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UNLABELLED: Cryptococcus neoformans causes cryptococcosis, one of the most prevalent fungal diseases, generally characterized by meningitis. There is a limited and not very effective number of drugs available to combat this disease. In this manuscript, we show the host defense peptide mimetic brilacidin (BRI) as a promising antifungal drug against C. neoformans. BRI can affect the organization of the cell membrane, increasing the fungal cell permeability. We also investigated the effects of BRI against the model system Saccharomyces cerevisiae by analyzing libraries of mutants grown in the presence of BRI. In S. cerevisiae, BRI also affects the cell membrane organization, but in addition the cell wall integrity pathway and calcium metabolism. In vivo experiments show BRI significantly reduces C. neoformans survival inside macrophages and partially clears C. neoformans lung infection in an immunocompetent murine model of invasive pulmonary cryptococcosis. We also observed that BRI interacts with caspofungin (CAS) and amphotericin (AmB), potentiating their mechanism of action against C. neoformans. BRI + CAS affects endocytic movement, calcineurin, and mitogen-activated protein kinases. Our results indicate that BRI is a novel antifungal drug against cryptococcosis. IMPORTANCE: Invasive fungal infections have a high mortality rate causing more deaths annually than tuberculosis or malaria. Cryptococcosis, one of the most prevalent fungal diseases, is generally characterized by meningitis and is mainly caused by two closely related species of basidiomycetous yeasts, Cryptococcus neoformans and Cryptococcus gattii. There are few therapeutic options for treating cryptococcosis, and searching for new antifungal agents against this disease is very important. Here, we present brilacidin (BRI) as a potential antifungal agent against C. neoformans. BRI is a small molecule host defense peptide mimetic that has previously exhibited broad-spectrum immunomodulatory/anti-inflammatory activity against bacteria and viruses. BRI alone was shown to inhibit the growth of C. neoformans, acting as a fungicidal drug, but surprisingly also potentiated the activity of caspofungin (CAS) against this species. We investigated the mechanism of action of BRI and BRI + CAS against C. neoformans. We propose BRI as a new antifungal agent against cryptococcosis.

Cryptococcus neoformans is an opportunistic fungal pathogen with a polysaccharide capsule that becomes greatly enlarged in the mammalian host and during in vitro growth under host-like conditions. To understand how individual environmental signals affect capsule size and gene expression, we grew cells in all combinations of 5 signals implicated in capsule size and systematically measured cell and capsule sizes. We also sampled these cultures over time and performed RNA-seq in quadruplicate, yielding 881 RNA-seq samples. Analysis of the resulting data sets showed that capsule induction in tissue culture medium, typically used to represent host-like conditions, requires the presence of either CO2 or exogenous cyclic AMP. Surprisingly, adding either of these pushes overall gene expression in the opposite direction from tissue culture media alone, even though both are required for capsule development. Another unexpected finding was that rich medium blocks capsule growth completely. Statistical analysis further revealed many genes whose expression is associated with capsule thickness; deletion of one of these significantly reduced capsule size. Beyond illuminating capsule induction, our massive, uniformly collected data set will be a significant resource for the research community. [ABSTRACT FROM AUTHOR]

The antimicrobial peptide (AMP) circularized bacteriocin enterocin AS-48 produced by Enterococcus sp. exhibits broad-spectrum antibacterial activity via dimer insertion into the plasma membrane to form membrane pore structures, compromising membrane integrity and leading to bactericidal activity. A specific alpha-helical region of enterocin AS-48 has been shown to be responsible for the membrane-penetrating activity of the peptide. The canon syn-enterocin peptide library, generated using rational design techniques to have ninety-five synthetic peptide variants from the truncated, linearized, membrane-interacting domain of enterocin AS-48, was screened against three clinically relevant fungal strains: Cryptococcus neoformans , Candida albicans , and Candida auris for potential antifungal activity. Twelve peptides exhibited antifungal activity against C. neoformans , and two peptides exhibited activity against C. albicans. The fourteen active antifungal peptides were minimally cytotoxic to an immortalized human keratinocyte cell line (HaCats). Four select peptides were identified with minimum inhibitory concentrations (MICs) below 8 µM against C. neoformans. In 36-hour cell growth tests with these fungicidal peptides, fungicidal peptide no. 32 displayed inhibitory properties comparable to the leading antifungal medication fluconazole against C. neoformans. Screening of peptide no. 32 against a deletion library of C. neoformans mutants revealed that the mechanism of action of peptide no. 32 may relate to multivesicular bodies (MVBs) or polysaccharide capsule targeting. These findings importantly demonstrate that naturally derived AMPs produced by bacteria can be sourced, engineered, and modified to exhibit potent antifungal activity. Our results will contribute to the development of broad treatment alternatives to fungal infections and lend themselves to direct implications for possible treatment options for C. neoformans infections. [ABSTRACT FROM AUTHOR]

Cryptococcosis is a lethal mycosis instigated by the pathogenic species Cryptococcus neoformans and Cryptococcus gattii, primarily affects the lungs, manifesting as pneumonia, and the brain, where it presents as meningitis. Mortality rate could reach 100% if infections remain untreated in cryptococcal meningitis. Treatment options for cryptococcosis are limited and and there are no licensed vaccines clinically available to treat or prevent cryptococcosis. Our study utilizes an integrated bioinformatics approaches to develop a polyvalent multiepitope subunit vaccine focusing on the key virulent proteins Heat shock transcription factor and Chaperone DnaK of both C. neoformans and C. gatti. Then in silico analysis was done to predict highly antigenic epitopes by assessing antigenicity, transmembrane topology screening, allergenecity, toxicity, and molecular docking approaches. Following this analysis, we designed two vaccine constructs integrating a compatible adjuvant and suitable linkers. These constructs exhibited notable characteristics including high antigenicity, non-toxicity, solubility, stability, and compatibility with Toll-like receptors (TLRs). The interaction between both vaccine constructs and TLR2, TLR3, and TLR9 was assessed through molecular docking analysis. Molecular dynamics simulations and MM-PBSA calculations suggest the substantial stabilizing property and binding affinity of Vaccine Construct V1 against TLR9. Both the vaccines revealed to have a higher number of interchain hydrogen bond with TLR9. These findings serve as a crucial stepping stone towards a comprehensive solution for combating cryptococcus infections induced by both C. neoformans and C. gattii. Further validation through in vivo studies is crucial to confirm the effectiveness and potential of the vaccine to curb the spread of cryptococcosis. Subsequent validation through in vivo studies is paramount to confirm the effectiveness and potential of the vaccine in reducing the spread of cryptococcosis. [ABSTRACT FROM AUTHOR]

Background: Cryptococcosis is an opportunistic fungal infection in immunocompromised patients. The major species include Cryptococcus grubii, Cryptococcus neoformans, and rarely, Cryptococcus gattii. Here we present a disseminated Cryptococcus gattii infection in a patient with elevated granulocyte-macrophage colony-stimulating-factor autoantibody which was successfully treated with antifungal therapy. Case presentation: A 61-year-old healthy man presented with a 3-week history of blurred vision, low-grade fever, headache, and a one-year history of low back pain following a fall on his farm. Physical examination revealed lower back tenderness and diplopia. He was tested negative for hepatitis B, C, and human immunodeficiency virus. Chest X-ray revealed a focal opacity in the right retrocardiac paraspinal region and pleural effusion. Magnetic resonance imaging showed a mass located at the L1 prevertebral region and multiple rim-enhancing lesions in bilateral cerebral hemispheres. Thoracoscopy demonstrated cystic lesions at the right costopleural angle. Pathology and microbiology studies confirmed the diagnosis of disseminated Cryptococcus gatti infection. Autoantibodies to granulocyte-macrophage colony-stimulating factor were detected and were considered to cause disseminated cryptococcosis. The patient was started on amphotericin B followed by fluconazole treatment. One month later, the symptoms ameliorated and repeated image studies after 1 year of follow-up showed the resolution of lesions. Conclusion: This report describes the first case of disseminated Cryptococcus gattii infection involving the musculoskeletal system, respiratory system, and central nervous system with granulocyte-macrophage colony-stimulating-factor autoantibody by evidence of histology and microbiology. [ABSTRACT FROM AUTHOR]

