Changes in antiparasitical activity of gold nanorods according to the chosen synthesis.

Gold nanorods (GNRs) are increasingly being studied for diagnostic and therapeutic purposes. Green synthesis based methods with natural compounds as additives stand out as a hope in terms of better synthesis methodology, with advantages of producing potentially less toxic and, perhaps, biologically active GNRs due to influence of natural additives used during synthesis. Exploring green chemistry using different natural phenolic compounds, the present work reveals different in vitro activity of GNRs evaluated against different parasites that causes skin infectious diseases compared to GNRs produced by convencional seed mediated method. This approach brings advantages in producing active GNRs, with ease calling, less cytotoxic and with a better selectivity index (SI) than GNRs synthesized by conventional seed mediated synthesis, opening new possibilities for therapies. Natural compounds used in green syntheses were gallic acid (GA), resveratrol (RSV) and a purified fraction of the hydroalcoholic extract of Stryphnodendron obovatum. GNRs exhibited great activity against Leishmania braziliensis, and the dermatophytes Tricophyton rubrum, T. interdigitale and Microsporum gypseum. The anti-Leishmania and antidermatophytic activity of GNRs reinforce the applicability of GNRs in biomedical field and the influence of synthesis method in biological activity, showing benefits related to the seedless synthesis with natural compounds. In addition, these preliminary results indicate the possibility of exploring at maximum the physical and chemical properties of GNRs in addition to the biological activity itself, such as the development of topical antiparasitic formulations for association with phototherapy.

Collateral consequences of agricultural fungicides on pathogenic yeasts: A One Health perspective to tackle azole resistance.

Candida and Cryptococcus affect millions of people yearly, being responsible for a wide array of clinical presentations, including life-threatening diseases. Interestingly, most human pathogenic yeasts are not restricted to the clinical setting, as they are also ubiquitous in the environment. Recent studies raise concern regarding the potential impact of agricultural use of azoles on resistance to medical antifungals in yeasts, as previously outlined with Aspergillus fumigatus. Thus, we undertook a narrative review of the literature and provide lines of evidence suggesting that an alternative, environmental route of azole resistance, may develop in pathogenic yeasts, in addition to patient route. However, it warrants sound evidence to support that pathogenic yeasts cross border between plants, animals and humans and that environmental reservoirs may contribute to azole resistance in Candida or other yeasts for humans. As these possibilities could concern public health, we propose a road map for future studies under the One Health perspective.

Effect of non-antifungal agrochemicals on the pathogenic fungus Cryptococcus gattii.

The chemical control of pests and weeds is employed to improve crop production and the quality of agricultural products. The intensive use of pesticides, however, may cause environmental contamination, thus altering microbial communities. Cryptococcus gattii is an environmental yeast and the causative agent of cryptococcosis in both humans and animals. Up to this day, the effects of agrochemicals on human pathogens living in nature are still widely unknown. In this work, we analyzed the susceptibility of C. gattii to nonfungicide agrochemicals (herbicides and insecticides). Microdilution and drug-combination susceptibility tests were performed for the herbicides flumioxazin (FLX), glyphosate (GLY), isoxaflutole (ISO), pendimethalin (PEND), and also for the insecticide fipronil (FIP). Moreover, these compounds were combined with the clinical antifungals amphotericin B and fluconazole. The MIC values found for the agrochemicals were the following: < 16 µg/ml, for flumioxazin; 128 to 256 µg/ml, for FIP, ISO, and PEND; and >256 µg/ml, for GLY. Synergistic and antagonistic interactions, depending on the strain and concentration tested, were also observed. All strains had undergone adaptation to increasing levels of agrochemicals, in order to select the less susceptible subpopulations. During this process, one C. gattii strain (196 L/03) tolerated high concentrations (50 to 900 µg/ml) of all pesticides assessed. Subsequently, the strain adapted to flumioxazin, isoxaflutole and pendimethalin showed a reduction in the susceptibility to agrochemicals and clinical antifungals, suggesting the occurrence of cross-resistance. Our data point to the risk of exposing C. gattii to agrochemicals existing in the environment, once it might impact the susceptibility of clinical antifungals.

N-acetylcysteine reduces amphotericin B deoxycholate nephrotoxicity and improves the outcome of murine cryptococcosis.

