Connecting Cryptococcal Meningitis and Gut Microbiome.

Fungal pathogens of the Cryptococcus neoformans species complex (C. neoformans SC) are a major cause of fungal meningitis in immunocompromised individuals. As with other melanotic microorganisms associated with human diseases, the cell-wall-associated melanin of C. neoformans SC is a major virulence factor that contributes to its ability to evade host immune responses. The levels of melanin substrate and the regulation of melanin formation could be influenced by the microbiota-gut-brain axis. Moreover, recent studies show that C. neoformans infections cause dysbiosis in the human gut microbiome. In this review, we discuss the potential association between cryptococcal meningitis and the gut microbiome. Additionally, the significant potential of targeting the gut microbiome in the diagnosis and treatment of this debilitating disease is emphasized.

Cryptococcal meningitis and cerebral vasculitis in a patient with primary intestinal lymphangiectasia: a case report.

Primary intestinal lymphangiectasia (Waldmann's disease) is a rare exudative enteropathy without precisely assessed infectious risk. We report the case of a 49-year-old male patient with meningitis and cerebral vasculitis due to Cryptococcus neoformans complicating Waldmann's disease diagnosed 12 years ago. The treatment combined liposomal amphotericin B, 3 mg/kg daily plus flucytosine 25 mg/kg/6 h, both intravenously during 15 days, then fluconazole 800 mg daily during 8 weeks, and finally 200 mg daily indefinitely. Dexamethasone 0.4 mg/kg daily during the first week was gradually decreased over 2 months. The outcome was good, and the patient is still followed 3 years later without any recurrence.

Novel spiro[pyrrolidine-2,3'-quinoline]-2'-one derivatives containing piperazine fragment as potential chitin synthase inhibitors and antifungal agents: Design, synthesis and biological evaluation.

A series of spiro[pyrrolidine-2,3'-quinoline]-2'-one derivatives were designed and synthesized for the discovery of novel antifungal drugs. The bioactivities of all derivatives were screened by evaluating their inhibitory effects against chitin synthase (CHS) and antimicrobial activities in vitro. Enzyme inhibition experiments showed that all the synthesized compounds inhibited the chitin synthase. Compounds 4d, 4k, 4n and 4o showed inhibitory effects against CHS with IC50 values which were close to that of the control drug (polyoxin B). The results of enzyme kinetics experiment showed that these compounds were non-competitive inhibitors of chitin synthase (Ki of compound 4o is 0.14 mM). Antimicrobial experiments showed that these compounds exhibited moderate to excellent antifungal activity against pathogenic fungal strains while the compounds showed little potency against bacteria. Among them, compounds 4d, 4f, 4k and 4n showed stronger antifungal activities against C. albicans than those of fluconazole and polyoxin B. Compounds 4f, 4n and 4o showed better antifungal activities against A. flavus than those of fluconazole and polyoxin B. Compound 4d showed similar activity to that of fluconazole and stronger activity than those of polyoxin B against C. neoformans and A. fumigatus. It is also showed that these compounds have the potency against drug-resistant fungal variants. The results of sorbitol protection assay and evaluation of antifungal activity against micafungin-resistant strains experiment further illustrated that these compounds inhibited the synthesis of chitin of fungal cell wall. Drug combination experiments showed that these compounds had synergistic or additive effects when combined with fluconazole or polyoxin B. The synergistic effects with polyoxin B further confirmed the compounds were non-competitive inhibitors of chitin synthase. Additionally, docking studies showed that these compounds had strong affinity with chitin synthase from C. albicans (CaChs2). These results indicate that the target of these synthesized compounds is chitin synthase, and these compounds had excellent antifungal activity while possessed the potency against drug-resistant fungal variants.

Fertile Cryptococcus neoformans var. neoformans (C. deneoformans) isolates from natural environment in Kosovo.

A total of 300 tree-associated samples were collected from green areas in three towns in Kosovo, and cultured to isolate Cryptococcus species. One soil sample from Prizren tested positive, yielding three isolates identified as C. neoformans var. neoformans (C. deneoformans), molecular type VNIV, two MATα, and one MATa. Mating experiments revealed that they were fertile when crossed together, and multilocus sequence typing analysis showed unique sequence types not found in the global database. The study reports the presence of C. neoformans strains in the environment in Kosovo and highlights the importance of monitoring pathogen distribution and the potential impacts of climate change.

