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.

Repurposing of Aspirin and Ibuprofen as Candidate Anti-Cryptococcus Drugs.

The usage of fluconazole and amphotericin B in clinical settings is often limited by, among other things, drug resistance development and undesired side effects. Thus, there is a constant need to find new drugs to better manage fungal infections. Toward this end, the study described in this paper considered the repurposing of aspirin (acetylsalicylic acid) and ibuprofen as alternative drugs to control the growth of cryptococcal cells. In vitro susceptibility tests, including a checkerboard assay, were performed to assess the response of Cryptococcus neoformans and Cryptococcus gattii to the above-mentioned anti-inflammatory drugs. Next, the capacity of these two drugs to induce stress as well as their mode of action in the killing of cryptococcal cells was determined. The studied fungal strains revealed a response to both aspirin and ibuprofen that was dose dependent, with ibuprofen exerting greater antimicrobial action. More importantly, the MICs of these drugs did not negatively (i) affect growth or (ii) impair the functioning of macrophages; rather, they enhanced the ability of these immune cells to phagocytose cryptococcal cells. Ibuprofen was also shown to act in synergy with fluconazole and amphotericin B. The treatment of cryptococcal cells with aspirin or ibuprofen led to stress induction via activation of the high-osmolarity glycerol (HOG) pathway, and cell death was eventually achieved through reactive oxygen species (ROS)-mediated membrane damage. The presented data highlight the potential clinical application of aspirin and ibuprofen as candidate anti-Cryptococcus drugs.

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 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.

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 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.

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.

The Repurposing of Anti-Psychotic Drugs, Quetiapine and Olanzapine, as Anti-Cryptococcus Drugs.

The management of cryptococcal infections is often difficult. This can, in part, be attributed to the fungistatic nature of fluconazole, which may result in cells disseminating to give rise to pathogen-emergent psychosis following brain inflammation. This chance at treatment failure has necessitated the current study wherein the antimicrobial quality of anti-psychotic drugs viz. quetiapine and olanzapine, was assessed. The response of test strains toward quetiapine or olanzapine alone and in combined therapy with fluconazole or amphotericn B was measured. In addition, the mode of action of the two anti-psychotic drugs in killing cryptococcal cells was determined. At the end, the ability of these anti-psychotic drugs to chemo-sensitize macrophages was also examined. The assessed strains were shown to be susceptible to the two anti-psychotic drugs, which possibly killed them via altering their membrane function. Additionally, these anti-psychotic drugs acted in synergy with fluconazole and amphotericin B in controlling the growth of the test strains. Importantly, these drugs improved the phagocytic efficiency of macrophages and, at the same time, stimulated them to produce pro-inflammatory cytokines (interleukin 6 and interferon gamma), said to be critical in the clearance of cryptococcal cells. The minimum inhibition concentration of each anti-psychotic drugs was calculated to be within its respective recommended therapeutic range. This study's findings highlight the potential clinical application of quetiapine and olanzapine as alternative anti-Cryptococcus drugs, which can be used to manage the fungal burden (infection) as well as the associated symptom (psychosis).

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.

Method for identification of Cryptococcus neoformans and Cryptococcus gattii useful in resource-limited settings.

AIMS: The high HIV/AIDS burden in Sub-Saharan Africa has led to cryptococcosis becoming a public health concern. In this resource-limited setting, conventional identification methods are mainly used to diagnose cryptococcal infections. However, these methods are often inconsistent, and importantly, cannot discriminate between the aetiological agents, Cryptococcus neoformans and C. gattii. Therefore, there is a need for an alternative reliable method to identify these species. METHODS: We examined the usefulness of a PCR method, including a restriction digest, in identifying clinical C. neoformans and C. gattii isolates. In addition, matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-ToF MS) was performed for validation purposes. RESULTS: The intraspecific variation between tested strains allowed for their delineation into three traditional varieties of C. neoformans, that is, varietal forms: neoformans, grubii and gattii. Furthermore, we uncovered a restriction site (signature sequence: 5'-AATATT-3') that is present only in the distinct species C. neoformans (varietal forms neoformans and grubii), and is, importantly, absent in the distinct species C. gattii (C. neoformans var. gattii). Thus, we were able to discriminate the distinct species by directly digesting the PCR amplicons using the endonuclease SspI. It was also possible to delineate some tested isolates as either C. neoformans or C. gattii using our MALDI-ToF MS data. CONCLUSIONS: The possibility of performing only a restriction digest makes the outlined method, similar to conventional techniques, economical and easy to optimise for routine use in resource-limited settings.

Cryptococcal 3-Hydroxy Fatty Acids Protect Cells Against Amoebal Phagocytosis.

We previously reported on a 3-hydroxy fatty acid that is secreted via cryptococcal capsular protuberances - possibly to promote pathogenesis and survival. Thus, we investigated the role of this molecule in mediating the fate of Cryptococcus (C.) neoformans and the related species C. gattii when predated upon by amoebae. We show that this molecule protects cells against the phagocytic effects of amoebae. C. neoformans UOFS Y-1378 (which produces 3-hydroxy fatty acids) was less sensitive toward amoebae compared to C. neoformans LMPE 046 and C. gattii R265 (both do not produce 3-hydroxy fatty acids) and addition of 3-hydroxy fatty acids to C. neoformans LMPE 046 and C. gattii R265 culture media, causes these strains to become more resistant to amoebal predation. Conversely, addition of aspirin (a 3-hydroxy fatty acid inhibitor) to C. neoformans UOFS Y-1378 culture media made cells more susceptible to amoebae. Our data suggest that this molecule is secreted at a high enough concentration to effect intracellular signaling within amoeba, which in turn, promotes fungal survival.