Extracellular Vesicles from Scedosporium apiospermum Mycelial Cells: Implication for Fungal-Host Interplays.

The release of extracellular vesicles (EVs) has been implicated as an alternative transport mechanism for the passage of macromolecules through the fungal cell wall, a phenomenon widely reported in yeasts but poorly explored in mycelial cells. In the present work, we have purified and characterized the EVs released by mycelia of the emerging, opportunistic, widespread and multidrug-resistant filamentous fungus Scedosporium apiospermum. Transmission electron microscopy images and light scattering measurements revealed the fungal EVs, which were observed individually or grouped with heterogeneous morphology, size and electron density. The mean diameter of the EVs, evaluated by the light scattering technique, was 179.7 nm. Overall, the structural stability of S. apiospermum EVs was preserved during incubation under various storage conditions. The lipid, carbohydrate and protein contents were quantified, and the EVs' protein profile was evidenced by SDS-PAGE, revealing proteins with molecular masses ranging from 20 to 118 kDa. Through immunoblotting, ELISA and immunocytochemistry assays, antigenic molecules were evidenced in EVs using a polyclonal serum (called anti-secreted molecules) from a rabbit inoculated with conditioned cell-free supernatant obtained from S. apiospermum mycelial cells. By Western blotting, several antigenic proteins were identified. The ELISA assay confirmed that the anti-secreted molecules exhibited a positive reaction up to a serum dilution of 1:3200. Despite transporting immunogenic molecules, S. apiospermum EVs slightly induced an in vitro cytotoxicity effect after 48 h of contact with either macrophages or lung epithelial cells. Interestingly, the pretreatment of both mammalian cells with purified EVs significantly increased the association index with S. apiospermum conidia. Furthermore, EVs were highly toxic to Galleria mellonella, leading to larval death in a typically dose- and time-dependent manner. Collectively, the results represent the first report of detecting EVs in the S. apiospermum filamentous form, highlighting a possible implication in fungal pathogenesis.

Antifungal potential of the new copper(II)-theophylline/1,10-phenanthroline complex against drug-resistant Candida species.

Candida spp. are the commonest fungal pathogens worldwide. Antifungal resistance is a problem that has prompted the discovery of novel anti-Candida drugs. Herein, 25 compounds, some of them containing copper(II), cobalt(II) and manganese(II) ions, were initially evaluated for inhibiting the growth of reference strains of Candida albicans and Candida tropicalis. Eight (32%) of the compounds inhibited the proliferation of these yeasts, displaying minimum inhibitory concentrations (MICs) ranging from 31.25 to 250 µg/mL and minimum fungicidal concentration (MFCs) from 62.5 to 250 µg/mL. Drug-likeness/pharmacokinetic calculated by SwissADME indicated that the 8 selected compounds were suitable for use as topical drugs. The complex CTP, Cu(theo)2phen(H2O).5H2O (theo = theophylline; phen = 1,10-phenanthroline), was chosen for further testing against 10 medically relevant Candida species that were resistant to fluconazole/amphotericin B. CTP demonstrated a broad spectrum of action, inhibiting the growth of all 20 clinical fungal isolates, with MICs from 7.81 to 62.5 µg/mL and MFCs from 15.62 to 62.5 µg/mL. Conversely, CTP did not cause lysis in erythrocytes. The toxicity of CTP was evaluated in vivo using Galleria mellonella and Tenebrio molitor. CTP had no or low levels of toxicity at doses ranging from 31.25 to 250 µg/mL for 5 days. After 24 h of treatment, G. mellonella larvae exhibited high survival rates even when exposed to high doses of CTP (600 µg/mL), with the 50% cytotoxic concentration calculated as 776.2 µg/mL, generating selectivity indexes varying from 12.4 to 99.4 depending on each Candida species. These findings suggest that CTP could serve as a potential drug to treat infections caused by Candida species resistant to clinically available antifungals.

Therapeutic Strategies against Leishmania and Trypanosoma.

Human African trypanosomiasis (also known as sleeping sickness, with Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense as etiological agents), American trypanosomiasis (also known as Chagas disease, with Trypanosoma cruzi as the etiological agent), and leishmaniasis (including cutaneous, mucocutaneous, and visceral forms, with multiple species belonging to the Leishmania genus as etiological agents) are recognized as neglected tropical diseases (NTDs) [...].

