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ABSTRACTFungal infections are a growing global health concern due to the limited number of available antifungal therapies as well as the emergence of fungi that are resistant to first-line antimicrobials, particularly azoles and echinocandins. Development of novel, selective antifungal therapies is challenging due to similarities between fungal and mammalian cells. An attractive source of potential antifungal treatments is provided by ecological niches co-inhabited by bacteria, fungi, and multicellular organisms, where complex relationships between multiple organisms have resulted in evolution of a wide variety of selective antimicrobials. Here, we characterized several analogs of one such natural compound, collismycin A. We show that NR-6226C has antifungal activity against several pathogenic Candida species, including C. albicans and C. glabrata, whereas it only has little toxicity against mammalian cells. Mechanistically, NR-6226C selectively chelates iron, which is a limiting factor for pathogenic fungi during infection. As a result, NR-6226C treatment causes severe mitochondrial dysfunction, leading to formation of reactive oxygen species, metabolic reprogramming, and a severe reduction in ATP levels. Using an in vivo model for fungal infections, we show that NR-6226C significantly increases survival of Candida-infected Galleria mellonella larvae. Finally, our data indicate that NR-6226C synergizes strongly with fluconazole in inhibition of C. albicans. Taken together, NR-6226C is a promising antifungal compound that acts by chelating iron and disrupting mitochondrial functions.IMPORTANCEDrug-resistant fungal infections are an emerging global threat, and pan-resistance to current antifungal therapies is an increasing problem. Clearly, there is a need for new antifungal drugs. In this study, we characterized a novel antifungal agent, the collismycin analog NR-6226C. NR-6226C has a favorable toxicity profile for human cells, which is essential for further clinical development. We unraveled the mechanism of action of NR-6226C and found that it disrupts iron homeostasis and thereby depletes fungal cells of energy. Importantly, NR-6226C strongly potentiates the antifungal activity of fluconazole, thereby providing inroads for combination therapy that may reduce or prevent azole resistance. Thus, NR-6226C is a promising compound for further development into antifungal treatment.

Cryptococcus neoformans is a pathogenic yeast that can form Titan cells in the lungs, which are fungal cells of abnormally large size. The factors that regulate Titan cell formation in vivo are still unknown, although an increased proportion of these fungal cells of infected mice correlates with induction of Th2-type responses. Here, we focused on the role played by the cytokine IL-17 in the formation of cryptococcal Titan cells using Il17a−/− knockout mice. We found that after 9 days of infection, there was a lower proportion of Titan cells in Il17a−/− mice compared to the fungal cells found in wild-type animals. Dissemination to the brain occurred earlier in Il17a−/− mice, which correlated with the lower proportion of Titan cells in the lungs. Furthermore, knockout-infected mice increased brain size more than WT mice. We also determined the profile of cytokines accumulated in the brain, and we found significant differences between both mouse strains. We found that in Il17a−/−, there was a modest increase in the concentrations of the Th1 cytokine TNF-α. To validate if the increase in this cytokine had any role in cryptococcal morphogenesis, we injected wild-type mice with TNF-α t and observed that fungal cell size was significantly reduced in mice treated with this cytokine. Our results suggest a compensatory production of cytokines in Il17a−/− mice that influences both cryptococcal morphology and dissemination.

Due to its location, the fungal cell wall is the compartment that allows the interaction with the environment and/or the host, playing an important role during infection as well as in different biological functions such as cell morphology, cell permeability and protection against stress. All these processes involve the activation of signaling pathways within the cell. The cell wall integrity (CWI) pathway is the main route responsible for maintaining the functionality and proper structure of the cell wall. This pathway is highly conserved in the fungal kingdom and has been extensively characterized in Saccharomyces cerevisiae. However, there are still many unknown aspects of this pathway in the pathogenic fungi, such as Cryptococcus neoformans. This yeast is of particular interest because it is found in the environment, but can also behave as pathogen in multiple organisms, including vertebrates and invertebrates, so it has to adapt to multiple factors to survive in multiple niches. In this review, we summarize the components of the CWI pathway in C. neoformans as well as its involvement in different aspects such as virulence factors, morphological changes, and its role as target for antifungal therapies among others.

The fungal cell wall is located outside the plasma membrane and is the cell compartment that mediates all the relationships of the cell with the environment. It protects the contents of the cell, gives rigidity and defines the cellular structure. The cell wall is a skeleton with high plasticity that protects the cell from different stresses, among which osmotic changes stand out. The cell wall allows interaction with the external environment since some of its proteins are adhesins and receptors. Since, some components have a high immunogenic capacity, certain wall components can drive the host’s immune response to promote fungus growth and dissemination. The cell wall is a characteristic structure of fungi and is composed mainly of glucans, chitin and glycoproteins. As the components of the fungal cell wall are not present in humans, this structure is an excellent target for antifungal therapy. In this article, we review recent data on the composition and synthesis, influence of the components of the cell wall in fungi-host interaction and the role as a target for the next generation of antifungal drugs in yeasts (Candida and Cryptococcus) and filamentous fungi (Aspergillus).