Cryptococcosis is a life-threatening fungal infection, and its current treatment is toxic and subject to resistance. Drug repurposing represents an interesting approach to find drugs to reduce the toxicity of antifungals. In this study, we evaluated the combination of N-acetylcysteine (NAC) with amphotericin B (AMB) for the treatment of cryptococcosis. We examined the effects of NAC on fungal morphophysiology and on the macrophage fungicidal activity 3 and 24 hours post inoculation. The therapeutic effects of NAC combination with AMB were investigated in a murine model with daily treatments regimens. NAC alone reduced the oxidative burst generated by AMB in yeast cells, but did not inhibit fungal growth. The combination NAC + AMB decreased capsule size, zeta potential, superoxide dismutase activity and lipid peroxidation. In macrophage assays, NAC + AMB did not influence the phagocytosis, but induced fungal killing with different levels of oxidative bursts when compared to AMB alone: there was an increased reactive oxygen species (ROS) after 3 hours and reduced levels after 24 hours. By contrast, ROS remained elevated when AMB was tested alone, demonstrating that NAC reduced AMB oxidative effects without influencing its antifungal activity. Uninfected mice treated with NAC + AMB had lower concentrations of serum creatinine and glutamate-pyruvate transaminase in comparison to AMB. The combination of NAC + AMB was far better than AMB alone in increasing survival and reducing morbidity in murine-induced cryptococcosis, leading to reduced fungal burden in lungs and brain and also lower concentrations of pro-inflammatory cytokines in the lungs. In conclusion, NAC + AMB may represent an alternative adjuvant for the treatment of cryptococcosis.

Hospitalisations for mycoses as an indicator of socio-environmental vulnerability in the Brazilian Amazon-Savanna transition region.

BACKGROUND: The infections caused by fungi represent a global concern and an important cause of hospital admissions in endemic areas. The influence of socio-environmental factors in infectious diseases has been documented; however, this phenomenon remains unclear regarding mycoses. OBJECTIVES: This study aimed to analyse the spatio-temporal dynamics of hospitalisations for mycoses (HM) and the association with socio-economic and climate data in the Amazon-Savanna Transition Region in the state of Maranhão, Brazil. METHODS: In this study, Spearman's correlation was applied to determine the correlation between HM, socio-economic and climatic data obtained from national databases in the period from 1998 to 2016. Hospitalisations for mycoses data were spatialised and analysed using the local Moran's index. RESULTS: Our data revealed a negative and significant correlation between HM and socio-economic data regarding population, demographic density, human development index, health facilities and sanitary sewage. Significant correlations were observed between HM and precipitation, maximum temperature and minimum temperature. The main modulating climatic variable was the minimum temperature. The spatial autocorrelation analysis showed the dynamics of HM in municipalities belonging to the different regions of the state influenced by socio-economic conditions. We observed the presence of municipalities with high incidence of HM surrounded by others with low HM cases and vice versa. CONCLUSIONS: Our results indicate that hospitalisations for mycoses represent an important indicator of socio-environmental vulnerability in the Amazon-Savanna transition region in Brazil. We encourage the adoption of measures to mitigate social and environmental impact on these diseases, especially in municipalities with low socio-economic status.

Thiazole derivatives act on virulence factors of Cryptococcus spp.

Cryptococcosis is an opportunistic or primary fungal infection considered to be the most prevalent fatal fungal disease worldwide. Owing to the limited number of available drugs, it is necessary to search for novel antifungal compounds. In the present work, we assessed the antifungal efficacy of three thiazole derivatives (1, 2, and 3). We conducted in vitro and in vivo assays to investigate their effects on important virulence factors, such as capsule and biofilm formation. In addition, the phagocytosis index of murine macrophages exposed to compounds 1, 2, and 3 and the in vivo efficacy of 1, 2, and 3 in Galleria mellonella infected with Cryptococcus spp. were evaluated. All compounds exhibited antifungal activity against biofilms and demonstrated a reduction in biofilm metabolic activity by 43-50% for C. gattii and 26-42% for C. neoformans. Thiazole compounds promoted significant changes in the capsule thickness of C. gattii compared to that of C. neoformans. Further examination of these compounds suggests that they can improve the phagocytosis process of peritoneal murine macrophages in vitro, causing an increase in the phagocytosis rate. Survival percentage was examined in the invertebrate model Galleria mellonella larvae, and only compound 3 could increase the survival at doses of 5 mg/kg after infection with C. gattii (P = .0001) and C. neoformans (P = .0007), similar to fluconazole at 10 mg/kg. The results demonstrated that thiazole compounds, mainly compound 3, have potential to be used for future studies in the search for new therapeutics for cryptococcosis.

