A Limited Number of Amino Acid Permeases Are Crucial for Cryptococcus neoformans Survival and Virulence.

One unique attribute of Cryptococcus neoformans is its ability to procure essential monomers from its surroundings to survive in diverse environments. Preferentially, sugars are the energy sources for this opportunistic pathogenic fungus under the carbon catabolite repression (CCR); however, sugar restriction induces alternative use of low molecular weight alcohol, organic acids, and amino acids. The expression of transmembrane amino acid permeases (Aaps) allows C. neoformans to utilize different amino acids and their conjugates, notwithstanding under the nitrogen catabolite repression (NCR). Being referred to as global permeases, there is a notion that all cryptococcal Aaps are important to survival and virulence. This functional divergence makes alternative drug targeting against Cryptococcus a challenge. We examine the functions and regulations of C. neoformans Aap variants with the aim of rationalizing their relevance to cryptococcal cell survival and virulence. Based on nutrient bioavailability, we linked the Cac1 pathway to Ras1 activation for thermotolerance that provides a temperature cushion for Aap activity under physiological conditions. Lastly, mutants of Aaps are examined for significant phenotypic deficiencies/advantages, which buttress the specific importance of limited numbers of Aaps involved in cryptococcal infections.

Aspergillus fumigatus secretes a protease(s) that displays in silico binding affinity towards the SARS-CoV-2 spike protein and mediates SARS-CoV-2 pseudovirion entry into HEK-293T cells.

BACKGROUND: The novel coronavirus disease of 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Data from the COVID-19 clinical control case studies showed that this disease could also manifest in patients with underlying microbial infections such as aspergillosis. The current study aimed to determine if the Aspergillus (A.) fumigatus culture media (i.e., supernatant) possessed protease activity that was sufficient to activate the SARS-CoV-2 spike protein. METHODS: The supernatant was first analysed for protease activity. Thereafter, it was assessed to determine if it possessed proteolytic activity to cleave a fluorogenic mimetic peptide of the SARS-CoV-2 spike protein that contained the S1/S2 site and a full-length spike protein contained in a SARS-CoV-2 pseudovirion. To complement this, a computer-based tool, HADDOCK, was used to predict if A. fumigatus alkaline protease 1 could bind to the SARS-CoV-2 spike protein. RESULTS: We show that the supernatant possessed proteolytic activity, and analyses of the molecular docking parameters revealed that A. fumigatus alkaline protease 1 could bind to the spike protein. To confirm the in silico data, it was imperative to provide experimental evidence for enzymatic activity. Here, it was noted that the A. fumigatus supernatant cleaved the mimetic peptide as well as transduced the HEK-293T cells with SARS-CoV-2 pseudovirions. CONCLUSION: These results suggest that A. fumigatus secretes a protease(s) that activates the SARS-CoV-2 spike protein. Importantly, should these two infectious agents co-occur, there is the potential for A. fumigatus to activate the SARS-CoV-2 spike protein, thus aggravating COVID-19 development.

Cryptococcal proteases exhibit the potential to activate the latent SARS-CoV-2 spike protein.

BACKGROUND: The COVID-19 pandemic has affected more than 650 million people and resulted in over 6.8 million deaths. Notably, the disease could co-manifest with microbial infections, like cryptococcosis, which also presents as a primary lung infection. OBJECTIVE: In this contribution, we sought to determine if cryptococcal supernatant (which contains secreted furin-like proteases) could activate the SARS-CoV-2 spike protein. METHODS: Molecular docking of the crystal structures of the SARS-CoV-2 spike protein (target) and selected cryptococcal proteases (ligands) was executed using the high ambiguity driven protein-protein docking (HADDOCK) server, with the furin protease serving as a reference ligand. The furin protease is found in human cells and typically activates the SARS-CoV-2 spike protein. Importantly, in order to provide experimental evidence for enzymatic activity, we also assessed the biochemical efficiency of cryptococcal proteases to initiate viral entry into HEK-293 T cells by SARS-CoV-2 spike pseudotyped Lentivirus. RESULTS: We show that the selected cryptococcal proteases could interact with the spike protein, and some had a better or comparable binding affinity for the spike protein than furin protease following an in silico comparative analysis of the molecular docking parameters. Furthermore, it was noted that the biochemical efficiency of the cryptococcal supernatant to transduce HEK-293 T cells with SARS-CoV-2 pseudovirions was comparable (p > 0.05) to that of recombinant furin. CONCLUSIONS: Taken together, these data show that cryptococcal proteases could activate the SARS-CoV-2 spike protein. In practice, it may be critical to determine if patients have an underlying cryptococcal infection, as this microbe could secrete proteases that may further activate the SARS-CoV-2 viral particles, thus undermining COVID-19 intervention measures.