We collected tree samples from three main towns in Kosovo to isolate the human fungal pathogen Cryptococcus. The results showed that C. neoformans var. neoformans isolates, able to produce basidiospores, were present in the environment, confirming that population at risk can be potentially infected.

Ethanolic Extract Propolis-Loaded Niosomes Diminish Phospholipase B1, Biofilm Formation, and Intracellular Replication of Cryptococcus neoformans in Macrophages.

Secretory phospholipase B1 (PLB1) and biofilms act as microbial virulence factors and play an important role in pulmonary cryptococcosis. This study aims to formulate the ethanolic extract of propolis-loaded niosomes (Nio-EEP) and evaluate the biological activities occurring during PLB1 production and biofilm formation of Cryptococcus neoformans. Some physicochemical characterizations of niosomes include a mean diameter of 270 nm in a spherical shape, a zeta-potential of -10.54 ± 1.37 mV, and 88.13 ± 0.01% entrapment efficiency. Nio-EEP can release EEP in a sustained manner and retains consistent physicochemical properties for a month. Nio-EEP has the capability to permeate the cellular membranes of C. neoformans, causing a significant decrease in the mRNA expression level of PLB1. Interestingly, biofilm formation, biofilm thickness, and the expression level of biofilm-related genes (UGD1 and UXS1) were also significantly reduced. Pre-treating with Nio-EEP prior to yeast infection reduced the intracellular replication of C. neoformans in alveolar macrophages by 47%. In conclusion, Nio-EEP mediates as an anti-virulence agent to inhibit PLB1 and biofilm production for preventing fungal colonization on lung epithelial cells and also decreases the intracellular replication of phagocytosed cryptococci. This nano-based EEP delivery might be a potential therapeutic strategy in the prophylaxis and treatment of pulmonary cryptococcosis in the future.

Immunization with a heat-killed prm1 deletion strain protects the host from Cryptococcus neoformans infection.

Systemic infection with Cryptococcus neoformans, a dangerous and contagious pathogen found throughout the world, frequently results in lethal cryptococcal pneumonia and meningoencephalitis, and no effective treatments and vaccination of cryptococcosis are available. Here, we describe Prm1, a novel regulator of C. neoformans virulence. C. neoformans prm1Δ cells exhibit extreme sensitivity to various environmental stress conditions. Furthermore, prm1Δ cells show deficiencies in the biosynthesis of chitosan and mannoprotein, which in turn result in impairment of cell wall integrity. Treatment of mice with heat-killed prm1Δ cells was found to facilitate the host immunological defence against infection with wild-type C. neoformans. Further investigation demonstrated that prm1Δ cells strongly promote pulmonary production of interferon-γ, leading to activation of macrophage M1 differentiation and inhibition of M2 polarization. Therefore, our findings suggest that C. neoformans Prm1 may be a viable target for the development of anti-cryptococcosis medications and, cells lacking Prm1 represent a promising candidate for a vaccine.

Mining for crypto protection: a search for Cryptosporidium antibodies reveals antigens associated with immunity.

Infectious diarrhea is a major cause of morbidity and mortality, particularly for children in low- and middle-income countries. Cryptosporidium is a diarrheal pathogen for which there is no vaccine and current therapies are only partially effective. In this issue of the JCI, Gilchrist, Campo, and colleagues surveyed a large cohort of Bangladeshi children to profile antibody responses against an array of Cryptosporidium proteins. They discovered 233 proteins to which children developed antibodies, identified seven as being associated with protection from reinfection, and provided insights regarding the longevity of Cryptosporidium antibodies and the development of antibody breadth. In this commentary, we discuss the burden of disease caused by Cryptosporidium and how these studies highlight the strategies to better manage this parasite.

CD146 deficiency promotes inflammatory type 2 responses in pulmonary cryptococcosis.