Cryptococcus neoformans is a pathogenic basidiomycetous yeast that primarily infects immunocompromised individuals. Fatal outcome of cryptococcosis depends on the ability of C. neoformans to sense and adapt to 37°C. A complex of conserved filament forming GTPases, called septins, composed of Cdc3, Cdc10, Cdc11, and Cdc12, assembles at the mother-bud neck in C. neoformans. Septins Cdc3 and Cdc12 are essential for proliferation of C. neoformans at 37°C and for virulence in the Galleria mellonella model of infection, presumably due to their requirement for septin complex formation, and the involvement in cytokinesis. However, how exactly Cdc3, and Cdc12 contribute to C. neoformans growth at 37°C remains unknown. Based on studies investigating roles of septins in Saccharomyces cerevisiae, septin complex at the mother-bud neck of C. neoformans is predicted to interact with proteins involved in cell cycle control, morphogenesis, and cytokinesis, but the septin-associated proteome in C. neoformans has not been investigated. Here, we utilized tandem mass spectrometry to define C. neoformans proteins that associate with either Cdc3 or Cdc10 at ∼25°C or after the shift to 37°C. Our findings unveil a diverse array of septin-associated proteins, highlighting potential roles of septins in cell division, and stress response. Two proteins, identified as associated with both Cdc3 and Cdc10, the actin-binding protein profilin, which was detected at both temperatures, and ATP-binding multi-drug transporter Afr1, which was detected exclusively at 37°C, were further confirmed by co-immunoprecipitation. We also confirmed that association of Cdc3 with Afr1 was enhanced at 37°C. Upon shift to 37°C, septins Cdc3 and Cdc10 exhibited altered localization and Cdc3 partially co-localized with Afr1. In addition, we also investigated changes to levels of individual C. neoformans proteins upon shift from ∼25 to 37°C in exponentially grown culture and when cells entered stationary phase at ∼25°C. Our study reveals changes to C. neoformans proteome associated with heat and nutrient deprivation stresses and provides a landscape of septin-associated C. neoformans proteome, which will facilitate elucidating the biology of septins and mechanisms of stress response in this fungal pathogen. [ABSTRACT FROM AUTHOR]

Immune responses during acute infection often contain canonical elements which are shared across the responses to an array of agents within a given pathogen class (i.e., respiratory viral infection). Identification of these shared, canonical elements across similar infections offers the potential for impacting development of novel diagnostics and therapeutics. In this way, analysis of host gene expression patterns ('signatures') in white blood cells has been shown to be useful for determining the etiology of some acute viral and bacterial infections. In order to study conserved immune elements shared across the host response to related pathogens, we performed in vitro human PBMC challenges with common fungal pathogens (Candida albicans, Cryptococcus neoformans and gattii); four strains of influenza virus (Influenza A/Puerto Rico/08/34 [H1N1, PR8], A/Brisbane/59/2007 [H1N1], A/Solomon Islands/3/2006 [H1N1], and A/Wisconsin/67/2005 [H3N2]); and gram-negative (Escherichia coli) and gram-positive (Streptococcus pneumoniae) bacteria. Exposed human cells were then analyzed for differential gene expression utilizing Affymetrix microarrays. Analysis of pathogen exposure of PBMCs revealed strong, conserved gene expression patterns representing these canonical immune response elements to each broad pathogen class. A 41-gene multinomial signature was developed which correctly classified fungal, viral, or bacterial exposure with 94–98% accuracy. Furthermore, a 21-gene signature consisting of a subset of the discriminatory PBMC-derived genes was capable of accurately differentiating human patients with invasive candidiasis, acute viral infection, or bacterial infection (AUC 0.94, 0.83, and 0.96 respectively). These data reinforce the conserved nature of the genomic responses in human peripheral blood cells upon exposure to infectious agents and highlight the potential for in vitro models to augment our ability to develop novel diagnostic classifiers for acute infectious diseases, particularly devastating fungal infections. [ABSTRACT FROM AUTHOR]

Studies across various pathogens highlight the importance of pathogen genetic differences in disease manifestation. In the human fungal pathogen Cryptococcus neoformans, sequence type (ST) associates with patient outcome. We performed a meta-analysis of four genomic studies and identified overlapping gene regions associated with virulence, suggesting the importance of these gene regions in cryptococcal disease in diverse clinical isolates. We explored the relationship between virulence and strain genetic differences using the cryptococcosis mouse model and a closely related library of ST93 clinical isolates. We identified four in vivo virulence phenotypes: hypervirulence, typical virulence with CNS disease, typical virulence with non-CNS disease, and latent disease. Hypervirulent isolates were clade specific and associated with an interferon gamma (IFNγ) dominated immune response. Using a genome wide association study (GWAS), we identified nine genes with polymorphisms associated with IFNγ production, including the inositol sensor ITR4. The itr4Δ mutant recapitulated the hypervirulence phenotype and ITR4 affects expression of two IFNγ associated genes. Finally, we showed that IFNγ production is associated with SNPs that downregulate ITR4 and with SNP accumulation in other IFNγ associated genes. These data highlight the complex role of pathogen genetics in virulence and identify genes associated with hypervirulence and IFNγ in Cryptococcus neoformans. Identification of virulence-associated genes in pathogens is important to understand mechanisms of disease. Here, Jackson et al. use a mouse model and clinical isolates of Cryptococcus neoformans to identify novel gene networks that impact virulence. [ABSTRACT FROM AUTHOR]

Thirty-six structurally diverse sesquiterpenoids, including caryolanes (1–12), germacranes (13–16), isodaucane (17), cadinanes (18–22), epicubenols (23, 24), oplopanane (25), pallenanes (26, 27), and eudesmanes (28–36), were isolated from the fermentation broth of Streptomyces fulvorobeus derived from Elephas maximus feces. Pallenane is a kind of rarely reported sesquiterpene with a distinctive C5/C3 bicyclic skeleton and was firstly found from microbial source. The structures of fifteen new compounds (1–4, 13–15, 17, 18, 22, 23, 25–28) were established through detailed spectroscopic data analysis, which included data from experimental and calculated ECD spectra as well as Mosher's reagent derivative method. Compound 34 exhibited moderate antifungal activity against Cryptococcus neoformans and Cryptococcus gattii with MIC values of 50 μg/mL. It effectively inhibited biofilm formation and destroyed the preformed biofilm, as well as hindered the adhesion of Cryptococcus species. The current work would enrich the chemical diversity of sesquiterpenoid family. [ABSTRACT FROM AUTHOR]

Cryptococcus neoformans is a globally distributed human fungal pathogen that can cause cryptococcal meningitis with high morbidity and mortality. In this study, we identified an anaphase-promoting complex (APC) activator, Cdh1, and examined its impact on the virulence of C. neoformans. Our subcellular localization analysis revealed that Cdh1 is situated in the nucleus of C. neoformans. Disrupting or overexpressing the CDH1 gene caused abnormal capsule formation in C. neoformans. The cdh1Δ mutant displayed slight sensitivity when grown at 37 °C, indicating that Cdh1 plays a role in maintaining the growth of C. neoformans at 37 °C. A fungal virulence assay showed that Cdh1 is closely associated with the virulence of C. neoformans, and both the cdh1Δ mutant and CDH1OE overexpression strains significantly diminished the virulence of C. neoformans. The Cryptococcus–macrophage interaction assay revealed that both the cdh1∆ mutant and the CDH1OE strains had significantly lower proliferation ability inside macrophages. Furthermore, the infection of the cdh1Δ mutant significantly activated neutrophil recruitment, as well as Th2 and Th17 immune responses, in lung tissue. In summary, our findings indicate that Cdh1 is crucial for producing virulence factors and fungal virulence in C. neoformans. The findings of this study can offer valuable insights and form the basis for further study of the regulatory mechanisms governing the pathogenicity of C. neoformans, potentially leading to the development of novel therapeutic strategies. [ABSTRACT FROM AUTHOR]

Cryptococcus neoformans is an opportunistic fungal pathogen that is a continuous global health concern, especially for immunocompromised populations. The World Health Organization recognized C. neoformans as one of four critical fungal pathogens, thus emphasizing the need for increased research efforts and clinical resource expansion. Currently, there are no fungal vaccines available for clinical use. Exciting new findings in cryptococcal vaccine development have identified whole cell-based and subunit-based vaccinations to help mitigate health risks and make commercialization attainable. Importantly, recent work has focused on how different cryptococcal cell-wall antigens modified in these vaccine candidates allow us to manipulate their immunogenicity to produce a desired long-term protective anti-fungal immune response. In this review, we discuss the different cryptococcal cell immunogens, namely the polysaccharide capsule, glucans, chitin/chitosan, mannoproteins, and extracellular vesicles, and their role in novel cryptococcal vaccination approaches. Additionally, we examine the immunological mechanisms responsible for protection in these vaccine candidates and the similar host response-stimulation pathways induced through different immunogen exposure. [ABSTRACT FROM AUTHOR]

