criptocócica/líquido cefalorraquidiano , Pessoa de Meia-Idade , Método de Monte Carlo , Estudos Prospectivos , Adulto JovemRESUMO
This study aimed to investigate the PD-1/ PD-L1 signaling pathway and its effects the activation of microglia/macrophage and balancing T cell subsets in cryptococcal meningitis (CM). A total of 126 CM patients and 126 healthy individuals were recruited for the study. The CM patients were treated with amphotericin B (AmB). Seventy five C57BL/6 mice were grouped into the normal control, CM model, CM + AmB, sham, and CM + PD-1 antibodies (Ab) groups. CD4+ and CD8+ T cells as well as microglia/macrophages were analyzed by means of flow cytometry. Ionized calcium-binding adaptor molecule 1 (Ibal) expression was detected using western blotting and immunohistochemistry techniques. And the expression of Rab5 and Rab11 were detected using an immunofluorescence assay. Both PD-1 and PD-L1 mRNA and protein expression among the mice in the study were evaluated by qRT-PCR and western blotting methods. Compared to the CM model group, the CM + AmB and CM + PD-1 Ab groups exhibited increased levels of Th1 cytokines and chemokines expression, and reduced levels of Th2 cytokines expressions. Elevated cell purity and viability of CD4+ T cell were recorded as well as increases in microglia, however, there were reductions in the number of CD8+ T cells. Depleted expressions of Ibal, Rab5, and Rab11 as well as reduced mRNA expressions of PD-1 and PD-L1 in CD4+ , microglia, and macrophage cells. The findings suggested that suppression of the PD-1/PD-L1 signaling pathway restricts the proliferation of CM by down-regulating the expressions of Th2 cells and suppressing microglia and macrophage activation.
Assuntos
Anfotericina B/administração & dosagem , Anticorpos/administração & dosagem , Antifúngicos/administração & dosagem , Antígeno B7-H1/metabolismo , Macrófagos/efeitos dos fármacos , Meningite Criptocócica/tratamento farmacológico , Microglia/efeitos dos fármacos , Receptor de Morte Celular Programada 1/metabolismo , Adolescente , Adulto , Idoso , Anfotericina B/farmacologia , Animais , Anticorpos/farmacologia , Antifúngicos/farmacologia , Antígeno B7-H1/genética , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/metabolismo , Modelos Animais de Doenças , Esquema de Medicação , Feminino , Humanos , Masculino , Meningite Criptocócica/genética , Meningite Criptocócica/imunologia , Meningite Criptocócica/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Receptor de Morte Celular Programada 1/genética , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Células Th2/efeitos dos fármacos , Células Th2/imunologia , Adulto JovemRESUMO
To make advances in the treatment of cryptococcal meningitis, it is crucial to know a given drug's free fraction that reaches the biophase. In the present study, we applied microdialysis (µD) as a tool to determine the free levels reached by voriconazole (VRC) in the brains of healthy and Cryptococcus neoformans-infected rats. The infection was induced by the intravenous (i.v.) administration of 1 × 105 CFU of yeast. The dose administered was 5 mg/kg (of body weight) of VRC, given i.v. Plasma and microdialysate samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and LC-UV methods. The free brain/free plasma ratio (fT) and population pharmacokinetic (popPK) analyses were performed to evaluate the impact of infection on PK parameters of the drug. The brain penetration ratio showed an increase on brain exposure in infected animals (fThealthy = 0.85 versus fTinfected = 1.86). The structural PK model with two compartments and Michaelis-Menten (MM) elimination describes the VRC concentration-time profile in plasma and tissue simultaneously. The covariate infection was included in volume of distribution in the peripheral compartment in healthy animals (V2) and maximum rate of metabolism (VM ). The levels reached in infected tissues were higher than the values described for MIC of VRC for Cryptococccus neoformans (0.03 to 0.5 µg ml-1), indicating its great potential to treat meningitis associated with C. neoformans.