Primaquine, an antimalarial drug that controls the growth of cryptococcal cells.

INTRODUCTION: The treatment of Cryptococcus neoformans with fluconazole and amphotericin B is, at times, characterised by clinical failure. Therefore, this study sought to re-purpose primaquine (PQ) as an anti-Cryptococcus compound. METHOD: The susceptibility profile of some cryptococcal strains towards PQ was determined using EUCAST guidelines, and PQ's mode of action was examined. In the end, the ability of PQ to enhance in vitro macrophage phagocytosis was also assessed. RESULTS: We show that PQ had a significant inhibitory effect on the metabolic activity of all tested cryptococcal strains, with 60 µM, defined as MIC50 in this preliminary study, as it reduced the metabolic activity by more than 50%. Moreover, at this concentration, the drug was able to affect mitochondrial function adversely, as treated cells displayed significant (p < 0.05) loss of mitochondrial membrane potential, cytochrome c (cyt c) leakage and overproduction of reactive oxygen species (ROS) when compared to non-treated cells. It is our reasoned summation that the produced ROS targeted the cell walls and cell membranes, inducing observable ultrastructural changes and a significant (p < 0.05) increase in membrane permeability when compared to non-treated cells. Concerning the PQ effect on macrophages, it was noted that it significantly (p < 0.05) enhanced macrophage phagocytic efficiency compared to non-treated macrophages. CONCLUSION: This preliminary study highlights the potential of PQ to inhibit the in vitro growth of cryptococcal cells. Moreover, PQ could control the proliferation of cryptococcal cells inside macrophages, which they often manipulate in a Trojan horse-like manner.

Cryptococcal Protease(s) and the Activation of SARS-CoV-2 Spike (S) Protein.

In this contribution, we report on the possibility that cryptococcal protease(s) could activate the SARS-CoV-2 spike (S) protein. The S protein is documented to have a unique four-amino-acid sequence (underlined, SPRRAR↓S) at the interface between the S1 and S2 sites, that serves as a cleavage site for the human protease, furin. We compared the biochemical efficiency of cryptococcal protease(s) and furin to mediate the proteolytic cleavage of the S1/S2 site in a fluorogenic peptide. We show that cryptococcal protease(s) processes this site in a manner comparable to the efficiency of furin (p > 0.581). We conclude the paper by discussing the impact of these findings in the context of a SARS-CoV-2 disease manifesting while there is an underlying cryptococcal infection.

The first survey of cryptococcal cells in bird droppings across Bloemfontein, South Africa.

BACKGROUND AND AIM: Cryptococcal yeast cells are spread across different ecosystems through bird movement and are deposited in bird guano. These cells may be inhaled by humans and lead to cryptococcal pneumonia. In individuals with reduced immune T-cell populations, cells may disseminate to the brain and cause the often-deadly cryptococcal meningitis. In this study, we surveyed cryptococcal cells in bird droppings across the city of Bloemfontein, South Africa. MATERIALS AND METHODS: We aseptically collected 120 bird dropping samples from 15 representative city sites. In the laboratory, samples were assessed with regards to location, weighed, and standardized to a mass of 1 g before suspension in 10 mL phosphate buffer saline. Samples were first screened usingCalcofluor-white stain as it is a rapid technique for the detection of fungi via binding to cell wall components such as chitin. After this, positive Calcofluor samples were serologically assayed for the cryptococcal antigen (CrAg). To confirm assay data, CrAg positive samples were then cultured on bird seed agar and resulting colonies were assessed using Indian ink. RESULTS: We determined that 10/15 locations were positive for the CrAg. Pathogenic cells were identified on bird seed agar as brown colonies. When examined using microscopy, brown colony cells exhibited characteristic thick capsules representative of cryptococcal cells. CONCLUSION: This is the first proximate analysis showing the ecological distribution of cryptococcal cells in Bloemfontein. This is important as associated infections are acquired from the environment. Similarly, given the threat posed by cryptococcal cells to immunocompromised individuals, local authorities must initiate measures curbing the spread of these cells.

