Thais F.F. Magalhães

Curcumin as a promising antifungal of clinical interest

OBJECTIVES The antifungal activity of curcumin was evaluated against 23 fungi strains and its in vitro inhibitory effect on the adhesion of Candida species to human buccal epithelial cells (BEC) was also investigated. METHODS The antifungal susceptibility was evaluated by broth microdilution assay following the CLSI (formerly the NCCLS) guidelines. The inhibitory effect of curcumin on the cell adhesion was performed with Candida species and BEC. RESULTS Paracoccidioides brasiliensis isolates were the most susceptible to curcumin while the growth of Aspergillus isolates was not affected. Curcumin was much more efficient than fluconazole in inhibiting the adhesion of Candida species to BEC, particularly those strains isolated from the buccal mucosa of AIDS patients. CONCLUSIONS The lack of antifungal compounds with reduced side effects highlights the importance of studying natural products for this purpose. Curcumin was a more potent antifungal than fluconazole against P. brasiliensis, the causal agent of the neglected disease paracoccidioidomycosis. Curcumin dramatically inhibited the adhesion of Candida species isolated from AIDS patients to BEC, demonstrating that curcumin is a promising lead compound that warrants further investigation into its therapeutical use in immunocompromised patients.

In vitro studies of anticandidal activity of goniothalamin enantiomers.

Aims:  The antifungal activity of (R)‐goniothalamin (1) and (S)‐goniothalamin (ent‐1) was evaluated against six Candida species. The in vitro effect of these compounds on yeast adhesion to human buccal epithelial cells (BEC) and Candida albicans and C. dubliniensis biofilms progression were also investigated.

Mimivirus Fibrils Are Important for Viral Attachment to the Microbial World by a Diverse Glycoside Interaction Repertoire.

ABSTRACT Acanthamoeba polyphaga mimivirus (APMV) is a giant virus from the Mimiviridae family. It has many unusual features, such as a pseudoicosahedral capsid that presents a starfish shape in one of its vertices, through which the ∼1.2-Mb double-stranded DNA is released. It also has a dense glycoprotein fibril layer covering the capsid that has not yet been functionally characterized. Here, we verified that although these structures are not essential for viral replication, they are truly necessary for viral adhesion to amoebae, its natural host. In the absence of fibrils, APMV had a significantly lower level of attachment to the Acanthamoeba castellanii surface. This adhesion is mediated by glycans, specifically, mannose and N-acetylglucosamine (a monomer of chitin and peptidoglycan), both of which are largely distributed in nature as structural components of several organisms. Indeed, APMV was able to attach to different organisms, such as Gram-positive bacteria, fungi, and arthropods, but not to Gram-negative bacteria. This prompted us to predict that (i) arthropods, mainly insects, might act as mimivirus dispersers and (ii) by attaching to other microorganisms, APMV could be ingested by amoebae, leading to the successful production of viral progeny. To date, this mechanism has never been described in the virosphere. IMPORTANCE APMV is a giant virus that is both genetically and structurally complex. Its size is similar to that of small bacteria, and it replicates inside amoebae. The viral capsid is covered by a dense glycoprotein fibril layer, but its function has remained unknown, until now. We found that the fibrils are not essential for mimivirus replication but that they are truly necessary for viral adhesion to the cell surface. This interaction is mediated by glycans, mainly N-acetylglucosamine. We also verified that APMV is able to attach to bacteria, fungi, and arthropods. This indicates that insects might act as mimivirus dispersers and that adhesion to other microorganisms could facilitate viral ingestion by amoebae, a mechanism never before described in the virosphere.

Synthesis of aryl aldimines and their activity against fungi of clinical interest.

Aldimines are aldehyde‐derived compounds that contain a C=N group. Besides its broad industrial applications, this class of non‐naturally occurring compounds are found to possess antibacterial, antifungal, antimalarial, antiproliferative, anti‐inflammatory, antiviral, and antipyretic properties. Based on this, six aryl aldimines were synthesized from the condensation of aromatic amines with benzaldehydes. The antifungal activities of synthesized compounds were evaluated against nineteen fungal strains that included Candida and Aspergillus species, Cryptococcus neoformans. The aryl aldimines 2‐(benzylideneamino)phenol (3) and 4‐(benzylideneamino)phenol (8) were the most active compounds against the fungi studied. Compounds 3 and 8 efficiently inhibited the metabolism of C. neoformans mature biofilm.