Antimicrobial compounds play a critical role in combating microbial infections. However, the emergence of antibiotic and antifungal resistance and the scarcity of new antibiotic developments pose a significant threat and demand the discovery of new antimicrobials for both bacterial and fungal pathogens. Our previous work described the first generation (G1) of organoantimony-based compounds that showed antimicrobial activity against several bacterial and fungal pathogens. Here, we present our efforts in modifying these compounds by replacing the tetraphenyl backbone in G1 compounds with a trimethyl group, thereby generating a new series of compounds we refer to as "generation 2", G2. In addition to the novel backbone structure, we introduced three new anionic chloro-cyanoxime ligand groups, namely 2,4-diCl-PhCO−, 2,6-diCl-PhCO− and 2Cl-PhCO−, which were found to be biologically active in the past. Nine new compounds of SbMe3L2 composition were obtained in high yields and characterized by NMR, IR spectroscopies, thermogravimetric TG/DSC and X-ray single crystal analyses. The antibacterial activity of the cyanoximates was tested against three bacterial (Pseudomonas aeruginosa PAO1, Escherichia coli S17 and methicillin-resistant Staphylococcus aureus (MRSA) NRS70) and two fungal (Candida albicans strain SC5314 and Cryptococcus neoformans strain H99) pathogens. Two compounds, SbMe3(MCO)2 and SbMe3(2,4-diClPhCO)2, were active against bacterial strains and inhibited the growth of PAO1 and MRSA with MICs of 50 and 100 µg/mL, respectively. Three compounds, SbMe3(MCO)2, SbMe3(ECO)2 and SbMe3(TCO)2, were active against fungal strains and inhibited either one of or both C. albicans and C. neoformans at MICs of 2.6–66.67 μg/mL. In addition, SbMe3(TCO)2 and SbMe3(MCO)2 were fungicidal at MFC 33.33–66.67 μg/mL. Ultra-thin-layer TEM imaging suggested that SbMe3(MCO)2 targets the integrity of bacterial membranes. Overall, four of the studied G2 series compounds possess antimicrobial activity against a broad range of microbial pathogens, with particular potential against fungal pathogens, which will be explored in further studies. [ABSTRACT FROM AUTHOR]

Fungal pathogens cause over a billion human infections annually, leading to more than 1.6 million deaths each year. The scarcity of available antifungal drugs intensifies the public health threat posed by human pathogenic fungal infections. Therefore there is a critical demand for novel, safe, and effective antifungal agents. Although chitinases are established as effective antifungal agents against phytopathogenic fungi, research on their activity against human pathogenic fungi is limited. The present study seeks to investigate the anti-inflammatory and antifungal activity of bacterial and fungal chitinase against human pathogenic fungi. The antifungal efficacy of bacterial chitinase from Streptomyces griseus, fungal chitinase from Trichoderma viride, and a combination of both was determined by calculating the inhibition percentage in fungal growth, indicated by the reduction in the dry mass of the fungi. Additionally, the anti-inflammatory activity of these chitinases was assessed by measuring the inhibition of albumin denaturation. Results revealed that chitinases exhibited greater antifungal activity compared to the standard. Notably, bacterial chitinase demonstrated higher effectiveness than fungal chitinase against Aspergillus fumigatus, while the bacterial and fungal chitinase had similar effects against different Cryptococcus neoformans and Candida species. The combination of bacterial and fungal chitinase demonstrated the highest antifungal activity against all tested fungi. Furthermore, the anti-inflammatory activity indicated that chitinases prevented 98% of albumin denaturation, marking the first study reporting the anti-inflammatory role of chitinases in preventing albumin denaturation. Additional in-vivo studies are necessary to explore the antifungal activity of chitinases against human pathogenic fungi and investigate the anti-inflammatory mechanisms of chitinase. [ABSTRACT FROM AUTHOR]

Purpose: The diagnosis of pulmonary cryptococcosis (PC) remains challenging, particularly in patients presenting with lobar or patchy consolidation on chest radiographs. Biopsies are sometimes performed for histopathologic examination and microbiological culture to differentiate infections, including PC, from lung cancers. However, to date, the clinical value of small biopsy samples and their reasonable processing methods for detecting Cryptococcus are rarely evaluated. Furthermore, the cryptococcal antigen (CrAg) test has been widely used in cryptococcosis diagnosis due to its high specificity. This 6-year retrospective study aimed to assess the efficacy of four tests commonly used for detecting Cryptococcus in the diagnosis of pulmonary cryptococcosis, and reveal that the combination of 2 or 3 methods would raise diagnosis sensitivity. Methods: The results of CrAg test, histopathologic examination and routine cryptococcal culture of sputum/bronchoalveolar lavage fluid (BALF) were collected from hospitalized patients between June 2019 to May 2024. Additionally, the results of 4 above-mentioned methods were analyzed to compare their effectiveness in PC diagnosis. Results: Among 1508 patients whose biopsy specimens were sent for pathogen detection, 63 PC cases were diagnosed, and 24 C. neoformans strains were cultivated using the Myco/F Lytic culture, which was more than those by sputum/BALF culture (9 strains). CrAg was positive in 82.5% (52/63) PC patients and remained the most sensitive method. The combination of CrAg and biopsy culture will increase the overall diagnostic yield to 95.2% (60/63). Conclusions: In summary, for those having biopsy tissue collected, the combination of CrAg and biopsy culture using Myco/F could effectively identify most PC cases. [ABSTRACT FROM AUTHOR]

Purpose: To report a rare case of fungal keratitis caused by Cryptococcus neoformans, highlighting its unique morphological features using in vivo confocal microscopy (IVCM). Methods: This was a retrospective case report. A 66-year-old man presented with foreign body sensation and blurred vision in his left eye for over 10 months. Results: His best-corrected visual acuity was 20/20. Slit-lamp examination revealed a gray-white lesion approximately 4–5 mm in the superficial layer of the central cornea without epithelial defects. The IVCM images revealed numerous round or round-like pathogens, each with a central highly reflective body surrounded by a dark ring, ranging in size from 5 to 30 µm, and to a maximum of 85 µm, observed in the corneal epithelium and superficial stroma. No obvious inflammatory cell infiltration was observed in the lesions or endothelium. C. neoformans infection was confirmed. The round pathogens completely disappeared after 8 weeks of treatment with topical amphotericin B and voriconazole eye drops. Conclusion: Fungal keratitis caused by C. neoformans is rare and easily overlooked due to atypical clinical signs and symptoms. This case reports the unique morphological features of C. neoformans in the cornea using IVCM for the first time, facilitating rapid, noninvasive auxiliary diagnosis of C. neoformans keratitis and treatment follow-up. [ABSTRACT FROM AUTHOR]

Background and Aims: Meningitis in immunocompromised patients is a significant cause of mortality and morbidity. In HIV-infected cases, it is majorly of infectious origin, with Cryptococcus neoformans and Mycobacterium tuberculosis being two opportunistic pathogens together accounting for about 75% of meningitis cases. However, other variants like bacterial, aseptic, fungal or acanthamoeba meningitis can also occur in patients with HIV. The present study aimed to observe the clinical profile and aetiology of different types of meningitis in HIV-infected patients. Methods: A cross-sectional observational study included HIV-infected patients with symptoms and signs suggestive of meningitis. Patients were then assessed according to clinical, blood and CSF biochemical, blood and CSF microbiological and/special test parameter and/or radiological parameters. Data was statistically analysed. Results: Of 120 patients included, a majority belonged to the age group 26–49 years. Common presenting features included headache, fever, photophobia, neck stiffness and altered mental status. Various aetiologies of the cases included 55% cases of cryptococcal meningitis, 38.33% cases of tubercular meningitis, 3.33% cases of tubercular/cryptococcal co-infection meningitis and 1.66% cases of syphilis and pyogenic infection each. CSF findings suggested elevated opening pressure in 64.16% cases, followed by positive serum CrAg in 63.33% cases. CSF CrAg and India ink were positive, and budding yeast cell was seen in 58.33% cases. Conclusion: Tuberculosis and cryptococcal meningitis continue to be the prevailing culprits of meningitis in individuals with HIV. As treatment is readily available for these infections, early suspicion and prompt diagnosis coupled with effective early initiation of treatment can improve the lifespan of these patients. [ABSTRACT FROM AUTHOR]