The Possible Role of Microbial Proteases in Facilitating SARS-CoV-2 Brain Invasion.

SARS-CoV-2 has been shown to display proclivity towards organs bearing angiotensin-converting enzyme (ACE2) expression cells. Of interest herein is the ability of the virus to exhibit neurotropism. However, there is limited information on how this virus invades the brain. With this contribution, we explore how, in the context of a microbial co-infection using a cryptococcal co-infection as a model, SARS-CoV-2 could reach the brain. We theorise that the secretion of proteases by disseminated fungal cells might also activate the S2 domain of the viral spike glycoprotein for membrane fusion with brain endothelial cells leading to endocytosis. Understanding this potential invasion mechanism could lead to better SARS-CoV-2 intervention measures, which may also be applicable in instances of co-infection, especially with protease-secreting pathogens.

Inhibitory effect of polyunsaturated fatty acids alone or in combination with fluconazole on Candida krusei biofilms in vitro and in Caenorhabditis elegans.

The incidence of infections by non-albicans Candida species, including Candida krusei, is increasing. Candida krusei exhibits intrinsic resistance to fluconazole and rapidly develops acquired resistance to other antifungals. Moreover, this yeast can form biofilm with increased resistance. Hence, there is a need to develop novel therapeutic strategies to combat infections caused by this pathogen. One such approach is through combination therapy with natural compounds, such as polyunsaturated fatty acids (PUFAs). This study aims to investigate the effect of PUFAs on fluconazole susceptibility of C. krusei biofilms, as well as the conserved nature of these effects in the Caenorhabditis elegans infection model. C. krusei biofilms were exposed to various fatty acids as well as combinations of fluconazole and linoleic acid (LA) or gamma-linolenic acid (GLA). The effect of these treatments on biofilm formation, cell ultrastructure, membrane integrity, oxidative stress and efflux pump activity was evaluated. In addition, the ability of the PUFAs to prolong survival and reduce the fungal burden of infected C. elegans, in the absence and presence of fluconazole, was assessed. Two PUFAs, LA and GLA had displayed significant inhibition of C. krusei biofilms and both of them increased the susceptibility of C. krusei biofilm to fluconazole in vitro via induction of oxidative stress, cell membrane damage, and disruption of efflux pump activity. These PUFAs also extended the lifespan of infected nematodes and displayed a potentiating effect with fluconazole in this model. This may pave the way for future studies into novel antifungal drug targets and treatment options. LAY SUMMARY: The pathogenic yeast, Candida krusei, is naturally resistant to the antifungal drug, fluconazole. This study finds that polyunsaturated fatty acids, linoleic and gamma-linolenic acid, can inhibit C. krusei and overcome this resistance of in vitro biofilms, as well as in a nematode infection model.

The role of lipid droplets in microbial pathogenesis.

The nonpolar lipids present in cells are mainly triacylglycerols and steryl esters. When cells are provided with an abundance of nutrients, these storage lipids accumulate. As large quantities of nonpolar lipids cannot be integrated into membranes, they are isolated from the cytosolic environment in lipid droplets. As specialized, inducible cytoplasmic organelles, lipid droplets have functions beyond the regulation of lipid metabolism, in cell signalling and activation, membrane trafficking and control of inflammatory mediator synthesis and secretion. Pathogens, including fungi, viruses, parasites, or intracellular bacteria can induce and may benefit from lipid droplets in infected cells. Here we review biogenesis of lipid droplets as well as the role of lipid droplets in the pathogenesis of selected viruses, bacteria, protists and yeasts.

Candida albicans SET3 Plays a Role in Early Biofilm Formation, Interaction With Pseudomonas aeruginosa and Virulence in Caenorhabditis elegans.