Hydroxyaldimines as potent in vitro anticryptococcal agents.

Cryptococcosis, a fungal infection that affects both immunocompromised and immunocompetent individuals, contributes to increasing indices of mortality and morbidity. The development of resistance by Cryptococcus spp., the limited number of commercial antifungal drugs and the various side effects of these drugs cause the treatment of cryptococcosis to be a challenge. The in vitro anticryptococcal activity of nine hydroxyaldimines was evaluated against 24 strains of Cryptococcus spp. Antifungal susceptibility was evaluated using a broth microdilution assay following the Clinical and Laboratory Standards Institute guidelines, using fluconazole as a positive control. Parameters such as the minimum inhibitory concentration and the minimum fungicidal concentration (MIC and MFC, respectively) were also determined. Antiproliferative activity on the normal cell line VERO was assessed 48 h post‐compound exposure to determine the selectivity index (SI) of the hydroxyaldimines and fluconazole. All hydroxyaldimines were active against Cryptococcus spp. strains. Compounds 3A9 and 3B7 were the most potent against the Cryptococcus gattii and Cryptococcus neoformans strains. Selectivity indices also revealed that 3B10, 3C3, 3D3 and 3D9 are good candidates for in vivo studies. The in vitro anticryptococcal activity of hydroxyaldimines against various strains of C. gattii and C. neoformans indicates the potential of this class of molecules as lead compound for the development of selective and efficient anticryptococcal agents.

Synthesis and Anti-Paracoccidioides Activity of Calix(n)arenes

Fungi are some of the most neglected pathogens in terms of new drug discovery. Paracoccidioidomycosis, an endemic disease to South America, is caused by dimorphic fungi of Paracoccidioides genus. Reasonable citotoxicity, limited efficacy, and frequent use of current commercial antifungals have led to the emergence of resistance. Thus, the development of new antifungals with more selectivity and large spectrum of action is mandatory. In this work, six calix(n)arenes were synthesized and their activity explored against seven isolates of P. brasiliensis and P. lutzii. p-tert- Butylcalix(4)arene (CX1) was the most potent compound against fungal cells and exhibited the lowest toxicity on healthy mammalian cells (V79, hamster lung and MDCK, dog kidney). This is the first report on the effect of calix(n)arenes as growth inhibitors of Paracoccidioides fungi.

In vitro studies of the activity of dithiocarbamate organoruthenium complexes against clinically relevant fungal pathogens.

The in vitro antifungal activity of nine dirutheniumpentadithiocarbamate complexes C1–C9 was investigated and assessed for its activity against four different fungal species with clinical interest and related to invasive fungal infections (IFIs), such as Candida spp. [C. albicans (two clinical isolates), C. glabrata, C. krusei, C. parapsolisis, C. tropicalis, C.dubliniensis (six clinical isolates)], Paracoccidioides brasiliensis (seven clinical isolates), Cryptococcus neoformans and Sporothrix schenckii. All synthesized complexes C1–C9 and also the free ligands L1–L9 were submitted to in vitro tests against those fungi and the results are very promising, since some of the obtained MIC (minimal inhibitory concentration) values were very low (from 10−6 mol mL−1 to 10−8 mol mL−1) against all investigated clinically relevant fungal pathogens, except for C. glabrata, that the MIC values are close to the ones obtained for fluconazole, the standard antifungal agent tested. Preliminary structure-activity relations (SAR) might be suggested and a strong influence from steric and lipophilic parameters in the antifungal activity can be noticed. Cytotoxicity assays (IC50) showed that the complexes are not as toxic (IC50 values are much higher—30 to 200 fold—than MIC values). These ruthenium complexes are very promising lead compounds for novel antifungal drug development, especially in IFIs, one of most harmful emerging infection diseases (EIDs).