Cryptococcus neoformans (C. neoformans) is an important opportunistic fungal pathogen for pulmonary cryptococcosis. Previously, we demonstrated that CD146 mediated the adhesion of C. neoformans to the airway epithelium. CD146 is more than an adhesion molecule. In the present study, we aimed to explore the roles of CD146 in the inflammatory response in pulmonary cryptococcosis. CD146 was decreased in lung tissues from patients with pulmonary cryptococcosis. Similarly, C. neoformans reduced pulmonary CD146 expression in mice following intratracheal inoculation. To explore the pathological roles of CD146 reduction in pulmonary cryptococcosis, CD146 knockout (KO) mice were inoculated with C. neoformans via intratracheal instillation. CD146 deficiency aggravated C. neoformans infection, as evidenced by a shortened survival time and increased fungal burdens in the lung. Inflammatory type 2 cytokines (IL-4, IL-5, and TNF-α) and alternatively activated macrophages were increased in the pulmonary tissues of CD146 KO-infected mice. CD146 is expressed in immune cells (macrophages, etc.) and nonimmune cells, i.e., epithelial cells and endothelial cells. Bone marrow chimeric mice were established and infected with C. neoformans. CD146 deficiency in immune cells but not in nonimmune cells increased fungal burdens in the lung. Mechanistically, upon C. neoformans challenge, CD146 KO macrophages produced more neutrophil chemokine KC and inflammatory cytokine TNF-α. Meanwhile, CD146 KO macrophages decreased the fungicidity and production of reactive oxygen species. Collectively, C. neoformans infection decreased CD146 in pulmonary tissues, leading to inflammatory type 2 responses, while CD146 deficiency worsened pulmonary cryptococcosis.

Toll-like receptor 4 and macrophage scavenger receptor 1 crosstalk regulates phagocytosis of a fungal pathogen.

The opportunistic fungal pathogen Cryptococcus neoformans causes lethal infections in immunocompromised patients. Macrophages are central to the host response to cryptococci; however, it is unclear how C. neoformans is recognised and phagocytosed by macrophages. Here we investigate the role of TLR4 in the non-opsonic phagocytosis of C. neoformans. We find that loss of TLR4 function unexpectedly increases phagocytosis of non-opsonised cryptococci by murine and human macrophages. The increased phagocytosis observed in Tlr4-/- cells was dampened by pre-treatment of macrophages with oxidised-LDL, a known ligand of scavenger receptors. The scavenger receptor, macrophage scavenger receptor 1 (MSR1) (also known as SR-A1 or CD204) was upregulated in Tlr4-/- macrophages. Genetic ablation of MSR1 resulted in a 75% decrease in phagocytosis of non-opsonised cryptococci, strongly suggesting that it is a key non-opsonic receptor for this pathogen. We go on to show that MSR1-mediated uptake likely involves the formation of a multimolecular signalling complex involving FcγR leading to SYK, PI3K, p38 and ERK1/2 activation to drive actin remodelling and phagocytosis. Altogether, our data indicate a hitherto unidentified role for TLR4/MSR1 crosstalk in the non-opsonic phagocytosis of C. neoformans.

Efficacy and non-toxicity of ciclopirox olamine-loaded liposomes against Cryptococcus neoformans clinical isolates.

The aim of this study was to evaluate the efficacy and non-toxicity of ciclopirox olamine-loaded liposomes against Cryptococcus neoformans clinical isolates. Initially, 24-1 fractional experimental design was carried out to obtain an optimized formulation of liposomes containing CPO (CPO-LipoC), which were then used to prepare stealth liposomes (CPO-LipoS). Liposomal formulations were characterized by their mean size diameter, polydispersity index (PDI), and drug encapsulation efficiency (EE%). Immunosuppressed mice were exposed to CPO-LipoS at 0.5 mg/kg/day for 14 days to verify possible histopathological alterations in the liver and kidneys. Immunosuppressed mice infected with C. neoformans were treated with CPO-LipoS at 0.5 mg/kg/day for 14 days to quantify the fungal burden in spleen, liver, lungs, and brain. CPO-LipoS presented a mean size diameter, PDI, and EE% of 101.4 ± 0.7 nm, 0.307, and 96.4 ± 0.9%, respectively. CPO-LipoS was non-toxic for the liver and kidneys of immunosuppressed mice. At the survival curve, all infected animals submitted to treatment with CPO-LipoS survived until the end of the experiment. Treatment with CPO-LipoS reduced C. neoformans cells in the spleen (59.3 ± 3.4%), liver (75.0 ± 3.6%), lungs (75.7 ± 6.7%), and brain (54.2 ± 3.2%). CPO-LipoS exhibit antifungal activity against C. neoformans, and the encapsulation of CPO into stealth liposomes allows its use as a systemic drug for treating cryptococcosis.

CRYPTOCOCCOSIS: A bibliographic narrative review on antifungal resistance.