The yeast Candida albicans exhibits multiple morphologies dependent on environmental cues. Candida albicans biofilms are frequently polymicrobial, enabling interspecies interaction through proximity and contact. The interaction between C. albicans and the bacterium, Pseudomonas aeruginosa, is antagonistic in vitro, with P. aeruginosa repressing the yeast-to-hyphal switch in C. albicans. Previous transcriptional analysis of C. albicans in polymicrobial biofilms with P. aeruginosa revealed upregulation of genes involved in regulation of morphology and biofilm formation, including SET3, a component of the Set3/Hos2 histone deacetylase complex (Set3C). This prompted the question regarding the involvement of SET3 in the interaction between C. albicans and P. aeruginosa, both in vitro and in vivo. We found that SET3 may influence early biofilm formation by C. albicans and the interaction between C. albicans and P. aeruginosa. In addition, although deletion of SET3 did not alter the morphology of C. albicans in the presence of P. aeruginosa, it did cause a reduction in virulence in a Caenorhabditis elegans infection model, even in the presence of P. aeruginosa.

The Repurposing of Acetylsalicylic Acid as a Photosensitiser to Inactivate the Growth of Cryptococcal Cells.

Photodynamic treatment (PDT) is often successful when used against aerobic microbes, given their natural susceptibility to oxidative damage. To this end, the current study aimed to explore the photodynamic action of acetylsalicylic acid (ASA; aspirin, which is commonly used to treat non-infectious ailments), when administered to respiring cryptococcal cells. The treatment of cryptococcal cells, i.e., exposure to 0.5 or 1 mM of ASA in the presence of ultraviolet light (UVL) for 10 min, resulted in a significant (p < 0.05) reduction in the growth of tested cells when compared to non-treated (non-Rx) cells, i.e., no ASA and no UVL. The treated cells were also characterised by diseased mitochondria, which is crucial for the survival of respiring cells, as observed by a significant (p < 0.05) loss of mitochondrial membrane potential (ΔΨM) and significant (p < 0.05) accumulation of reactive oxygen species (ROS) when compared to non-Rx cells. Moreover, the photolytic products of acetylsalicylic acid altered the ultrastructural appearance of treated cells as well as limited the expression levels of the capsular-associated gene, CAP64, when compared to non-Rx cells. The results of the study highlight the potential use of ASA as a photosensitiser that is effective for controlling the growth of cryptococcal cells. Potentially, this treatment can also be used as an adjuvant, to complement and support the usage of current anti-microbial agents.

Role of the high-affinity reductive iron acquisition pathway of Candida albicans in prostaglandin E2 production, virulence, and interaction with Pseudomonas aeruginosa.

Components of the iron reductive pathway of Candida albicans have been implicated in the production of prostaglandin E2 (PGE2) and virulence. However, it is unknown whether other components of this pathway influence PGE2. We investigated the role of the iron reductive pathway of C. albicans in biofilm formation, PGE2 production, and virulence in Caenorhabditis elegans. Additionally, as the co-occurrence of C. albicans and Pseudomonas aeruginosa in host tissues is frequent and involves competition for host-associated iron, we examined the effects of this interaction. Deletion of multicopper oxidase gene, FET99, and iron permease genes, FTH1 and FTH2, affected biofilm metabolic activity, and for the FTH2 mutant, also biofilm morphology. Deletion of CCC1 (vacuolar iron transporter) and CCC2 (P-type ATPase copper importer) also influenced biofilm morphology. For PGE2 production, deletion of FET99, FTH1, FTH2, CCC1, and CCC2 caused a significant reduction by monomicrobial biofilms, while FTH2deletion caused the highest reduction in polymicrobial biofilms. URA3 positive mutants of FET99 and FTH2 demonstrated attenuated virulence in C. elegans, potentially due to the inability of mutants to form hyphae in vivo. Deductively, the role of the iron reductive pathway in PGE2 synthesis is indirect, possibly due to their role in iron homeostasis. LAY SUMMARY: Iron uptake is vital for disease-causing microbes like Candida albicans. Using strains deficient in some iron-uptake genes, we show that iron-uptake genes, especially FET99 and FTH2, play a role in biofilm formation, prostaglandin production, and virulence in the nematode infection model.