The venoms of Theraphosidae spiders have evolved into diverse natural pharmacopeias through selective pressures. Cryptococcus neoformans is a global health threat that frequently causes life-threatening meningitis and fungemia, particularly in immunocompromised patients. In this study, we identify a novel anti-C. neoformans peptide, QS18 (QCFKVCFRKRCFTKCSRS), from the venom gland of China's native spider species Chilobrachys liboensis by utilizing bioinformatic tools. QS18 shares over 50% sequence similarity with tachyplesin peptides, previously identified only in horseshoe crab hemocytes, expanding the known repertoire of the tachyplesin family to terrestrial arachnids. The oxidative folding of QS18 notably enhances its antifungal activity and stability, resulting in a minimum inhibitory concentration of 1.4 µM. The antimicrobial mechanism of QS18 involves cell membrane disruption. QS18 exhibits less than 5% hemolysis in human erythrocytes, indicating microbial selectivity and a favorable safety profile for therapeutic use. Furthermore, mouse model studies highlight QS18's ability as an antifungal agent with notable anti-inflammatory activity. Our study demonstrates QS18 as both a promising template for spider venom peptide research and a novel candidate for the development of peptide antifungals. [ABSTRACT FROM AUTHOR]

This study focuses on the synthesis of a series of oligo- α -(1→6)-D-galactopyranosides bearing β -D-galactofuranosyl residues at O-2 and/or O-3, which relate structurally to fragments of glucuronoxylomannogalactan (GXMGal) from the fungal pathogen Cryptococcus neoformans that causes severe diseases in immunocompromised patients. The preparation of target compounds is based on the use of a selectively O-protected N-phenyltrifluoroacetimidoyl galactopyranoside donor with an allyl group at O-2, levulinoyl group (Lev) at O-3, pentafluorobenzoyl (PFB) group at O-4, and fluorenylmethoxycarbonyl (Fmoc) group at O-6. The choice of protecting groups for this donor ensures the stereospecific formation of α- (1→6)-glycosidic bonds due to the stereodirecting effect of acyls at O-3, O-4, and O-6. At the same time, this combination of O-substituents permits the selective recovery of free OH groups at O-2, O-3, and O-6 for chain elongation via the introduction of β- D-galactofuranosyl and α- D-galactopyranosyl residues. The reported compounds are obtained as aminopropyl glycosides, which are transformed into biotinylated conjugates for further use as coating antigens in immunological studies. The obtained oligosaccharides were subjected to detailed 13C NMR analysis to show the spatial similarity of the obtained hexasaccharide with the corresponding fragment in the GXMGal chain, making this compound suitable for further immunological studies of C. neoformans. [ABSTRACT FROM AUTHOR]

While increased mutation rates typically have negative consequences in multicellular organisms, hypermutation can be advantageous for microbes adapting to the environment. Previously, we identified two hypermutator Cryptococcus neoformans clinical isolates that rapidly develop drug resistance due to transposition of a retrotransposon, Cnl1. Cnl1-mediated hypermutation is caused by a nonsense mutation in a gene encoding an RNA interference (RNAi) component, ZNF3, combined with a tremendous transposon burden. To elucidate adaptive mechanisms following RNAi loss, two bioinformatic pipelines were developed to identify RNAi loss-of-function (LOF) mutations in a collection of 387 sequenced C. neoformans isolates. Remarkably, several RNAi-loss isolates were identified that are not hypermutators and have not accumulated transposons. To test whether these RNAi LOF mutations can cause hypermutation, the mutations were introduced into a nonhypermutator strain with a high transposon burden, which resulted in a hypermutator phenotype. To further investigate whether RNAi-loss isolates can become hypermutators, in vitro passaging was performed. Although no hypermutators were found in two C. neoformans RNAi-loss strains after short-term passage, hypermutation was observed in a passaged Cryptococcus deneoformans strain with an increased transposon burden. Consistent with a two-step evolution, when an RNAi-loss isolate was crossed with an isolate containing a high Cnl1 burden, F1 hypermutator progeny inheriting a high transposon burden were identified. In addition to Cnl1 transpositions, insertions of a gigantic DNA transposon KDZ1 (~11 kb) contributed to hypermutation in the progeny. Our results suggest that RNAi loss is relatively common (7/387, ~1.8%) and enables distinct evolutionary trajectories: hypermutation following transposon accumulation or survival without hypermutation. [ABSTRACT FROM AUTHOR]

The eukaryotic serine/threonine protein phosphatase PP2A is a heterotrimeric enzyme composed of a scaffold A subunit, a regulatory B subunit, and a catalytic C subunit. Of the four known B subunits, the B"' subunit (known as striatin) interacts with the multi-protein striatin-interacting phosphatase and kinase (STRIPAK) complex. Orthologs of STRIPAK components were identified in Cryptococcus neoformans, namely PP2AA/Tpd3, PP2AC/Pph22, PP2AB/Far8, STRIP/Far11, SLMAP/Far9, and Mob3. Structural modeling, protein domain analysis, and detected protein-protein interactions suggest C. neoformans STRIPAK is assembled similarly to the human and fungal orthologs. Here, STRIPAK components Pph22, Far8, and Mob3 were functionally characterized. Whole-genome sequencing revealed that mutations in STRIPAK complex subunits lead to increased segmental and chromosomal aneuploidy, suggesting STRIPAK functions in maintaining genome stability. We demonstrate that PPH22 is a haploinsufficient gene: heterozygous PPH22/pph22Δ mutant diploid strains exhibit defects in hyphal growth and sporulation and have a significant fitness disadvantage when grown in competition against a wild-type diploid. Deletion mutants pph22Δ, far8Δ, and mob3Δ exhibit defects in mating and sexual differentiation, including impaired hyphae, basidia, and basidiospore production. Loss of either PPH22 or FAR8 in a haploid background leads to growth defects at 30°C, severely reduced growth at elevated temperature, abnormal cell morphology, and impaired virulence. Additionally, pph22Δ strains frequently accumulate suppressor mutations that result in overexpression of another putative PP2A catalytic subunit, PPG1. The pph22Δ and far8Δ mutants are also unable to grow in the presence of the calcineurin inhibitors cyclosporine A or FK506, and thus these mutations are synthetically lethal with loss of calcineurin activity. Conversely, mob3Δ mutants display increased thermotolerance, capsule production, and melanization, and are hypervirulent in a murine infection model. Taken together, these findings reveal that the C. neoformans STRIPAK complex plays an important role in genome stability, vegetative growth, sexual development, and virulence in this prominent human fungal pathogen. Author summary: This study focused on a highly conserved protein signaling complex known as STRIPAK, which is important for various developmental processes in fungi and humans. By investigating the functions of this complex in Cryptococcus neoformans, it was discovered to play crucial roles in maintaining genome stability, sexual development, and pathogenesis. In particular, mutations in the genes encoding two subunits of the STRIPAK complex were found to lead to significant defects, including abnormal growth and cell morphology, compromised stress response, and impaired virulence. Interestingly, mutation of a third STRIPAK complex subunit resulted in hypervirulence, characterized by increased thermotolerance, enhanced production of melanin pigment and polysaccharide capsule, and reduced survival in infected animals. Our findings reveal that the STRIPAK complex is an important regulator of growth and virulence of C. neoformans, highlighting its potential as a target for therapies aimed at combating fungal infections. This work furthers understanding of how the STRIPAK complex functions in Cryptococcus, and also in other organisms including humans, where related protein phosphatase complexes govern key cellular processes. [ABSTRACT FROM AUTHOR]

Cryptococcus neoformans , a critical priority pathogen designated by the World Health Organization, poses significant therapeutic challenges due to the limited availability of treatment options. The emergence of antifungal resistance, coupled with cross-resistance, further hampers treatment efficacy. Aneuploidy, known for its ability to induce diverse traits, including antifungal resistance, remains poorly understood in C. neoformans. We investigated the impact of tunicamycin, a well-established ER stress inducer, on aneuploidy formation in C. neoformans. Our findings show that both mild and severe ER stress induced by tunicamycin lead to the formation of aneuploid strains in C. neoformans. These aneuploid strains exhibit diverse karyotypes, with some conferring resistance or cross-resistance to antifungal drugs fluconazole and 5-flucytosine. Furthermore, these aneuploid strains display instability, spontaneously losing extra chromosomes in the absence of stress. Transcriptome analysis reveals the simultaneous upregulation of multiple drug resistance-associated genes in aneuploid strains. Our study reveals the genome plasticity of C. neoformans as a major mechanism contributing to non-antifungal-induced antifungal resistance. [ABSTRACT FROM AUTHOR]