Cryptococcosis is an infectious fungal disease widely studied for its epidemiological importance in the context of public health, given the high morbidity and mortality associated with this invasive fungal infection. Many cases of the disease present clinical resistance and progress to death, even in the presence of antifungal therapy. The prolonged use of triazole drugs to maintain the treatment of cryptococcosis in AIDS patients, can lead to selective pressure from mutant strains, among other resistance mechanisms, justifying the poor clinical evolution of some cases. In this study, a narrative review of the literature on the occurrence of antifungal resistance in cryptococcosis agents was performed. Publications from 2010 to 2022 that address this topic were selected using Google Scholars and Scopus website. Data from the studies were analyzed for the values of minimum inhibitory concentration (MIC) of drugs used in the management of cryptococcosis. The review showed that the highest MIC values occurred for voriconazole, especially against C. neoformans. It is concluded that there is a lack of studies with statistical analysis of the data obtained, in order to provide a better dimensioning of the resistance rates of cryptococcosis agents to different antifungal agents, both in geographical and temporal context.

Searching for new antifungals for the treatment of cryptococcosis.

There is a consensus that the antifungal repertoire for the treatment of cryptococcal infections is limited. Standard treatment involves the administration of an antifungal drug derived from natural sources (i.e., amphotericin B) and two other drugs developed synthetically (i.e., flucytosine and fluconazole). Despite treatment, the mortality rates associated with fungal cryptococcosis are high. Amphotericin B and flucytosine are toxic, require intravenous administration, and are usually unavailable in low-income countries because of their high cost. However, fluconazole is cost-effective, widely available, and harmless with regard to its side effects. However, fluconazole is a fungistatic agent that has contributed considerably to the increase in fungal resistance and frequent relapses in patients with cryptococcal meningitis. Therefore, there is an unquestionable need to identify new alternatives or adjuvants to conventional drugs for the treatment of cryptococcosis. A potential antifungal agent should be able to kill cryptococci and "bypass" the virulence mechanism of the yeast. Furthermore, it should have fungicidal action, low toxicity, high selectivity, easily penetrate the central nervous system, and widely available. In this review, we describe cryptococcosis, its conventional therapy, and failures arising from the use of drugs traditionally considered to be the reference standard. Additionally, we present the approaches used for the discovery of new drugs to counteract cryptococcosis, ranging from the conventional screening of natural products to the inclusion of structural modifications to optimize anticryptococcal activity, as well as drug repositioning and combined therapies.

ATP sulfurylase atypical leucine zipper interacts with Cys3 and calcineurin A in the regulation of sulfur amino acid biosynthesis in Cryptococcus neoformans.

Fungal pathogens are a major cause of death, especially among immunocompromised patients. Therapies against invasive fungal infections are restricted to a few antifungals; therefore, novel therapies are necessary. Nutritional signaling and regulation are important for pathogen establishment in the host. In Cryptococcus neoformans, the causal agent of fungal meningitis, amino acid uptake and biosynthesis are major aspects of nutritional adaptation. Disruptions in these pathways lead to virulence attenuation in an animal model of infection, especially for sulfur uptake and sulfur amino acid biosynthesis. Deletion of Cys3, the main transcription factor that controls these pathways, is the most deleterious gene knockout in vitro and in vivo, making it an important target for further application. Previously, we demonstrated that Cys3 is part of a protein complex, including calcineurin, which is necessary to maintain high Cys3 protein levels during sulfur uptake and sulfur amino acid biosynthesis. In the current study, other aspects of Cys3 regulation are explored. Two lines of evidence suggest that C. neoformans Cys3 does not interact with the F-box WD40 protein annotated as Met30, indicating another protein mediates Cys3 ubiquitin degradation. However, we found another level of Cys3 regulation, which involves protein interactions between Cys3 and ATP sulfurylase (MET3 gene). We show that an atypical leucine zipper at the N-terminus of ATP sulfurylase is essential for physical interaction with Cys3 and calcineurin. Our data suggests that Cys3 and ATP sulfurylase interact to regulate Cys3 transcriptional activity. This work evidences the complexity involved in the regulation of a transcription factor essential for the sulfur metabolism, which is a biological process important to nutritional adaptation, oxidative stress response, nucleic acid stability, and methylation. This information may be useful in designing novel therapies against fungal infections.

Pomegranate sarcotesta lectin (PgTeL) inhibits planktonic growth and disrupts biofilm formed by Cryptococcus neoformans.