Overview of the Development, Impacts, and Challenges of Live-Attenuated Oral Rotavirus Vaccines.

Safety, efficacy, and cost-effectiveness are paramount to vaccine development. Following the isolation of rotavirus particles in 1969 and its evidence as an aetiology of severe dehydrating diarrhoea in infants and young children worldwide, the quest to find not only an acceptable and reliable but cost-effective vaccine has continued until now. Four live-attenuated oral rotavirus vaccines (LAORoVs) (Rotarix®, RotaTeq®, Rotavac®, and RotaSIIL®) have been developed and licensed to be used against all forms of rotavirus-associated infection. The efficacy of these vaccines is more obvious in the high-income countries (HIC) compared with the low- to middle-income countries (LMICs); however, the impact is far exceeding in the low-income countries (LICs). Despite the rotavirus vaccine efficacy and effectiveness, more than 90 countries (mostly Asia, America, and Europe) are yet to implement any of these vaccines. Implementation of these vaccines has continued to suffer a setback in these countries due to the vaccine cost, policy, discharging of strategic preventive measures, and infrastructures. This review reappraises the impacts and effectiveness of the current live-attenuated oral rotavirus vaccines from many representative countries of the globe. It examines the problems associated with the low efficacy of these vaccines and the way forward. Lastly, forefront efforts put forward to develop initial procedures for oral rotavirus vaccines were examined and re-connected to today vaccines.

Environmental Factors That Contribute to the Maintenance of Cryptococcus neoformans Pathogenesis.

The ability of microorganisms to colonise and display an intracellular lifestyle within a host body increases their fitness to survive and avoid extinction. This host-pathogen association drives microbial evolution, as such organisms are under selective pressure and can become more pathogenic. Some of these microorganisms can quickly spread through the environment via transmission. The non-transmittable fungal pathogens, such as Cryptococcus, probably return into the environment upon decomposition of the infected host. This review analyses whether re-entry of the pathogen into the environment causes restoration of its non-pathogenic state or whether environmental factors and parameters assist them in maintaining pathogenesis. Cryptococcus (C.) neoformans is therefore used as a model organism to evaluate the impact of environmental stress factors that aid the survival and pathogenesis of C. neoformans intracellularly and extracellularly.

Caenorhabditis elegans as a model animal for investigating fungal pathogenesis.

The morbidity and mortality associated with systemic fungal infections in humans cannot be underestimated. The nematode Caenorhabditis elegans has become popular for the in vivo study of the pathogenesis of human fungal pathogens and as an antifungal drug-screening tool. C. elegans offers many advantages as a model organism for the study of human fungal diseases, including lack of ethics requirements, easy maintenance in the laboratory, fully sequenced genome, availability of genetic mutants, and the possibility of liquid assays for high-throughput antifungal screening. Its major drawbacks include the inability to grow at 37 °C and absence of an adaptive immune response. However, several virulence factors involved in the pathogenesis of medically important fungal pathogens have been identified using the C. elegans model, consequently providing new leads for drug discovery and potential drug targets. We review the use of C. elegans as a model animal to understand the pathogenesis of medically important human fungal pathogens and the discovery of novel antifungal compounds. The review makes a case for C. elegans as a suitable invertebrate model for a plethora of practical applications in the investigation of fungal pathogenesis as well as its amenability for liquid-based high-throughput screening of potential antifungal compounds.

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions.

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for macrophages. This predatory organism, similar to macrophages, employs phagocytosis to kill internalized cells. With the aid of a confocal laser-scanning microscope, images depicting interactive moments between cryptococcal cells and amoeba are captured. The resolution power of the electron microscope also helps to reveal the ultrastructural detail of cryptococcal cells when trapped inside the amoeba food vacuole. Since phagocytosis is a continuous process, quantitative data is then integrated in the analysis to explain what happens at the timepoint when an image is captured. To be specific, relative fluorescence units are read in order to quantify the efficiency of amoeba in internalizing cryptococcal cells. For this purpose, cryptococcal cells are stained with a dye that makes them fluoresce once trapped inside the acidic environment of the food vacuole. When used together, information gathered through such techniques can provide critical information to help draw conclusions on the behavior and fate of cells when internalized by amoeba and, possibly, by other phagocytic cells.