Follow-up of previously healthy patients surviving cryptococcal meningitis found that cryptococcal antigen could be detected for >1 year in serum from 38 of 44 (86%) patients and in cerebrospinal fluid (CSF) from 20 of 31 patients (67%), far beyond the time of culture conversion. The speed of titer decline, measured as the number of days for a 2-fold drop in titer to occur, was slower in serum than in CSF. The speed of decline of antigen titers was much slower in serum and CSF for patients infected with Cryptococcus gattii than Cryptococcus neoformans. The speed of decline in CSF and serum titers was also much slower in patients who had received a ventriculoperitoneal shunt for increased intracranial pressure. The variable and extraordinarily slow rate of clearance in our patients did not appear to reflect differences in disease control but rather differences in species and shunting for increased intracranial pressure. [ABSTRACT FROM AUTHOR]

The leaves of Citrus deliciosa Tenore were collected in southern Brazil, dried, and subjected to the hydrodistillation process to obtain the essential oil. The extraction of essential oil yielded 0.97% ± 0.04. The chromatographic profile of this compound revealed 11 substances, being methyl N-methyl-anthranilate (75.1%), γ-terpinene (13.8%), and Limonene (7%) as major substances. The essential oil shows antifungal action against all tested yeasts, with promising action against Cryptococcus neoformans, Cryptococcus gattii, and Trichosporon asahii. This compound was also able to inhibit the biofilm production of Candida albicans, Candida glabrata, Candida parapsilosis, and T. asahii. The essential oil of tangerine showed weak antioxidant action. It did not show cytotoxicity in human mononuclear cells. It is hoped that these results may guide future studies for the production of formulations that can be used in the treatment of biofilms caused by yeasts, as well as in candidiasis, cryptococcosis, and/or trichosporonosis. [ABSTRACT FROM AUTHOR]

Objective: To evaluate the diagnostic value of metagenomic next-generation sequencing (mNGS) in pulmonary cryptococcosis (PC) using bronchoalveolar lavage fluid (BALF) and lung biopsy tissue specimens. Methods: In this retrospective study, 321 patients diagnosed with lower respiratory tract diseases who underwent mNGS using BALF and LBT samples, between January 2021 and December 2023 were included. Individuals were classified into PC and non-PC groups according to the diagnostic criteria for PC, and conventional fungal cultures were performed. A serum/BALF cryptococcal antigen (CrAg) test was performed in some patients with PC. The diagnostic efficiencies of three methods for PC (mNGS, conventional culture, and CrAg) were compared. Additionally, two mNGS methods were used in this study: original mNGS (OmNGS, testing time from January 2021 to December 2022) and modified mNGS (MmNGS, testing time from January to December 2023). The diagnostic efficiency of the two mNGS methods on PC was simultaneously compared. Results: Among the 321 patients, 23 (7.2%) had PC and 298 (92.8%) did not. Compared with the composite reference standard for PC diagnosis, the sensitivity, specificity, and accuracy of mNGS for PC were 78.3% (95% confidence interval [CI], 55.8%–91.7%), 98.7% (95% CI, 96.4%–99.6%), and 97.2% (95% CI, 94.7%–98.7%), respectively. The sensitivity of mNGS was similar to that of CrAg (80.0%, 12/15) (P > 0.05). The diagnostic sensitivity of both mNGS and CrAg was higher than that of conventional culture (35.0%, 7/20) (P = 0.006, P = 0.016), and the combined detection of mNGS and CrAg further improved the diagnostic sensitivity of PC (93.3%, 14/15). The area under the receiver operating characteristic curve of mNGS was superior to that of conventional culture (0.885 vs. 0.675). In addition, the diagnostic sensitivity of PC was higher than that of OmNGS (P = 0.046). Conclusion: The sensitivity of mNGS is better than that of conventional culture. The combination of mNGS and CrAg improves the testing sensitivity of Cryptococcus. MmNGS could further improve the detection of Cryptococcus. Conventional PC detection methods are indispensable and mNGS can be used as a rapid and accurate auxiliary diagnostic method for PC. [ABSTRACT FROM AUTHOR]

Cryptococcus neoformans is a widespread fungal pathogen that can infect the human central nervous system (CNS) and cause fungal meningitis, leading to hundreds of thousands of deaths worldwide each year. Previous studies have demonstrated that many signal transduction pathways are crucial for the morphological development and virulence of C. neoformans. In this review, data from over 116 research articles have been compiled to show that many signaling pathways control various characteristics of C. neoformans, individually or in association with other pathways, and to establish strong links among them to better understand C. neoformans pathogenesis. Every characteristic of C. neoformans is closely linked to these signaling pathways, making this a rich area for further research. It is essential to thoroughly explore these pathways to address questions that remain and apply a molecular mechanistic approach to link them. Targeting these pathways is crucial for understanding the exact mechanism of infection pathogenesis and will facilitate the development of antifungal drugs as well as the diagnosis and prevention of cryptococcosis. [ABSTRACT FROM AUTHOR]

Fungal infections present a significant global health challenge, prompting ongoing research to discover innovative antifungal agents. The 110 kDa heat shock proteins (Hsp110s) are molecular chaperones essential for maintaining cellular protein homeostasis in eukaryotes. Fungal Hsp110s have emerged as a promising target for innovative antifungal strategies. Notably, 2H stands out as a promising candidate in the endeavor to target Hsp110s and combat fungal infections. Our study reveals that 2H exhibits broad-spectrum antifungal activity, effectively disrupting the in vitro chaperone activity of Hsp110 from Candida auris and inhibiting the growth of Cryptococcus neoformans. Pharmacokinetic analysis indicates that oral administration of 2H may offer enhanced efficacy compared to intravenous delivery, emphasizing the importance of optimizing the AUC/MIC ratio for advancing its clinical therapy. [ABSTRACT FROM AUTHOR]

Background/Objective: Abortions in livestock herds are a major contributor to economic losses. The incidence of fungal aetiology has been reported thus far. The purpose of this study is to investigate the prevalence of abortions due to fungi in referred cases to the Center of Excellence in Ruminant Abortion and Neonatal Mortality, Veterinary Hospital of Ferdowsi University of Mashhad, Mashhad, Iran, from different provinces of Iran. Methods: A total of 200 calves were referred between 2018 and 2022. Samples were taken from the lungs, liver, spleen and abomasum. The fungi present in the cultures were analysed microscopically and morphologically. Results: There were varying contamination levels in the liver, spleen and lungs, with prevalence ranging from 15% to 17%. Abdomasum had a 1% prevalence rate. Different fungal species, including Cryptococcus neoformans (C. neoformans), Aspergillus spp., Rhodotorula spp., Trichosporon spp., Candida spp., Geotrichum spp., Penicillium spp. and Mucor spp., were identified, indicating a diverse range of pathogens affecting calves. The specificity of fungal contaminants in certain organs, such as C. neoformans, Rhodotorula spp. and Trichosporon spp., highlighted the organ's microenvironment as a potential factor influencing fungal growth. Conclusion: As fungi are emerging as a significant cause of morbidity and mortality in animals, the growing role of fungi in livestock abortions should be investigated. As clinical signs are not pathognomonic, laboratory help is imperative to confirm fungal abortion diagnosis. [ABSTRACT FROM AUTHOR]

Fungal infections present a significant health risk, particularly in immunocompromised individuals. Berberine, a natural isoquinoline alkaloid, has demonstrated broad-spectrum antimicrobial activity, though its antifungal potential and underlying mechanisms against both yeast-like and filamentous fungi are not fully understood. This study investigates the antifungal efficacy of berberine against Candida albicans, Cryptococcus neoformans, Trichophyton rubrum, and Trichophyton mentagrophytes in vitro, as well as its therapeutic potential in a murine model of cryptococcal infection. Berberine showed strong antifungal activity, with MIC values ranging from 64 to 128 µg/mL. SEM and TEM analyses revealed that berberine induced notable disruptions to the cell wall and membrane in C. neoformans. No signs of cell necrosis or apoptosis were observed in fungal cells treated with 2 × MIC berberine, and it did not increase intracellular ROS levels or affect mitochondrial membrane potential. Molecular docking and binding affinity assays demonstrated a strong interaction between berberine and the fungal enzyme CYP51, with a dissociation constant (KD) of less than 1 × 10−12 M, suggesting potent inhibition of ergosterol biosynthesis. In vivo studies further showed that berberine promoted healing in guinea pigs infected with T. mentagrophytes, and in a murine cryptococcal infection model, it prolonged survival and reduced lung inflammation, showing comparable efficacy to fluconazole. These findings indicate that berberine exerts broad-spectrum antifungal effects through membrane disruption and CYP51 inhibition, highlighting its potential as a promising therapeutic option for fungal infections. [ABSTRACT FROM AUTHOR]