Cryptococcus neoformans is an encapsulated pathogenic yeast that can change the size of the cells during infection. In particular, this process can occur by enlarging the size of the capsule without modifying the size of the cell body, or by increasing the diameter of the cell body, which is normally accompanied by an increase of the capsule too. This last process leads to the formation of cells of an abnormal enlarged size denominated titan cells. Previous works characterized titan cell formation during pulmonary infection but research on this topic has been hampered due to the difficulty to obtain them in vitro. In this work, we describe in vitro conditions (low nutrient, serum supplemented medium at neutral pH) that promote the transition from regular to titan-like cells. Moreover, addition of azide and static incubation of the cultures in a CO2 enriched atmosphere favored cellular enlargement. This transition occurred at low cell densities, suggesting that the process was regulated by quorum sensing molecules and it was independent of the cryptococcal serotype/species. Transition to titan-like cell was impaired by pharmacological inhibition of PKC signaling pathway. Analysis of the gene expression profile during the transition to titan-like cells showed overexpression of enzymes involved in carbohydrate metabolism, as well as proteins from the coatomer complex, and related to iron metabolism. Indeed, we observed that iron limitation also induced the formation of titan cells. Our gene expression analysis also revealed other elements involved in titan cell formation, such as calnexin, whose absence resulted in appearance of abnormal large cells even in regular rich media. In summary, our work provides a new alternative method to investigate titan cell formation devoid the bioethical problems that involve animal experimentation.

Cryptococcus neoformans is an environmental encapsulated yeast that behaves as an opportunistic pathogen in immunocompromised individuals. The capsule is the main virulence factor of this pathogen. This structure is highly dynamic, and it can change its size and structure according to the environmental conditions. During infection, C. neoformans significantly enlarges the size of the capsule by the addition of new polysaccharide. It is believed that capsule growth is an energy-cost process, but this aspect has never been addressed. In this work, we have evaluated the role of mitochondrial activity on capsule growth using specific inhibitors of the electron respiratory chain. We observed that capsule growth was impaired in the presence of inhibitors of the respiratory chain as salicylhydroxamic acid or antimycin A. Furthermore, capsule growth correlated with an increase of the mitochondrial membrane potential and higher production of reactive oxygen species. Our results confirm that capsule growth depends on mitochondrial activity, and open new insights to understand the regulation of this process.

In many aspects, the immune response against pathogens in insects is similar to the innate immunity in mammals. This has caused a strong interest in the scientific community for the use of this model in research of host–pathogen interactions. In recent years, the use of Galleria mellonella larvae, an insect belonging to the Lepidoptera order, has emerged as an excellent model to study the virulence of human pathogens. It is a model that offers many advantages; for example, it is easy to handle and establish in every laboratory, the larvae have a low cost, and they tolerate a wide range of temperatures, including human temperature 37 °C. The immune response of G. mellonella is innate and is divided into a cellular component (hemocytes) and humoral component (antimicrobial peptides, lytic enzymes, and peptides and melanin) that work together against different intruders. It has been shown that the immune response of this insect has a great specificity and has the ability to distinguish between different classes of microorganisms. In this review, we delve into the different components of the innate immune response of Galleria mellonella, and how these components manifest in the infection of fungal pathogens including Candida albicans, Aspergillus fumigatus, Cryptococcus neoformans, and Histoplasma capsulatum.

ABSTRACT The fungal pathogen Cryptococcus neoformans has several virulence factors, among which the most important is a polysaccharide capsule. The size of the capsule is variable and can increase significantly during infection. In this work, we investigated the relationship between capsular enlargement and the cell cycle. Capsule growth occurred primarily during the G1 phase. Real-time visualization of capsule growth demonstrated that this process occurred before the appearance of the bud and that capsule growth arrested during budding. Benomyl, which arrests the cells in G2/M, inhibited capsule growth, while sirolimus (rapamycin) addition, which induces G1 arrest, resulted in cells with larger capsule. Furthermore, we have characterized a mutant strain that lacks a putative G1/S cyclin. This mutant showed an increased capacity to enlarge the capsule, both in vivo (using Galleria mellonella as the host model) and in vitro. In the absence of Cln1, there was a significant increase in the production of extracellular vesicles. Proteomic assays suggest that in the cln1 mutant strain, there is an upregulation of the glyoxylate acid cycle. Besides, this cyclin mutant is avirulent at 37°C, which correlates with growth defects at this temperature in rich medium. In addition, the cln1 mutant showed lower intracellular replication rates in murine macrophages. We conclude that cell cycle regulatory elements are involved in the modulation of the expression of the main virulence factor in C. neoformans. IMPORTANCE Cryptococcus neoformans is a pathogenic fungus that has significant incidence worldwide. Its main virulence factor is a polysaccharide capsule that can increase in size during infection. In this work, we demonstrate that this process occurs in a specific phase of the cell cycle, in particular, in G1. In agreement, mutants that have an abnormal longer G1 phase show larger capsule sizes. We believe that our findings are relevant because they provide a link between capsule growth, cell cycle progression, and virulence in C. neoformans that reveals new aspects about the pathogenicity of this fungus. Moreover, our findings indicate that cell cycle elements could be used as antifungal targets in C. neoformans by affecting both the growth of the cells and the expression of the main virulence factor of this pathogenic yeast.