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

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.

A new method for studying cryptococcosis in a murine model using 99mTc-Cryptococcus gattii.

Cryptococcus gattii is one of the etiologic agents of cryptococcosis, a systemic mycosis that occurs in healthy and immunosuppressed humans and animals worldwide. Primary pulmonary infection caused by C. gattii is usually followed by fungal dissemination to the central nervous system, resulting in high mortality rates. In this context, animal models of cryptococcosis are useful in the study of fungal pathogenesis and host response against the pathogen, and for testing novel therapeutic options. The most frequently applied method to study fungal dissemination from the lungs to other organs is by culturing tissues, which is not accurate for the detection and quantification of fungal load at early stages of the infection. To overcome this problem, the purpose of this study was to develop a new method for the quantification of Cryptococcus dissemination. One C. gattii strain was efficiently radiolabeled with technetium-99m (99mTc), without affecting viability of the cells. Further, the 99mTc-C. gattii (111 MBq) strain was used to infect mice by intratracheal and intravenous route for biodistribution studies. 99mTc-C. gattii was successfully used in detection of the yeast in the brain of mice 6 hours postinoculation, while the detection using colony forming units was possible only 24 hours postinfection. Our results provided an alternative method that could be applied in further investigations regarding the efficacy of antifungals, fungal virulence, and host-pathogen interactions.

Heterocycle Thiazole Compounds Exhibit Antifungal Activity through Increase in the Production of Reactive Oxygen Species in the Cryptococcus neoformans-Cryptococcus gattii Species Complex.

Human cryptococcosis can occur as a primary or opportunistic infection and develops as an acute, subacute, or chronic systemic infection involving different organs of the host. Given the limited therapeutic options and the occasional resistance to fluconazole, there is a need to develop novel drugs for the treatment of cryptococcosis. In this report, we describe promising thiazole compounds 1, 2, 3, and 4 and explore their possible modes of action against Cryptococcus To this end, we show evidence of interference in the Cryptococcus antioxidant system. The tested compounds exhibited MICs ranging from 0.25 to 2 µg/ml against Cryptococcus neoformans strains H99 and KN99α. Interestingly, the knockout strains for Cu oxidase and sarcosine oxidase were resistant to thiazoles. MIC values of thiazole compounds 1, 2, and 4 against these mutants were higher than for the parental strain. After the treatment of C. neoformans ATCC 24067 (or C. deneoformans) and C. gattii strain L27/01 (or C. deuterogattii) with thiazoles, we verified an increase in intracellular reactive oxygen species (ROS). Also, we verified the synergistic interactions among thiazoles and menadione, which generates superoxides, with fractional inhibitory concentrations (FICs) equal to 0.1874, 0.3024, 0.25, and 0.25 for the thiazole compounds 1, 2, 3, and 4, respectively. In addition, thiazoles exhibited antagonistic interactions with parasulphonatephenyl porphyrinato ferrate III (FeTPPS). Thus, in this work, we showed that the action of these thiazoles is related to an interference with the antioxidant system. These findings suggest that oxidative stress may be primarily related to the accumulation of superoxide radicals.

A subdose of fluconazole alters the virulence of Cryptococcus gattii during murine cryptococcosis and modulates type I interferon expression.

Cryptococcosis is an invasive infection caused by yeast-like fungus of the genera Cryptococcus spp. The antifungal therapy for this disease provides some toxicity and the incidence of infections caused by resistant strains increased. Thus, we aimed to assess the consequences of fluconazole subdoses during the treatment of cryptococcosis in the murine inflammatory response and in the virulence factors of Cryptococcus gattii. Mice infected with Cryptococcus gattii were treated with subdoses of fluconazole. We determined the behavior of mice and type 1 interferon expression during the treatment; we also studied the virulence factors and susceptibility to fluconazole for the colonies recovered from the animals. A subdose of fluconazole prolonged the survival of mice, but the morbidity of cryptococcosis was higher in treated animals. These data were linked to the increase in: (i) fluconazole minimum inhibitory concentration, (ii) capsule size and (iii) melanization of C. gattii, which probably led to the increased expression of type I interferons in the brains of mice but not in the lungs. In conclusion, a subdose of fluconazole altered fungal virulence factors and susceptibility to this azole, leading to an altered inflammatory host response and increased morbidity.

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

UNLABELLED: 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.

The absence of microbiota delays the inflammatory response to Cryptococcus gattii.