Background/Objectives: Amphotericin B is indicated in deep systemic fungal infections. The aim was to determine the sociodemographic, clinical and pharmacological variables of a group of Colombian patients treated with amphotericin B and factors associated with mo rtality. Methods: A longitudinal observational retrospective study on the use of amphotericin B in Colombia was conducted between January 2015 and December 2022. The multivariate analysis sought to identify variables related to mortality. Results: A total of 310 patients were identified, with a median age of 44.0 years, and 71.0% were women. Conventional amphotericin B was the most used (74.8%). The main uses were cryptococcosis (38.7%), histoplasmosis (31.9%) and candidiasis (29.4%). More than a third of patients died during hospitalization (40.3%). An increase in the Charlson Comorbidity Index score (HR: 1.13; 95% CI: 1.05–1.22) and in the qSOFA score (HR: 1.34; 95% CI: 1.04–1.73), coinfection by Mycobacterium tuberculosis (HR: 2.09; 95% CI: 1.32–3.31) and the requirement of vasopressors (HR: 4.20; 95% CI: 2.16–8.15) or invasive mechanical ventilation (HR: 2.73; 95% CI: 1.40–5.33) increased the probability of in-hospital death. In contrast, those who received systemic corticosteroids (HR: 0.43; 95% CI: 0.26–0.70) had a lower risk. Conventional amphotericin B is the most used drug mainly treating Cryptococcus neoformans infections. Conclusions: The use of amphotericin B was consistent with clinical practice guideline recommendations. In-hospital mortality was common, and factors such as increased comorbidities, higher qSOFA scores, coinfection with Mycobacterium tuberculosis and invasive procedures like mechanical ventilation were linked to increased mortality. [ABSTRACT FROM AUTHOR]

Cryptococcal infection commonly begins as an opportunistic infection in humans, however, this can escalate to a systemic or life-threatening form in immunocompromised individuals. Here, we aim to identify novel antifungal molecules from plants resources. Sclareolide, a phytochemical classified as a sesquiterpene lactone, was assessed against Cryptococcus neoformans H99. Sclareolide exhibited promising antifungal properties with a minimum inhibitory concentration (MIC) of 16 µg/mL. Additionally, the C. neoformans growth rate was significantly affected by sclareolide treatment in a concentration-dependent manner, as observed through a time killing assay, with a significant reduction at MIC × 8 compared to the control by 48 h. To elucidate the underlying mechanisms of sclareolide antifungal activity, fluorescence-based methods were employed. Propidium iodide (PI) accumulation assay indicated a reduction in C. neoformans membrane integrity, with values as low as 6.62 ± 0.18% after treatment. Moreover, sclareolide at MIC × 4 and MIC × 8 significantly increased the production of reactive oxygen species (ROS) and reduced the mitochondrial membrane potential (MMP), suggesting oxidative stress and mitochondrial dysfunction in C. neoformans. Sclareolide did not induce caspase-dependent apoptosis, suggesting a non-apoptotic mechanism. Further, a checkerboard experiment was performed to assess potential synergistic interaction with Amphotericin B, however, no synergism was observed. Moving on, sclareolide at 128 µg/mL did not exhibit toxicity in Galleria mellonella, further supporting its potential as a safe antifungal agent. These findings suggest that the antifungal activity of sclareolide against C. neoformans is mediated by oxidative stress. Further in vivo and pharmacokinetic studies are recommended to explore the potential of sclareolide as a prototype for the development of novel anti-cryptococcal therapies. [ABSTRACT FROM AUTHOR]

We present a patient with human immunodeficiency virus who developed multiple painful lesions that previously in the past had speciated as Cryptococcus neoformans cutaneously, and in the lung. Despite induction therapy for presumed re-infection, the patient did not improve so a biopsy was performed and this was speciated as Mycobacterium avium complex, with final diagnosis being disseminated Mycobacterium avium complex. This case highlights the importance of considering a broad differential diagnosis for any new lesions regardless of prior culture data.

Yao Qin,1 Xingwu Zou,1 Yanghui Jin,2 Jinmeng Li,3 Qingshan Cai1 1Department of Tuberculosis, Hangzhou Red Cross Hospital, Hangzhou, People’s Republic of China; 2Department of Orthopaedic, Hangzhou Red Cross Hospital, Hangzhou, People’s Republic of China; 3Department of Pharmacy, Hangzhou Red Cross Hospital, Hangzhou, People’s Republic of ChinaCorrespondence: Qingshan Cai, Department of Tuberculosis, Hangzhou Red Cross Hospital, Hangzhou, People’s Republic of China, Email caiqs66@163.comAim: Cryptococcus neoformans osteomyelitis coupled with tuberculosis and tuberculous lymphadenitis, is a rare occurrence in clinical. Diagnostic challenges arise due to the clinical radiological similarity of this condition to other lung infections and the limited and sensitive nature of traditional approaches. Here, we present a case of co-infection diagnosed using Metagenomic Next-Generation Sequencing, highlighting the effectiveness of advanced genomic techniques in such complex scenarios.Case Presentation: We present a case of a 67-year-old female infected with cryptococcal osteomyelitis and presented with swelling and pain in the right ankle. Following a biopsy of the right ankle joint, Metagenomic Next-Generation Sequencing (mNGS) of the biopsy tissue revealed Cryptococcus neoformans infection. Positive results for Cryptococcus capsular antigen and pathological findings confirmed the presence of Cryptococcus neoformans. The patient underwent surgical debridement, coupled with oral fluconazole treatment (300mg/day), leading to the resolution of symptoms.Conclusion: Cryptococcus neoformans is an uncommon cause of ankle infection. Metagenomic Next-Generation Sequencing (mNGS) serves as a valuable diagnostic tool, aiding clinicians in differentiating cryptococcal osteomyelitis from other atypical infections.Keywords: Cryptococcus neoformans, tuberculosis, diagnosis, metagenomic next-generation sequencing, mNGS

Abstract Cryptococcus neoformans (Cn) is an opportunistic encapsulated fungal pathogen that causes life-threatening meningoencephalitis in immunosuppressed individuals. Since IL-6 is important for blood-brain barrier support and its deficiency has been shown to facilitate Cn brain invasion, we investigated the impact of IL-6 on systemic Cn infection in vivo, focusing on central nervous system (CNS) colonization and glial responses, specifically microglia and astrocytes. IL-6 knock-out (IL-6−/−) mice showed faster mortality than C57BL/6 (Wild-type) and IL-6−/− supplemented with recombinant IL-6 (rIL-6; 40 pg/g/day) mice. Despite showing early lung inflammation but no major histological differences in pulmonary cryptococcosis progression among the experimental groups, IL-6−/− mice had significantly higher blood and brain tissue fungal burden at 7-days post infection. Exposure of cryptococci to rIL-6 in vitro increased capsule growth. In addition, IL-6−/− brains were characterized by an increased dystrophic microglia number during Cn infection, which are associated with neurodegeneration and senescence. In contrast, the brains of IL-6-producing or -supplemented mice displayed high numbers of activated and phagocytic microglia, which are related to a stronger anti-cryptococcal response or tissue repair. Likewise, culture of rIL-6 with microglia-like cells promoted high fungal phagocytosis and killing, whereas IL-6 silencing in microglia decreased fungal phagocytosis. Lastly, astrogliosis was high and moderate in infected brains removed from Wild-type and IL-6−/− supplemented with rIL-6 animals, respectively, while minimal astrogliosis was observed in IL-6−/− tissue, highlighting the potential of astrocytes in containing and combating cryptococcal infection. Our findings suggest a critical role for IL-6 in Cn CNS dissemination, neurocryptococcosis development, and host defense.