Functional Characterization of Cryptococcal Genes: Then and Now.

Site-directed mutagenesis enables researchers to switch a gene of interest off for functional characterization of the gene. In the pathogenic yeasts, Cryptococcus neoformans and sister species C. deneoformans, this is almost exclusively achieved by introducing DNA into cells through either biolistic transformation or electroporation. The targeted gene is then disrupted by homologous recombination (HR) between the gene and the transforming DNA. Both techniques have downsides; biolistic transformation equipment is very expensive, limiting the use thereof to well-resourced laboratories, and HR occurs at extremely low frequencies in electroporated cryptococcal cells, making this method unappealing for gene targeting when not making use of additional modifications or methods to enhance HR in these cells. One approach to increase the frequency of HR in electroporated cryptococcal cells have recently been described. In this approach, CRISPR-Cas9 technology is utilized to form a double strand break in the targeted gene where after the occurrence of HR seems to be higher. The less expensive electroporation technique can therefore be used to deliver the CRISPR-Cas9 components into cells to disrupt a gene of interest, but only if the CRISPR components can be maintained for long enough in cells to enable their expression. Maintenance of episomal DNA occurs readily in C. deneoformans, but only under certain conditions in C. neoformans. In addition, CRISPR-Cas9 allows for gene complementation in order to fulfill Falkow's molecular Koch's postulates and adds other novel methods for studying genes as well, such as the addition of a fluorophore to an inactive Cas9 enzyme to highlight the location of a gene in a chromosome. These developments add less expensive alternatives to current methods, which could lead to more research on this yeast in developing countries where cryptococcal infections are more prevalent and researchers have access to more clinical isolates.

Copper Acyl Salicylate Has Potential as an Anti-Cryptococcus Antifungal Agent.

The in vitro antifungal activity of aspirin against cryptococcal cells has been reported. However, the unwanted effects of aspirin may limit its clinical application. Conceivably, a derivative of aspirin could overcome this challenge. Toward this end, this study considered the usage of an aspirinate-metal complex, namely, copper acyl salicylate (CAS), as an anti-Cryptococcus antifungal agent. Additionally, the study examined the effects of this compound on macrophage function. The in vitro susceptibility results revealed that cryptococcal cells were vulnerable (in a dose-dependent manner) to CAS, which might have effected growth inhibition by damaging cryptococcal cell membranes. Interestingly, when CAS was used in combination with fluconazole or amphotericin B, synergism was observed. Furthermore, CAS did not negatively affect the growth or metabolic activity of macrophages; rather, it sensitized those immune cells to produce interferon gamma and interleukin 6, which, in turn, might have aided in the phagocytosis of cryptococcal cells. Compared to our aspirin data, CAS was noted to be more effective in killing cryptococcal cells (based on susceptibility results) and less toxic toward macrophages (based on growth inhibition results). Taking these findings together, it is reasonable to conclude that CAS may be a better anti-Cryptococcus drug that could deliver better therapeutic outcomes, compared to aspirin.

Elucidation of the Role of 3-Hydroxy Fatty Acids in Cryptococcus-amoeba Interactions.

We previously reported that 3-hydroxy fatty acids promoted the survival of cryptococcal cells when acted upon by amoebae. To expand on this, the current study sought to explain how these molecules may protect cells. Our data suggest that 3-hydroxy fatty acids may subvert the internalization of cryptococcal cells via suppression of the levels of a fetuin A-like amoebal protein, which may be important for enhancing phagocytosis. Additionally, we show that an acapsular strain (that is devoid of 3-hydroxy fatty acids) was protected against the effects of hydrogen peroxide when exogenous 3-hydroxy fatty acids were present, but not in the absence of 3-hydroxy fatty acids. A similar response profile was noted when a strain with a capsule was challenged with hydrogen peroxide. We also show that cryptococcal cells that naturally produce 3-hydroxy fatty acids were more resistant to the effects of amoebapore (an amoeba-specific hydrolytic enzyme), compared to cells that do not produce these molecules. Taken together, our findings suggest that 3-hydroxy fatty acids possess an anti-phagocytic activity that may be expressed when cells interact with macrophages. This may allow the yeast cells to evade immuno-processing.