The inflammatory response plays a crucial role in infectious diseases, and the intestinal microbiota is linked to maturation of the immune system. However, the association between microbiota and the response against fungal infections has not been elucidated. Our aim was to evaluate the influence of microbiota on Cryptococcus gattii infection. Germ-free (GF), conventional (CV), conventionalized (CVN-mice that received feces from conventional animals), and LPS-stimulated mice were infected with C. gattii. GF mice were more susceptible to infection, showing lower survival, higher fungal burden in the lungs and brain, increased behavioral changes, reduced levels of IFN-γ, IL-1ß and IL-17, and lower NFκBp65 phosphorylation compared to CV mice. Low expression of inflammatory cytokines was associated with smaller yeast cells and polysaccharide capsules (the main virulence factor of C. gattii) in the lungs, and less tissue damage. Furthermore, macrophages from GF mice showed reduced ability to engulf, produce ROS, and kill C. gattii. Restoration of microbiota (CVN mice) or LPS administration made GF mice more responsive to infection, which was associated with increased survival and higher levels of inflammatory mediators. This study is the first to demonstrate the influence of microbiota in the host response against C. gattii.

Squamous cell carcinoma derived from chronic chromoblastomycosis in Brazil.

BACKGROUND: Chromoblastomycosis (CBM) is a chronic fungal infection caused mainly by the melanized fungi Fonsecaea species. The chronic lesions may be predisposed to develop into cancer, the most serious complication of the disease. METHODS: In this report, 7 cases of squamous cell carcinoma (SCC) resulting from chronic CBM in patients from Maranhão in the Brazilian Amazon are described. RESULTS: The 7 patients presented with SCC that resulted from chronic CBM, caused by Fonsecaea species >10 years' duration. The malignant lesions occurred independent of the antifungal therapy and all patients underwent curative amputation, except for 1 patient who developed metastases in the inguinal and intra-abdominal lymph nodes and thigh muscles. A majority of previous reports have focused on the malignant transformation of CBM described in only 1 patient each. This is a first report describing a group of patients from a single Brazilian state. CONCLUSIONS: Here, we provide new epidemiologic data on malignant CBM lesions, an endemic disease that is seemingly neglected worldwide. We reinforce the idea that typically chronic lesions may be predisposed to turn malignant.

Melanin protects Paracoccidioides brasiliensis from the effects of antimicrobial photodynamic inhibition and antifungal drugs.

Paracoccidioidomycosis (PCM) is a public health concern in Latin America and South America that when not correctly treated can lead to patient death. In this study, the influence of melanin produced by Paracoccidioides spp. on the effects of treatment with antimicrobial photodynamic inhibition (aPI) and antifungal drugs was evaluated. aPI was performed using toluidine blue (TBO) as a photosensitizer and a 630-nm light-emitting diode (LED) light. The antifungals tested were itraconazole and amphotericin B. We evaluated the effects of each approach, aPI or antifungals, against nonmelanized and melanized yeast cells by performing susceptibility tests and by quantifying oxidative and nitrosative bursts during the experiments. aPI reduced nonmelanized cells by 3.0 log units and melanized cells by 1.3 log units. The results showed that melanization protects the fungal cell, probably by acting as a scavenger of nitric oxide and reactive oxygen species, but not of peroxynitrite. Melanin also increased the MICs of itraconazole and amphotericin B, and the drugs were fungicidal for nonmelanized and fungistatic for melanized yeast cells. Our study shows that melanin production by Paracoccidioides yeast cells serves a protective function during aPI and treatment with itraconazole and amphotericin B. The results suggest that melanin binds to the drugs, changing their antifungal activities, and also acts as a scavenger of reactive oxygen species and nitric oxide, but not of peroxynitrite, indicating that peroxynitrite is the main radical that is responsible for fungal death after aPI.

Heteroresistance to Itraconazole Alters the Morphology and Increases the Virulence of Cryptococcus gattii.