Atorvastatin as a promising anticryptococcal agent

Cryptococcosis caused by Cryptococcus gattii leads to pneumonia and meningoencephalitis, and has a high mortality rate worldwide due to the inadequacy of available therapy and increasing drug resistance. There is a need to develop effective treatments, and drug repositioning is an interesting alternative to achieve new strategies to treat cryptococcosis. Atorvastatin (ATO), a statin currently used to treat hypercholesterolaemia, was tested in this study as an adjuvant to control infections caused by C. gattii. Several aspects of the effect of ATO on the host and the yeast were evaluated, with particular focus on the association of ATO with fluconazole (FLC), which (i) reduced ergosterol content in the cell membrane and altered properties of the polysaccharide capsule of C. gattii; (ii) increased the production of reactive oxygen species by macrophages; and (iii) reduced yeast phagocytosis and the intracellular proliferation rate. In an animal model, infected mice treated with ATO + FLC showed increased survival, improved clinical condition, and reduced fungal burden in the lungs and brain. This study is the first to perform in vivo tests with ATO + FLC for the treatment of cryptococcosis. The results suggest that ATO may be an important adjuvant for the treatment of cryptococcosis.

Treatment with pCramoll Alone and in Combination with Fluconazole Provides Therapeutic Benefits in C. gattii Infected Mice

Cryptococcus gattii is one of the main causative agents of cryptococcosis in immunocompetent individuals. Treatment of the infection is based on the use of antimycotics, however, the toxicity of these drugs and the increase of drug-resistant strains have driven the search for more effective and less toxic therapies for cryptococcosis. pCramoll are isolectins purified from seeds of Cratylia mollis, a native forage plant from Brazil, which has become a versatile tool for biomedical application. We evaluated the effect of pCramoll alone and in combination with fluconazole for the treatment of mice infected with C. gatti. pCramoll alone or in combination with fluconazole increased the survival, reduced the morbidity and improved mice behavior i.e., neuropsychiatric state, motor behavior, autonomic function, muscle tone and strength and reflex/sensory function. These results were associated with (i) decreased pulmonary and cerebral fungal burden and (ii) increased inflammatory infiltrate and modulatory of IFNγ, IL-6, IL-10, and IL-17A cytokines in mice treated with pCramoll. Indeed, bone marrow-derived macrophages pulsed with pCramoll had increased ability to engulf C. gattii, with an enhanced production of reactive oxygen species and decrease of intracellular fungal proliferation. These findings point toward the use of pCramoll in combination with fluconazole as a viable, alternative therapy for cryptococcosis management.

Environmental Triazole Induces Cross-Resistance to Clinical Drugs and Affects Morphophysiology and Virulence of Cryptococcus gattii and C. neoformans

ABSTRACT Cryptococcus gattii and Cryptococcus neoformans are environmental fungi that cause cryptococcosis, which is usually treated with amphotericin B and fluconazole. However, therapeutic failure is increasing because of the emergence of resistant strains. Because these species are constantly isolated from vegetal materials and the usage of agrochemicals is growing, we postulate that pesticides could be responsible for the altered susceptibility of these fungi to clinical drugs. Therefore, we evaluated the influence of the pesticide tebuconazole on the susceptibility to clinical drugs, morphophysiology, and virulence of C. gattii and C. neoformans strains. The results showed that tebuconazole exposure caused in vitro cross-resistance (CR) between the agrochemical and clinical azoles (fluconazole, itraconazole, and ravuconazole) but not with amphotericin B. In some strains, CR was observed even after the exposure ceased. Further, tebuconazole exposure changed the morphology, including formation of pseudohyphae in C. neoformans H99, and the surface charge of the cells. Although the virulence of both species previously exposed to tebuconazole was decreased in mice, the tebuconazole-exposed colonies recovered from the lungs were more resistant to azole drugs than the nonexposed cells. This in vivo CR was confirmed when fluconazole was not able to reduce the fungal burden in the lungs of mice. The tolerance to azoles could be due to increased expression of the ERG11 gene in both species and of efflux pump genes (AFR1 and MDR1) in C. neoformans. Our study data support the idea that agrochemical usage can significantly affect human pathogens present in the environment by affecting their resistance to clinical drugs.