Cryptococcus neoformans is the highest‐ranked fungal pathogen in the Fungal Priority Pathogens List (FPPL) released by the World Health Organization (WHO). In this study, through in silico simulations, a multi‐epitope vaccine against Cryptococcus neoformans was developed using the mannoprotein antigen (MP88) as a vaccine candidate. Following the retrieval of the MP88 protein sequences, these were used to predict antigenic B‐cell and T‐cell epitopes via the bepipred tool and the artificial neural network, respectively. Conserved B‐cell epitopes AYSTPA, AYSTPAS, PASSNCK, and DSAYPP were identified as the most promising B‐cell epitopes. While YMAADQFCL, VSYEEWMNY, and FQQRYTGTF were identified as the best candidates for CD8+ T‐cell epitopes; and YARLLSLNA, ISYGTAMAV, and INQTSYARL were identified as the most promising CD4+ T‐cell epitopes. The vaccine construct was modeled along with adjuvant and peptide linkers and the expasy protparam tool was used to predict the physiochemical properties. According to this, the construct vaccine was predicted to be antigenic, nontoxic, nonallergenic, soluble, stable, hydrophilic, and thermostable. Furthermore, the three‐dimensional structure was also used in docking analyses with Toll‐like receptor (TLR4). Finally, the cDNA of vaccine was successfully cloned into the E. coli pET‐28a (+) expression vector. The results presented here could contribute towards the design of an effective vaccine against Cryptococcus neoformans.

Abstract Introduction Intracranial fungal infections’ (IcFIs) varying clinical manifestations lead to difficulties in diagnosis and treatment. African populations are disproportionately affected by the high burden of the disease. There is a lack of clarity as to the diagnostic and treatment modalities employed across the continent. In this review, we aim to detail the management, and outcome of IcFIs across Africa. Methods This scoping review was conducted using the Arksey and O'Malley framework. MEDLINE, EMBASE, Cochrane Library, African Index Medicus, and African Journals Online were searched for relevant articles from database inception to August 10th, 2021. The Preferred Reporting Items for Systematic Review and Meta-Analysis extension for Scoping Reviews guidelines were used to report the findings of the review. Results Of the 5,779 records identified, 131 articles were included. The mean age was 35.6 years, and the majority (56.4%) were males. The majority (n = 8,433/8,693, 97.0%) of IcFIs presented as a meningitis, the most common communicable predisposing factor of IcFIs was HIV/AIDS (n = 7,815/8,693, 89.9%), and the most common non-communicable risk factor was diabetes mellitus (n = 32/8,693, 0.4%). Cryptococcus species was the most common (n = 8,428/8,693, 97.0%) causative organism. The most commonly used diagnostic modality was cerebrospinal (CSF) cultures (n = 4,390/6,830, 64.3%) for diffuse IcFIs, and MRI imaging (n = 12/30, 40%) for focal IcFIs. The most common treatment modality was medical management with antifungals only (n = 4,481/8,693, 51.6%). The most commonly used antifungal agent in paediatric, and adult patients was amphotericin B and fluconazole dual therapy (51.5% vs 44.9%). The overall mortality rate was high (n = 3,475/7,493, 46.3%), and similar for both adult and paediatric patients (47.8% vs 42.1%). Conclusion Most IcFIs occurred in immunosuppressed individuals, and despite the new diagnostic techniques, CSF culture was mostly used in Africa. Antifungals regimens used was similar between children and adults. The outcome of IcFIs in Africa was poor for both paediatric and adult patients.

Abstract 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.

Abstract 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.

Nearly two million people die each year from fungal infections. Additionally, fungal crop infections jeopardize the global food supply. The use of 254 nm UVC radiation from mercury vapor lamps is a disinfection technique known to be effective against all microorganisms, and there are surveys of published UVC sensitivities. However, these mainly focus on bacteria and viruses. Therefore, a corresponding overview for fungi will be provided here, including far-UVC, UVB, UVA, and visible light, in addition to the conventional 254 nm UVC inactivation. The available literature was searched for photoinactivation data for fungi in the above-mentioned spectral ranges. To standardize the presentation, the mean log-reduction doses were retrieved and sorted by fungal species, spectral range, wavelength, and medium, among others. Additionally, the median log-reduction dose was determined for fungi in transparent liquid media. Approximately 400 evaluable individual data sets from publications over the last 100 years were compiled. Most studies were performed with 254 nm radiation from mercury vapor lamps on Aspergillus niger, Candida albicans, and Saccharomyces cerevisiae. However, the data found were highly scattered, which could be due to the experimental conditions. Even though the number of individual data sets seems large, many important fungi have not been extensively studied so far. For example, UV irradiation data does not yet exist for half of the fungal species classified as “high priority” or “medium priority” by the World Health Organization (WHO). In addition, researchers should measure the transmission of their fungal suspensions at the irradiation wavelength to avoid the undesirable effects of either absorption or scattering on irradiation results.

Cryptococcus neoformans (Cn) is an opportunistic fungus that causes severe central nervous system (CNS) disease in immunocompromised individuals. Brain parenchyma invasion requires fungal traversal of the blood-brain barrier. In this study, we describe that Cn alters the brain endothelium by activating small GTPase RhoA, causing reorganization of the actin cytoskeleton and tight junction modulation to regulate endothelial barrier permeability. We confirm that the main fungal capsule polysaccharide glucuronoxylomannan is responsible for these alterations. We reveal a therapeutic benefit of RhoA inhibition by CCG-1423 in vivo. RhoA inhibition prolonged survival and reduced fungal burden in a murine model of disseminated cryptococcosis, supporting the therapeutic potential of targeting RhoA in the context of cryptococcal infection. We examine the complex virulence of Cn in establishing CNS disease, describing cellular components of the brain endothelium that may serve as molecular targets for future antifungal therapies to alleviate the burden of life-threatening cryptococcal CNS infection. [ABSTRACT FROM AUTHOR]

The research on actinobacteria isolated from traditional medicinal plants is limited. Here, four new Streptomyces isolates (Ha1, Pp1, UzK and UzM) were obtained from the rhizospheres of Helianthus annuus, Pongamia pinnata and Ziziphus mauritiana, frequently utilized in Indian traditional medicine. The Streptomyces isolates aqueous extracts were studied alone against the growth of the Cryptococcus neoformans H99 reference strain, the fluconazole-tolerant T1-5796 and 89–610 strains, three histone deacetylase (HDAC) genes mutant strains, C. gattii NIH198, Candida albicans, C. glabrata, C. parapsilosis and C. tropicalis to determine minimum inhibitory concentration (MIC). Next, the extracts were employed in combination with aluminium-phthalocyanine chloride nanoemulsion-mediated photodynamic therapy to evaluate a possible interaction. We demonstrated that the C. neoformans T1-5796 fluconazole-tolerant strain was more severely inhibited by the Pp1 isolate extract (MIC: 6 mg mL−1) than H99, which was not inhibited. Growth inhibition of the HDAC null mutants was more prominent for the extract of the UzM isolate, showing inhibition at 2 mg mL−1. The UzM extract was also the most effective in hindering the Candida species proliferation, with MIC values ranging from 10 to 40 mg mL−1. The four Streptomyces extracts, especially UzK and UzM, significantly enhanced the antifungal effect of the photodynamic therapy. Our results indicate these Streptomyces isolates as sources of novel metabolites which could potentiate the effect of photodynamic therapy in controlling yeasts superficial infections. [ABSTRACT FROM AUTHOR]

Cryptococcosis, an infection caused by Cryptococcus neoformans and Cryptococcus gattii, predominantly targets the central nervous system (CNS) in patients with AIDS but is not limited to this group. The disease can also occur in individuals with various immunosuppressive conditions, frequently involving the brain or lungs. Cryptococcal meningitis (CM) is the most common form of fungal meningoencephalitis, leading to intracerebral infections, cerebral infarction, or hydrocephalus. The clinical presentation of CM is nonspecific, and imaging features can vary significantly. This case report presents a patient with cerebral infarction, who was HIV‐negative but had been on long‐term cortisone therapy. Notably, the patient had a history of pulmonary cryptococcosis 15 years prior to cerebral involvement. When initially at our clinic, histology and culture results from brain biopsies were negative and the earlier pulmonary cryptococcosis history was unknown. Subsequently, cryptococcal antigen was detected in both serum and cerebrospinal fluid (CSF), and C. neoformans was cultivated from CSF. This case highlights the critical importance of maintaining a high index of suspicion for CM, particularly in patients with a history of previous cryptococcal infections, and it also demonstrates the possibility of false‐negative brain biopsy results due to secondary vascular events associated with CM. [ABSTRACT FROM AUTHOR]