Cryptococcus gattii is the main etiological agent of cryptococcosis in immunocompetent individuals. The triazole drug itraconazole is one of the antifungals used to treat patients with cryptococcosis. Heteroresistance is an adaptive mechanism to counteract the stress of increasing drug concentrations, and it can enhance the ability of a microorganism to survive under antifungal pressure. In this study, we evaluated the ability of 11 C. gattii strains to develop itraconazole heteroresistance. Heteroresistant clones were analyzed for drug susceptibility, alterations in cell diameter, capsule properties, and virulence in a murine model. Heteroresistance to itraconazole was intrinsic in all of the strains analyzed, reduced both the capsule size and the cell diameter, induced molecular heterogeneity at the chromosomal level, changed the negatively charged cells, reduced ergosterol content, and improved the antioxidant system. A positive correlation between surface/volume ratio of original cells and the level of heteroresistance to itraconazole (LHI) was observed in addition to a negative correlation between capsule size of heteroresistant clones and LHI. Moreover, heteroresistance to itraconazole increased the engulfment of C. gattii by macrophages and augmented fungal proliferation inside these cells, which probably accounted for the reduced survival of the mice infected with the heteroresistant clones and the higher fungal burden in lungs and brain. Our results indicate that heteroresistance to itraconazole is intrinsic and increases the virulence of C. gattii. This phenomenon may represent an additional mechanism that contributes to relapses of cryptococcosis in patients during itraconazole therapy.

Antifungal activity of 6-quinolinyl N-oxide chalcones against Paracoccidioides.

BACKGROUND: Chalcones are an important class of natural compounds that have been widely applied as synthons in synthetic organic chemistry and possess diverse and interesting biological properties. METHODS: We conducted tests with the synthetic substances 6-quinolinyl N-oxide chalcones 4c and 4e to determine their antifungal activity against several isolates of Paracoccidioides spp. and their activity in a murine model. We also determined whether the chalcones interacted with other drugs or interfered with the morphology of Paracoccidioides brasiliensis (Pb18) yeast cells. RESULTS: We verified that the substances were active against Paracoccidioides spp., but we did not show an interaction with the drugs tested when only the fractional inhibitory concentration index values were considered individually. We observed that the substances induced in vitro morphological changes. Compounds 4c and 4e showed activity similar to itraconazole in treated mice, as demonstrated by their ability to reduce the number of cfu recovered from the lungs. Histopathological analysis showed that animals treated with 4c presented fewer areas containing inflammatory infiltrate and larger areas of preserved lung tissue, whereas animals treated with itraconazole showed accumulation of inflammatory infiltrate and some granulomas. Mice treated with 4e exhibited inflammation that compromised the tissue. CONCLUSIONS: The results presented in this paper confirm the antifungal potential of the chalcones tested. The chalcone 4c was the more effective at controlling the disease in mice and this compound could be a candidate for future studies of the treatment of paracoccidioidomycosis.

2-(benzylideneamino)phenol: a promising hydroxyaldimine with potent activity against dermatophytoses.

Infections caused by dermatophytes, mainly Trichophyton rubrum,are often vulnerable to relapses upon cessation of antifungal therapy, reinforcing the need of new antifungals. Aldimines have potential biological activities, but there are few reports on their antifungal profile. The aim of this study was to evaluate the antifungal activity of 2-(benzylideneamino)phenol (3A3) and 4-(benzylideneamino)phenol (3A4) against dermatophytes. We determined the minimum inhibitory concentration, minimum fungicidal concentration, time-kill curves and fractional inhibitory concentration of the combination of 3A3, 3A4 and itraconazole against a set of isolates of T. rubrum and T. interdigitale. 3A3 was tested in a murine model of dermatophytoses caused by T. rubrum, and the effect on phagocytosis was assessed. The MIC values ranged from 8 to 32 µg/mL for 3A3 and from 64 to 256 µg/mL for 3A4. The interaction between 3A3 and 3A4 with itraconazole proved to be synergistic and indifferent, respectively. 3A3 was as efficient as itraconazole in reducing the fungal burden on the skin of mice, being this effect associated with the influx of neutrophil and macrophage. Also, 3A3 was able to increase the internalization of conidia by macrophages. Altogether, our data encourage future clinical studies with 3A3 to treat dermatophytoses.

IFN-γ impairs Trichophyton rubrum proliferation in a murine model of dermatophytosis through the production of IL-1ß and reactive oxygen species.