Assuntos
Encéfalo/metabolismo , Voriconazol/farmacocinética , Voriconazol/uso terapêutico , Animais , Encéfalo/efeitos dos fármacos , Cryptococcus neoformans/efeitos dos fármacos , Cryptococcus neoformans/patogenicidade , Masculino , Meningite Criptocócica/tratamento farmacológico , Meningite Criptocócica/metabolismo , Microdiálise , Ratos , Ratos WistarRESUMO
Cryptococcus neoformans is an encapsulated basidiomycetous yeast commonly associated with pigeon droppings and soil. The opportunistic pathogen infects humans through the respiratory system and the metabolic implications of C. neoformans infection have yet to be explored. Studying the metabolic profile associated with the infection could lead to the identification of important metabolites associated with pulmonary infection. Therefore, the aim of the study was to simulate cryptococcal infection at the primary site of infection, the lungs, and to identify the metabolic profile and important metabolites associated with the infection at low and high multiplicity of infections (MOI). The culture supernatant of lung epithelial cells infected with C. neoformans at MOI of 10 and 100 over a period of 18 hours were analysed using gas chromatography mass spectrometry. The metabolic profiles obtained were further analysed using multivariate analysis and the pathway analysis tool, MetaboAnalyst 2.0. Based on the results from the multivariate analyses, ten metabolites were selected as the discriminatory metabolites that were important in both the infection conditions. The pathways affected during early C. neoformans infection of lung epithelial cells were mainly the central carbon metabolism and biosynthesis of amino acids. Infection at a higher MOI led to a perturbance in the ß-alanine metabolism and an increase in the secretion of pantothenic acid into the growth media. Pantothenic acid production during yeast infection has not been documented and the ß-alanine metabolism as well as the pantothenate and CoA biosynthesis pathways may represent underlying metabolic pathways associated with disease progression. Our study suggested that ß-alanine metabolism and the pantothenate and CoA biosynthesis pathways might be the important pathways associated with cryptococcal infection.
Assuntos
Adenocarcinoma/metabolismo , Cryptococcus neoformans/fisiologia , Neoplasias Pulmonares/metabolismo , Meningite Criptocócica/metabolismo , Metabolômica , Adenocarcinoma/microbiologia , Adenocarcinoma/patologia , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Técnicas In Vitro , Neoplasias Pulmonares/microbiologia , Neoplasias Pulmonares/patologia , Meningite Criptocócica/microbiologia , Meningite Criptocócica/patologia , Células Tumorais CultivadasRESUMO
Cryptococcal meningitis (CM) is a global disease with significant morbidity and mortality. Although low peripheral blood cluster of differentiation 4 (CD4) cell counts are found to be related to a high burden of cryptococcus in HIV-infected patients, little is known about possible immune defects in previously healthy patients (PHPs). We performed a retrospective study of 41 CM patients treated from January 2005 to December 2014 who did not have HIV-infection. There were 33 PHPs and 8 not previously healthy patients (non-PHPs). We analyzed clinical test data pertaining to peripheral blood T cells, antibodies, inflammation markers, and cerebral spinal fluid (CSF) completed during the disease onset phase and 5 years following diagnosis. PHPs had significantly higher counts of cluster of differentiation 3 (CD3), cluster of differentiation 4 (CD4), and cluster of differentiation 45 (CD45) cells, and lower percentages of CD8 cells than non-PHPs (Pâ<â0.05). Measurements of inflammatory markers and immunoglobulin in blood were comparable except for lower immunoglobulin A (IgA) levels in non-PHPs (Pâ=â0.0410). Examination of CSF revealed lower white blood cell (WBC) counts in non-PHPs. Five-year mortality in PHPs was higher than in non-PHPs (22.0% vs 12.5%) but this was not statistically significant (Pâ>â0.05). Multivariate analysis revealed that higher immunoglobulin G (IgG) levels in serum during disease onset may be an independent predictor of mortality (Pâ=â0.015). In conclusion, PHPs demonstrate an immunophenotype that is distinct from that of non-PHPs, leading to an improved understanding of the immunology of cryptococcal meningitis.