AIMS: We investigated the putative fungistatic and fungicidal activities of pomegranate sarcotesta lectin (PgTeL) against Cryptococcus neoformans B3501 (serotype D), specifically the ability of PgTeL to inhibit yeast capsule and biofilm formation in this strain. METHODS AND RESULTS: PgTeL showed a minimum inhibitory concentration of 172.0 µg ml-1, at which it did not exhibit a fungicidal effect. PgTeL concentrations of 4.0-256.0 µg ml-1 reduced biofilm biomass by 31.0%-64.0%. Furthermore, 32.0-256.0 µg ml-1 PgTeL decreased the metabolic activity of the biofilm by 32.0%-93.0%. Scanning electron microscopy images clearly revealed disruption of the biofilm matrix. Moreover, PgTeL disrupted preformed biofilms. At concentrations of 8.0-256.0 µg ml-1, PgTeL reduced metabolic activity in C. neoformans by 36.0%-92.0%. However, PgTeL did not inhibit the ability of B3501 cells to form capsules under stress conditions. CONCLUSIONS: PgTeL inhibited biofilm formation and disrupted preformed biofilms, demonstrating its potential for use as an anticryptococcal agent.

New cyclic arylguanidine scaffolds as a platform for development of antimicrobial and antiviral agents.

According to WHO, infectious diseases are still a significant threat to public health. The combine effects of antibiotic resistance, immunopressure, and mutations within the bacterial and viral genomes necessitates the search for new molecules exhibiting antimicrobial and antiviral activities. Such molecules often contain cyclic guanidine moiety. As part of this work, we investigated the selected antimicrobial and antiviral activity of compounds from the cyclic arylguanidine group. Molecules were designed using molecular modeling and obtained using microwave radiation (MW) and sonochemical ()))) methods, in accordance with the previously developed pathways. The obtained compounds were screened for the ability to inhibit the growth of Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans, and Cryptococcus neoformans. The capacity to block the entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into the host cell was probed using a bioluminescence immunoassay. The cytotoxicity and hemolytic properties of the most active molecules were also evaluated. The N-[2-(naphthalen-1-yl)ethyl]-5-phenyl-1,4,5,6-tetrahydro-1,3,5-triazin-2-amine 12j showed a high inhibition of Staphylococcus aureus and Cryptococcus neoformans (MIC ≤ 0.25 µg/mL), with no cytotoxic nor hemolytic effect (CC50, HC10 > 32 µm/mL). The CO-ADD platform identified many potentially useful molecules. A particularly rich population was examined in the database of the N.D. Zelinsky Institute of Organic Chemistry, in which 2517 active molecules (MIC ≤ 32 mg/mL) were found, of which about 10% are active at very low concentrations (MIC ≤ 1 mg/mL).

Fungal mechanisms of intracellular survival: what can we learn from bacterial pathogens?

Fungal infections represent a major, albeit neglected, public health threat with serious medical and economic burdens globally. With unacceptably high mortality rates, invasive fungal pathogens are responsible for millions of deaths each year, with a steadily increasing incidence primarily in immunocompromised individuals. The poor therapeutic options and rise of antifungal drug resistance pose further challenges in controlling these infections. These fungal pathogens have adapted to survive within mammalian hosts and can establish intracellular niches to promote survival within host immune cells. To do that, they have developed diverse methods to circumvent the innate immune system attack. This includes strategies such as altering their morphology, counteracting macrophage antimicrobial action, and metabolic adaptation. This is reminiscent of how bacterial pathogens have adapted to survive within host cells and cause disease. However, relative to the great deal of information available concerning intracellular bacterial pathogenesis, less is known about the mechanisms fungal pathogens employ. Therefore, here we review our current knowledge and recent advances in our understanding of how fungi can evade and persist within host immune cells. This review will focus on the major fungal pathogens, including Cryptococcus neoformans, Candida albicans, and Aspergillus fumigatus, among others. As we discover and understand the strategies used by these fungi, similarities with their bacterial counterparts are becoming apparent, hence we can use the abundant information from bacteria to guide our studies in fungi. By understanding these strategies, new lines of research will open that can improve the treatments of these devastating fungal diseases.

Efficient splicing of the CPE intein derived from directed evolution of the Cryptococcus neoformans PRP8 intein.

Intein-mediated protein splicing has been widely used in protein engineering; however, the splicing efficiency and extein specificity usually limit its further application. Thus, there is a demand for more general inteins that can overcome these limitations. Here, we study the trans-splicing of CPE intein obtained from the directed evolution of Cne PRP8, which shows that its splicing rate is ~29- fold higher than that of the wild-type. When the +1 residue of C-extein is changed to cysteine, CPE also shows high splicing activity. Faster association and higher affinity may contribute to the high splicing rate compared with wild-type intein. These findings have important implications for the future engineering of inteins and provide clues for fundamental studies of protein structure and folding.