Cryptococcus neoformans is an environmental pathogen that causes life-threatening disease in immunocompromised persons. The majority of immunological studies have centered on CD4+ T-cell dysfunction and associated cytokine signaling pathways, optimization of phagocytic cell function against fungal cells, and identification of robust antigens for vaccine development. However, a growing body of literature exists regarding cytotoxic cells, specifically CD8+ T-cells, Natural Killer cells, gamma/delta T-cells, NK T-cells, and Cytotoxic CD4+ T-cells, and their role in the innate and adaptive immune response during C. neoformans and C. deneoformans infection. In this review, we (1) provide a comprehensive report of data gathered from mouse and human studies on cytotoxic cell function and phenotype, (2) discuss harmonious and conflicting results on cellular responses in mice models and human infection, (3) identify gaps of knowledge in the field ripe for exploration, and (4) highlight how innovative immunological tools could enhance the study of cytotoxic cells and their potential immunomodulation during cryptococcosis. [ABSTRACT FROM AUTHOR]

Cryptococcus neoformans is included in the World Health Organization fungal priority pathogen list, complied to expedite improved research and public-health interventions. The limited number of available antifungal drugs, their associated toxicity, and the emergence of drug-resistant strains make the development of new therapeutic strategies mandatory. Pattern-recognition receptors (PRRs) from the host's innate immune system constitute a potential source of new antimicrobial agents. CD5 and CD6 are lymphoid members of the ancient scavenger receptor cysteine-rich superfamily (SRCR-SF) which bind pathogen-associated molecular patterns (PAMPs) of fungal and bacterial origin. Evidence supports the concept that such binding maps to 11-mer sequences present in each of their three SRCR extracellular domains. Herein, we have designed synthetic peptides containing tandems of such 11-mer sequences (namely CD5-T and CD6-T) and analyzed their C. neoformans-binding properties in vitro. Our results show both inhibitory effects on fungal growth and an ability to impact capsule formation and titanization, two critical virulence factors of C. neoformans involved in immune evasion. These effects hold promise for CD5-T and CD6-T peptides as single or adjuvant therapeutic agents against cryptococcosis. [ABSTRACT FROM AUTHOR]

This work aimed to conduct a comparative analysis of the occurrence, relative density (RD) and abundance (RA) of simple sequence repeats (SSRs), within the genomic and transcriptomic sequences of four different isolates of Cryptococcus neoformans. Among the whole genome sequences, it was observed that the C. neoformans isolate JEC21 exhibited the most elevated RA (165.3) and RD (2471.8) of SSRs. Conversely, among the transcriptome sequences, the C. neoformans isolate KN99 displayed the greatest RA (145.5) of SSRs. Among the classes of SSRs, trinucleotide repeats exhibited a higher prevalence in both the genomic sequences (50%) and transcriptome sequences (65%) over other repeats. Motif conservation analysis among the isolates demonstrated that 41.1% of motifs were conserved among the isolates of C. neoformans. The study also found unique motifs that could potentially serve as molecular probes for the purpose of isolate identification. A total of 8117 primers were created for the isolates of Cryptococcus to enrich it with genomic resources. The genomic resources created in this study have the potential to aid in the diversity analysis and the creation of markers specific to individual isolates. [ABSTRACT FROM AUTHOR]

Cryptococcus neoformans and Cryptococcus gattii have been isolated from several different environmental sources, including several different tree species. This could indicate that the two species are ubiquitous in the same way as Saccharomyces cerevisiae. Laboratory infection studies have established that the two species can colonize the surface and inside of different plant species. However, the nature and number of studies make it difficult to form a general hypothesis about the relationship between C. neoformans/gattii and plants. Despite being reported, plant disease from infections is not well documented, and only from mated infection mixes. Interestingly, environmental isolates are often only of one of the two mating types. The profile of carbohydrate-active enzymes (CAZymes) encoded in the genomes of C. neoformans and C. gattii , makes it unlikely that they can degrade the plant cell wall as plant pathogens. Instead, they have the pectinolytic potential to live as commensal epi- or endophytes. While the opportunistic human pathogens Cryptococcus neoformans and Cryptococcus gattii are often isolated from plants and plant-related material, evidence suggests that these Cryptococcus species do not directly infect plants. Studies find that plants are important for Cryptococcus mating and dispersal. However, these studies have not provided enough detail about how plants and these fungi interact, especially in ways that could show the fungi are capable of causing disease. This review synthesizes recent findings from studies utilizing different plant models associated with the ecology of C. neoformans and C. gattii. Unanswered questions about their environmental role are highlighted. Overall, current research indicates that Cryptococcus utilizes plants as a substrate rather than harming them, arguing against Cryptococcus as a genuine plant pathogen. We hypothesize that plants represent reservoirs that aid dispersal, not hosts vulnerable to infection. [ABSTRACT FROM AUTHOR]

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.Key Points: • The importance of anticytokine autoantibodies in the pathogenesis of various infectious diseases, including opportunistic mycoses, is increasingly being reported. • Neutralizing auto-Abs against GM-CSF are present in Colombian patients with cryptococcosis caused by C. gattii or C. neoformans. [ABSTRACT FROM AUTHOR]

Opportunistic infections or pathogens that reactivate from latent infection in the renal transplant recipients often arise during the peak immunosuppression period and infections such as Pneumocystis jirovecii may arise in the late period of posttransplant. In developing countries, tuberculosis (TB) is a major concern among transplant patients. Here, we present a case of 42-year-old male who presented after 10 years of renal transplant with a history of fever. On evaluation, he was diagnosed to have concomitant multiple infections like disseminated TB, Cryptococcal meningitis, Cytomegalovirus viremia, and Pneumocystis infection. He was treated with appropriate antibiotics, antifungal, and antiviral with close monitoring of drug-related side effects and graft function. Detailed evaluation and to look for concomitant infections is required for transplant patients who had no response to one particular group of drugs after initial diagnosis. He had stable graft function and developed drug-related side effects which required dose reduction intermittently. This case taught us how to manage four major infections with balancing immunosuppression, antibiotics, and graft function that we see in day-to-day practice of transplant clinics. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Cryptococcus neoformans (Cn) is an opportunistic encapsulated fungal pathogen that causes life-threatening meningoencephalitis in immunosuppressed individuals. Since IL-6 is important for blood-brain barrier support and its deficiency has been shown to facilitate Cn brain invasion, we investigated the impact of IL-6 on systemic Cn infection in vivo, focusing on central nervous system (CNS) colonization and glial responses, specifically microglia and astrocytes. IL-6 knock-out (IL-6−/−) mice showed faster mortality than C57BL/6 (Wild-type) and IL-6−/− supplemented with recombinant IL-6 (rIL-6; 40 pg/g/day) mice. Despite showing early lung inflammation but no major histological differences in pulmonary cryptococcosis progression among the experimental groups, IL-6−/− mice had significantly higher blood and brain tissue fungal burden at 7-days post infection. Exposure of cryptococci to rIL-6 in vitro increased capsule growth. In addition, IL-6−/− brains were characterized by an increased dystrophic microglia number during Cn infection, which are associated with neurodegeneration and senescence. In contrast, the brains of IL-6-producing or -supplemented mice displayed high numbers of activated and phagocytic microglia, which are related to a stronger anti-cryptococcal response or tissue repair. Likewise, culture of rIL-6 with microglia-like cells promoted high fungal phagocytosis and killing, whereas IL-6 silencing in microglia decreased fungal phagocytosis. Lastly, astrogliosis was high and moderate in infected brains removed from Wild-type and IL-6−/− supplemented with rIL-6 animals, respectively, while minimal astrogliosis was observed in IL-6−/− tissue, highlighting the potential of astrocytes in containing and combating cryptococcal infection. Our findings suggest a critical role for IL-6 in Cn CNS dissemination, neurocryptococcosis development, and host defense. [ABSTRACT FROM AUTHOR]

We conducted a retrospective, observational study among acquired immune deficiency syndrome (AIDS) patients with cryptococcal meningitis or talaromycosis to assess AmB formulations-related adverse events (AEs). Total 205 eligible patients were enrolled. Of them, 139 received AmB therapy, 51 received liposomal AmB (L-AmB) therapy, and 15 received AmB cholesteryl sulfate complex (ABCD) therapy. The incidences of total AEs between the AmB, L-AmB and ABCD group had no significant differences. The ABCD group had significantly higher incidences of hepatotoxicity and hematological toxicity than the AmB and L-AmB groups. The incidence of grade 3–4 hematological toxicity in the ABCD group was significantly higher than that in the AmB and L-AmB groups. Multinomial logistic regression models showed that compared with AmB, ABCD had a higher risk for the occurrence of grade 3–4 hematological toxicity (aOR = 43.924, 95%CI 6.296-306.418; p < 0.001). We demonstrated that ABCD was more prone to hepatotoxicity and hematological toxicity than AmB and L-AmB among AIDS patients, which is worth noting. [ABSTRACT FROM AUTHOR]