Assuntos
Meningite Criptocócica/imunologia , Adolescente , Adulto , Idoso , Biomarcadores/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Criança , Pré-Escolar , Feminino , Humanos , Imunoglobulina G/sangue , Contagem de Linfócitos , Masculino , Meningite Criptocócica/metabolismo , Meningite Criptocócica/mortalidade , Pessoa de Meia-Idade , Fenótipo , Estudos Retrospectivos , Adulto JovemRESUMO
Cryptococcal meningitis is the most common opportunistic fungal infection causing morbidity and mortality (>60%) in HIV-associated immunocompromised individuals caused by Cryptococcus neoformans. Molecular mechanisms of cryptococcal infection in brain have been studied using experimental animal models and cell lines. There are limited studies for the molecular understanding of cryptococcal meningitis in human brain. The proteins involved in the process of invasion and infection in human brain still remains obscure. To this end we carried out mass spectrometry-based quantitative proteomics of frontal lobe brain tissues from cryptococcal meningitis patients and controls to identify host proteins that are associated with the pathogenesis of cryptococcal meningitis. We identified 317 proteins to be differentially expressed (≥2-fold) from a total of 3423 human proteins. We found proteins involved in immune response and signal transduction to be differentially expressed in response to cryptococcal infection in human brain. Immune response proteins including complement factors, major histocompatibility proteins, proteins previously known to be involved in fungal invasion to brain such as caveolin 1 and actin were identified to be differentially expressed in cryptococcal meningitis brain tissues co-infected with HIV. We also validated the expression status of 5 proteins using immunohistochemistry. Overexpression of major histocompatibility complexes, class I, B (HLA-B), actin alpha 2 smooth muscle aorta (ACTA2) and caveolin 1 (CAV1) and downregulation of peripheral myelin protein 2 (PMP2) and alpha crystallin B chain (CRYAB) in cryptococcal meningitis were confirmed by IHC-based validation experiments. This study provides the brain proteome profile of cryptococcal meningitis co-infected with HIV for a better understanding of the host response associated with the disease.
Assuntos
Coinfecção , Cryptococcus neoformans/fisiologia , Infecções por HIV/metabolismo , Interações Hospedeiro-Patógeno , Meningite Criptocócica/metabolismo , Proteoma , Proteômica , Biologia Computacional/métodos , Humanos , Imuno-Histoquímica , Meningite Criptocócica/genética , Meningite Criptocócica/microbiologia , Anotação de Sequência Molecular , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas , Proteômica/métodos , Reprodutibilidade dos Testes , Espectrometria de Massas em TandemRESUMO
Cryptococcus neoformans is an opportunistic yeast that kills over 625,000 people yearly through lethal meningitis. Host phagocytes serve as the first line of defense against this pathogen, but fungal engulfment and subsequent intracellular proliferation also correlate with poor patient outcome. Defining the interactions of this facultative intracellular pathogen with host phagocytes is key to understanding the latter's opposing roles in infection and how they contribute to fungal latency, dissemination, and virulence. We used high-content imaging and a human monocytic cell line to screen 1,201 fungal mutants for strains with altered host interactions and identified multiple genes that influence fungal adherence and phagocytosis. One of these genes was PFA4, which encodes a protein S-acyl transferase (PAT), one of a family of DHHC domain-containing proteins that catalyzes lipid modification of proteins. Deletion of PFA4 caused dramatic defects in cryptococcal morphology, stress tolerance, and virulence. Bioorthogonal palmitoylome-profiling identified Pfa4-specific protein substrates involved in cell wall synthesis, signal transduction, and membrane trafficking responsible for these phenotypic alterations. We demonstrate that a single PAT is responsible for the modification of a subset of proteins that are critical in cryptococcal pathogenesis. Since several of these palmitoylated substrates are conserved in other pathogenic fungi, protein palmitoylation represents a potential avenue for new antifungal therapeutics.
Assuntos
Aciltransferases/metabolismo , Criptococose/metabolismo , Cryptococcus neoformans/fisiologia , Proteínas Fúngicas/metabolismo , Interações Hospedeiro-Patógeno , Monócitos/microbiologia , Processamento de Proteína Pós-Traducional , Acilação , Aciltransferases/genética , Adesão Celular , Linhagem Celular , Parede Celular/imunologia , Parede Celular/metabolismo , Parede Celular/patologia , Criptococose/imunologia , Criptococose/microbiologia , Criptococose/patologia , Cryptococcus neoformans/citologia , Cryptococcus neoformans/enzimologia , Cryptococcus neoformans/patogenicidade , Proteínas Fúngicas/genética , Deleção de Genes , Humanos , Meningite Criptocócica/imunologia , Meningite Criptocócica/metabolismo , Meningite Criptocócica/microbiologia , Meningite Criptocócica/patologia , Viabilidade Microbiana , Monócitos/imunologia , Monócitos/metabolismo , Monócitos/patologia , Mutação , Fagocitose , Transdução de Sinais , Estresse Fisiológico , Especificidade por Substrato , Virulência , Latência ViralRESUMO