Trichophyton rubrum is the main etiological agent of dermatophytosis, an infection of the skin that affects millions of people worldwide. In this study, we developed a murine model of the dermatophytosis caused by T. rubrum in which C57BL/6 wild-type, interleukin (IL)-12(-/-), and interferon-gamma (IFN-γ(-/-)) mice were inoculated with 1 × 10(6) conidia/animal. The fungal burden, myeloperoxidase and N-acetylglucosaminidase activities, cytokine and chemokine profiles, and histopathology of the skin were evaluated on the seventh and fourteenth days post infection. Phagocytic indices, intracellular proliferation rates, and oxidative bursts generated by macrophages from WT and IFN-γ(-/-) mice were determined. On day 7 post infection, higher fungal burdens were observed comparison with burdens on day 14 post infection. The IL-12(-/-) and IFN-γ(-/-) mice showed higher fungal burdens on the skin and lower levels of IL-1ß. Conversely, the WT mice showed lower fungal burdens with higher production of TNF-α, IL-1ß, and chemokine ligand 1/keratinocyte chemoattractant (CXCL1/KC). The macrophages from WT mice proved to be more efficient at engulfing and killing T. rubrum conidia through the production of reactive oxygen species. The results show that our model is a useful tool for understanding the pathogenesis of dermatophytosis caused by T. rubrum and that IL-12 and IFN-γ are pivotal in controlling the infection through the recruitment and activation of neutrophils and macrophages.

The role of oxidative and nitrosative bursts caused by azoles and amphotericin B against the fungal pathogen Cryptococcus gattii.

OBJECTIVES: Although the most accepted mechanisms of action of amphotericin B and azoles are related to ergosterol, it is possible that these drugs have other effects on the fungal cell. In the present study, the role of endogenous reactive oxygen species (ROS) and peroxynitrite produced by azoles and amphotericin B in the fungus Cryptococcus gattii were examined. METHODS: We studied distinct parameters to evaluate the effect of oxidative and nitrosative stresses induced by these drugs in C. gattii cells: lipid peroxidation, ergosterol content, ROS and peroxynitrite production, enzymatic activity of the antioxidant system and the in vitro interaction of antifungal drugs with a peroxidase inhibitor, a superoxide dismutase inhibitor and a peroxynitrite scavenger. RESULTS: The data demonstrated that itraconazole led to ROS formation and lipid peroxidation in C. gattii cells in the early stages of the treatment; this did not occur with fluconazole. This phenomenon strongly increased the activities of enzymes of the antioxidant system. These results were confirmed by synergism observed between the catalase inhibitor and itraconazole. Amphotericin B caused lipid peroxidation in C. gattii cells through a greatly enhanced production of oxidative and nitrosative radicals with increased peroxidase activity. These data were confirmed by the synergism between the catalase/superoxide dismutase inhibitors and amphotericin B. In addition, the effect of this antifungal was antagonized by the peroxynitrite scavenger. CONCLUSIONS: Oxidative and nitrosative bursts play an important role in the antifungal activity of itraconazole and amphotericin B against C. gattii.

Proteasome inhibition as a therapeutic target for the fungal pathogen Cryptococcus neoformans.

The current therapeutic challenges for treating fungal diseases demand new approaches and new drugs. A promising strategy involves combination therapy with agents of distinct mechanisms of action to increase fungicidal activity and limit the impact of mutations leading to resistance. In this study, we evaluated the antifungal potential of bortezomib by examining the inhibition of proteasome activity, cell proliferation, and capsule production by Cryptococcus neoformans, the causative agent of fungal meningoencephalitis. Chemical genetic screens with collections of deletion mutants identified potential druggable targets for combination therapy with bortezomib. In vitro assays of combinations of bortezomib with flucytosine, chlorpromazine, bafilomycin A1, copper sulfate, or hydroxyurea revealed antifungal effects against C. neoformans. Furthermore, combination treatment with bortezomib and flucytosine in a murine inhalation model of cryptococcosis resulted in the improvement of neurological functions and reduced fungal replication and dissemination, leading to a delay in disease progression. This study therefore highlights the utility of chemical genetic screens to identify new therapeutic approaches as well as the antifungal potential of proteasome inhibition. IMPORTANCE Fungal diseases of humans are difficult to treat, and there is a clear need for additional antifungal drugs, better diagnostics, effective vaccines, and new approaches to deal with emerging drug resistance. Fungi are challenging to control because they share many common biochemical functions with their mammalian hosts and it is therefore difficult to identify fungal-specific targets for drug development. One approach is to employ existing antifungal drugs in combination with agents that target common cellular processes at levels that are (ideally) not toxic for the host. We pursued this approach in this study by examining the potential of the clinically approved proteasome inhibitor bortezomib to influence the proliferation and virulence of Cryptococcus neoformans. We found that the combination of bortezomib with the anti-cryptococcal drug flucytosine improved the survival of infected mice, thus demonstrating the potential of this strategy for antifungal therapy.