Brilacidin, a novel antifungal agent against Cryptococcus neoformans.

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.

Brilacidin, a host defense peptide mimetic, potentiates ibrexafungerp antifungal activity against the human pathogenic fungus Aspergillus fumigatus.

Aspergillus fumigatus is the primary etiological agent of aspergillosis. Here, we show that the host defense peptide mimetic, brilacidin (BRI) can potentiate ibrexafungerp (IBX) against clinical isolates of A. fumigatus. CAS-resistant strains with mutations in fks1 that encodes the 1,3-ß-D-glucan synthase are not IBX-resistant and BRI+IBX can inhibit their growth. The combination of BRI+IBX plays a fungicidal role, increases the fungal cell permeability and decreases the fungal survival in the presence of A549 epithelial cells.

Milteforan, a promising veterinary commercial product against feline sporotrichosis.

Sporotrichosis, the cutaneous mycosis most commonly reported in Latin America, is caused by the Sporothrix clinical clade species, including Sporothrix brasiliensis and Sporothrix schenckii sensu stricto. In Brazil, S. brasiliensis represents a vital health threat to humans and domestic animals due to its zoonotic transmission. Itraconazole, terbinafine, and amphotericin B are the most used antifungals for treating sporotrichosis. However, many strains of S. brasiliensis and S. schenckii have shown resistance to these agents, highlighting the importance of finding new therapeutic options. Here, we demonstrate that milteforan, a commercial veterinary product against dog leishmaniasis whose active principle is miltefosine, is a possible therapeutic alternative for the treatment of sporotrichosis, as observed by its fungicidal activity in vitro against different strains of S. brasiliensis and S. schenckii, and by its antifungal activity when used to treat infected epithelial cells and macrophages. Our results suggest milteforan as a possible alternative to treat feline sporotrichosis.

Transcriptomic analysis reveals that mTOR pathway can be modulated in macrophage cells by the presence of cryptococcal cells.

Cryptococcus neoformans and Cryptococcus gattii are the etiological agents of cryptococcosis, a high mortality disease. The development of such disease depends on the interaction of fungal cells with macrophages, in which they can reside and replicate. In order to dissect the molecular mechanisms by which cryptococcal cells modulate the activity of macrophages, a genome-scale comparative analysis of transcriptional changes in macrophages exposed to Cryptococcus spp. was conducted. Altered expression of nearly 40 genes was detected in macrophages exposed to cryptococcal cells. The major processes were associated with the mTOR pathway, whose associated genes exhibited decreased expression in macrophages incubated with cryptococcal cells. Phosphorylation of p70S6K and GSK-3ß was also decreased in macrophages incubated with fungal cells. In this way, Cryptococci presence could drive the modulation of mTOR pathway in macrophages possibly to increase the survival of the pathogen.

Zrg1, a cryptococcal protein associated with regulation of growth in nutrient deprivation conditions.

Cryptococcus gattii is one of the causes of cryptococcosis, a life-threatening disease generally characterized by pneumonia and/or meningitis. Zinc is an essential element for life, being required for the activity of many proteins with catalytic and structural roles. Here, we characterize ZRG1 (zinc-related gene 1), which codes a product involved in zinc metabolism. Transcriptional profiling revealed that zinc availability regulated the expression of ZRG1, and its null mutants demonstrated impaired growth in zinc- and nitrogen-limiting conditions. Moreover, zrg1 strains displayed alterations in the expression of the zinc homeostasis-related genes ZAP1 and ZIP1. Notably, cryptococcal cells lacking Zrg1 displayed upregulation of autophagy-like phenotypes. Despite no differences were detected in the classical virulence-associated traits; cryptococcal cells lacking ZRG1 displayed decreased capacity for survival inside macrophages and attenuated virulence in an invertebrate model. Together, these results indicate that ZRG1 plays an important role in proper zinc metabolism, and is necessary for cryptococcal fitness and virulence.

Participation of Zip3, a ZIP domain-containing protein, in stress response and virulence in Cryptococcus gattii.

Cryptococcus gattii is an etiologic agent of cryptococcosis, a potentially fatal disease that affects humans and animals. The successful infection of mammalian hosts by cryptococcal cells relies on their ability to infect and survive in macrophages. Such phagocytic cells present a hostile environment to intracellular pathogens via the production of reactive nitrogen and oxygen species, as well as low pH and reduced nutrient bioavailability. To overcome the low-metal environment found during infection, fungal pathogens express high-affinity transporters, including members of the ZIP family. Previously, we determined that functional zinc uptake driven by Zip1 and Zip2 is necessary for full C.gattiivirulence. Here, we characterized the ZIP3 gene of C. gattii, an ortholog of the Saccharomyces cerevisiae ATX2, which codes a manganese transporter localized to the membrane of the Golgi apparatus. Cryptococcal cells lacking Zip3 were tolerant to toxic concentrations of manganese and had imbalanced expression of intracellular metal transporters, such as the vacuolar Pmc1 and Vcx1, as well as the Golgi Pmr1. Moreover, null mutants of the ZIP3 gene displayed higher sensitivity to reactive oxygen species (ROS) and substantial alteration in the expression of ROS-detoxifying enzyme-coding genes. In line with these phenotypes, cryptococcal cells displayed decreased virulence in a non-vertebrate model of cryptococcosis. Furthermore, we found that the ZIP3 null mutant strain displayed decreased melanization and secretion of the major capsular component glucuronoxylomannan, as well as an altered extracellular vesicle dimensions profile. Collectively, our data suggest that Zip3 activity impacts the physiology, and consequently, several virulence traits of C. gattii.

Effects of zinc transporters on Cryptococcus gattii virulence.

Zinc is an essential nutrient for all living organisms because it is a co-factor of several important proteins. Furthermore, zinc may play an essential role in the infectiousness of microorganisms. Previously, we determined that functional zinc metabolism is associated with Cryptococcus gattii virulence. Here, we characterized the ZIP zinc transporters in this human pathogen. Transcriptional profiling revealed that zinc levels regulated the expression of the ZIP1, ZIP2 and ZIP3 genes, although only the C. gattii zinc transporter Zip1 was required for yeast growth under zinc-limiting conditions. To associate zinc uptake defects with virulence, the most studied cryptococcal virulence factors (i.e., capsule, melanin and growth at 37 °C) were assessed in ZIP mutant strains; however, no differences were detected in these classical virulence-associated traits among the mutant and WT strains. Interestingly, higher levels of reactive oxygen species were detected in the zip1Δ and in the zip1Δ zip2Δ double mutants. In line with these phenotypic alterations, the zip1Δ zip2Δ double mutant displayed attenuated virulence in a murine model of cryptococcosis. Together, these results indicate that adequate zinc uptake is necessary for cryptococcal fitness and virulence.