BACKGROUND: Cryptococcal meningitis (CM) is a leading cause of HIV-associated mortality globally. High fungal burden in cerebrospinal fluid (CSF) at diagnosis and poor fungal clearance during treatment are recognized adverse prognostic markers; however, the underlying pathogenic factors that drive these clinical manifestations are incompletely understood. We profiled a large set of clinical isolates for established cryptococcal virulence traits to evaluate the contribution of C. neoformans phenotypic diversity to clinical presentation and outcome in human cryptococcosis. METHODS: Sixty-five C. neoformans isolates from clinical trial patients with matched clinical data were assayed in vitro to determine murine macrophage uptake, intracellular proliferation rate (IPR), capsule induction, and laccase activity. Analysis of the correlation between prognostic clinical and host immune parameters and fungal phenotypes was performed using Spearman's r, while the fungal-dependent impact on long-term survival was determined by Cox regression analysis. RESULTS: High levels of fungal uptake by macrophages in vitro, but not the IPR, were associated with CSF fungal burden (r = 0.38, P = 0.002) and long-term patient survival (hazard ratio [HR] 2.6, 95% CI 1.2-5.5, P = 0.012). High-uptake strains were hypocapsular (r = -0.28, P = 0.05) and exhibited enhanced laccase activity (r = 0.36, P = 0.003). Fungal isolates with greater laccase activity exhibited heightened survival ex vivo in purified CSF (r = 0.49, P < 0.0001) and resistance to clearance following patient antifungal treatment (r = 0.39, P = 0.003). CONCLUSION: These findings underscore the contribution of cryptococcal-phagocyte interactions and laccase-dependent melanin pathways to human clinical presentation and outcome. Furthermore, characterization of fungal-specific pathways that drive clinical manifestation provide potential targets for the development of therapeutics and the management of CM. FUNDING: This work was made possible by funding from the Wellcome Trust (WT088148MF), the Medical Research Council (MR/J008176/1), the NIHR Surgical Reconstruction and Microbiology Research Centre and the Lister Institute for Preventive Medicine (to R.C. May), and a Wellcome Trust Intermediate fellowship (089966, to T. Bicanic). The C. neoformans isolates were collected within clinical trials funded by the British Infection Society (fellowship to T. Bicanic), the Wellcome Trust (research training fellowships WT069991, to A.E. Brouwer and WT081794, to J.N. Jarvis), and the Medical Research Council, United Kingdom (76201). The funding sources had no role in the design or conduct of this study, nor in preparation of the manuscript.
Assuntos
Infecções Oportunistas Relacionadas com a AIDS/metabolismo , Cryptococcus neoformans/metabolismo , Macrófagos/metabolismo , Meningite Criptocócica/metabolismo , Fagocitose , Infecções Oportunistas Relacionadas com a AIDS/microbiologia , Infecções Oportunistas Relacionadas com a AIDS/mortalidade , Infecções Oportunistas Relacionadas com a AIDS/patologia , Adulto , Animais , Linhagem Celular , Feminino , Humanos , Lacase , Macrófagos/microbiologia , Macrófagos/patologia , Masculino , Meningite Criptocócica/microbiologia , Meningite Criptocócica/mortalidade , Meningite Criptocócica/patologia , Camundongos , Taxa de SobrevidaRESUMO
The increase in immunosuppressed patient populations has correlated with a rise in clinical fungal infections, including cryptococcosis. Patient outcome following Cryptococcus infection is linked to initial fungal burden in cerebrospinal fluid (CSF) and fungal clearance following treatment; however, the role of the pathogen in disease prognosis is poorly defined. In this issue of the JCI, Sabiiti and colleagues have directly correlated phenotypic traits of Cryptococcus neoformans with clinical outcome of infected patients. A better understanding of both the host and pathogen contributions to disease etiology will provide more options for targeted treatment strategies.
Assuntos
Infecções Oportunistas Relacionadas com a AIDS/metabolismo , Cryptococcus neoformans/metabolismo , Macrófagos/metabolismo , Meningite Criptocócica/metabolismo , Fagocitose , Animais , Feminino , Humanos , MasculinoRESUMO
BACKGROUND AND AIMS: Cryptococcal meningitis (CM) has gradually increased in the recent 20 years in the whole world. Although the mortality decreased significantly in recent years, it was still high, especially in patients with persistent infection. Therefore, we compare differences of clinical features between persistent and nonpersistent CM patients. METHODS: We conducted a retrospective review of medical records of patients diagnosed with CM from January 2000 to December 2011 in four centers in China, including demographic features, underlying diseases, clinical presentations, laboratory data, and so on. RESULTS: Of 106 CM patients enrolled, 16 were identified as persistent cases. Among all variables, persistent CM patients were more like to be human immunodeficiency viruses (HIV) infection (P < 0.05), stiff neck (P < 0.01), a serum hemoglobin < 90 g/L (P < 0.01), a serum potassium concentration <2.7 mg/L (P < 0.01), an intracranial pressure (ICP) >400 mmH2 O (P < 0.01), and a latex agglutination cryptococcal antigen titer of cerebrospinal fluid (CSF LACT) >1:1024 (P < 0.01) than nonpersistent ones. A multivariate analysis showed that HIV infection (OR 7.49), stiff neck (OR 11.7), a serum potassium <2.7 mmol/L (OR 9.45), and an ICP >400 mmH2 O (OR 6.83) were closely correlated with persistent CM. CONCLUSIONS: Although it is difficult to deal with persistent CM nowadays, some cases could be predicted early enough in the future, so as to be treated appropriately and have relatively good outcomes.
Assuntos
Meningite Criptocócica/diagnóstico , Meningite Criptocócica/epidemiologia , Adulto , China/epidemiologia , Doença Crônica , Feminino , Seguimentos , Humanos , Masculino , Meningite Criptocócica/metabolismo , Pessoa de Meia-Idade , Estudos Retrospectivos , Fatores de Tempo , Adulto JovemRESUMO
BACKGROUND: Cryptococcal meningoencephalitis is a lethal infection with relatively few therapeutic options. The optimal dosage of liposomal amphotericin B (LAmB) alone or in combination with flucytosine is not known. METHODS: A murine model of cryptococcal meningoencephalitis was used. The fungal density in the brain was determined using quantitative cultures. Pharmacokinetic-pharmacodynamic relationships were determined for LAmB and flucytosine administered alone. The effect of the combination was described using the Greco model and a mathematical model. The results were bridged to humans. RESULTS: Inoculation resulted in hematogenous dissemination and logarithmic growth within the central nervous system. There was histological evidence of multifocal infection throughout the brain. Both LAmB and flucytosine produced a dose-dependent reduction in fungal burden. The effect of the combination of agents in the brain was additive. Bridging studies suggested that a human dosage of LAmB 3 mg/kg/d resulted in a submaximal antifungal effect. Regimens of LAmB 6 mg/kg/d alone, LAmB 3 mg/kg/d plus flucytosine 50 mg/kg/d, and LAmB 3 mg/kg/d plus flucytosine 100 mg/kg/d all resulted in near-maximal antifungal activity. CONCLUSIONS: Potential regimens for further study in clinical trials include LAmB 6 mg/kg/d alone, LAmB 3 mg/kg/d plus flucytosine 50 mg/kg/d, and LAmB 3 mg/kg/d plus flucytosine 100 mg/kg/d.
Assuntos
Anfotericina B/farmacologia , Antifúngicos/farmacologia , Cryptococcus neoformans/efeitos dos fármacos , Flucitosina/farmacologia , Meningite Criptocócica/tratamento farmacológico , Meningoencefalite/tratamento farmacológico , Anfotericina B/farmacocinética , Animais , Antifúngicos/farmacocinética , Encéfalo/efeitos dos fármacos , Encéfalo/microbiologia , Cryptococcus neoformans/isolamento & purificação , Quimioterapia Combinada , Flucitosina/farmacocinética , Humanos , Hospedeiro Imunocomprometido , Masculino , Meningite Criptocócica/imunologia , Meningite Criptocócica/metabolismo , Meningite Criptocócica/microbiologia , Meningoencefalite/microbiologia , Camundongos , Testes de Sensibilidade Microbiana , Modelos BiológicosRESUMO
Cryptococcus neoformans is the most common cause of fungal meningitis, with high mortality and morbidity. The reason for the frequent occurrence of Cryptococcus infection in the central nervous system (CNS) is poorly understood. The facts that human and animal brains contain abundant inositol and that Cryptococcus has a sophisticated system for the acquisition of inositol from the environment suggests that host inositol utilization may contribute to the development of cryptococcal meningitis. In this study, we found that inositol plays an important role in Cryptococcus traversal across the blood-brain barrier (BBB) both in an in vitro human BBB model and in in vivo animal models. The capacity of inositol to stimulate BBB crossing was dependent upon fungal inositol transporters, indicated by a 70% reduction in transmigration efficiency in mutant strains lacking two major inositol transporters, Itr1a and Itr3c. Upregulation of genes involved in the inositol catabolic pathway was evident in a microarray analysis following inositol treatment. In addition, inositol increased the production of hyaluronic acid in Cryptococcus cells, which is a ligand known to binding host CD44 receptor for their invasion. These studies suggest an inositol-dependent Cryptococcus traversal of the BBB, and support our hypothesis that utilization of host-derived inositol by Cryptococcus contributes to CNS infection.