Dechlorination of wastewater from shell-based glucosamine processing by mangrove wetland-derived fungi.

Wastewater from processing crustacean shell features ultrahigh chloride content. Bioremediation of the wastewater is challenging due to the high chloride ion content, making it inhospitable for most microorganisms to survive and growth. In this study, mangrove wetland-derived fungi were first tested for their salt tolerance, and the highly tolerant isolates were cultured in shrimp processing wastewater and the chloride concentration was monitored. Notably, the filamentous fungal species Aspergillus piperis could remove over 70% of the chloride in the wastewater within 3 days, with the fastest biomass increase (2.01 times heavier) and chloride removal occurring between day one and two. The chloride ions were sequestered into the fungal cells. The genome of this fungal species contained Cl- conversion enzymes, which may have contributed to the ion removal. The fungal strain was found to be of low virulence in larval models and could serve as a starting point for further considerations in bioremediation of shell processing wastewater, promoting the development of green technology in the shell processing industry.

Antimicrobial and Antibiofilm Activities of Copper(II)-1,10-phenanthroline-5,6-pione Against Commensal Bacteria and Fungi Responsible for Vaginal Microbiota Dysbiosis.

The disbalance of vaginal eubiotic microbiota can lead to overgrowth of Candida species and bacteria responsible for aerobic vaginitis, activating inflammatory pathways. The presence of Trichomonas vaginalis, a sexually transmitted protozoan pathogen, can be a predisposing factor for disordering the growth of bacterial/fungal pathogenic species due to the increase in pH and reduction of eubiotic microbiota. Herein, we evaluated the effects of the potent trichomonacidal compound, copper(II)-1,10-phenanthroline-5,6-dione (Cu-phendione), against pathogens responsible for candidiasis and aerobic vaginitis. Cu-phendione showed antimicrobial activity against Candida albicans, non-albicans Candida species (C. glabrata, C. krusei, C. parapsilosis, and C. tropicalis) and Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus, Enterococcus faecalis, and Streptococcus agalactiae) bacteria. Moreover, Cu-phendione was able to interfere with the fungal biofilm formation. These results highlight the antimicrobial potential of Cu-phendione against bacterial and fungal strains of vaginitis-causing infectious agents.

Development of Echinocandin Resistance in Candida haemulonii: An Emergent, Widespread, and Opportunistic Fungal Pathogen.

Echinocandins, used for the prevention and treatment of invasive fungal infections, have led to a rise in breakthrough infections caused by resistant Candida species. Among these species, those belonging to the Candida haemulonii complex are rare multidrug-resistant (MDR) yeasts that are frequently misidentified but have emerged as significant healthcare-associated pathogens causing invasive infections. The objectives of this study were to investigate the evolutionary pathways of echinocandin resistance in C. haemulonii by identifying mutations in the FKS1 gene and evaluating the impact of resistance on fitness. After subjecting a MDR clinical isolate of C. haemulonii (named Ch4) to direct selection using increasing caspofungin concentrations, we successfully obtained an isolate (designated Ch4'r) that exhibited a high level of resistance, with MIC values exceeding 16 mg/L for all tested echinocandin drugs (caspofungin, micafungin, and anidulafungin). Sequence analysis revealed a specific mutation in the resistant Ch4'r strain, leading to an arginine-histidine amino acid substitution (R1354H), occurring at the G4061A position of the HS2 region of the FKS1 gene. Compared to the wild-type strain, Ch4'r exhibited significantly reduced growth proliferation, biofilm formation capability, and phagocytosis ratio, indicating a decrease in fitness. Transmission electron microscopy analysis revealed alterations in cell wall components, with a notable increase in cell wall thickness. The resistant strain also exhibited higher amounts (2.5-fold) of chitin, a cell wall-located molecule, compared to the wild-type strain. Furthermore, the resistant strain demonstrated attenuated virulence in the Galleria mellonella larval model. The evolved strain Ch4'r maintained its resistance profile in vivo since the treatment with either caspofungin or micafungin did not improve larval survival or reduce the fungal load. Taken together, our findings suggest that the acquisition of pan-echinocandin resistance occurred rapidly after drug exposure and was associated with a significant fitness cost in C. haemulonii. This is particularly concerning as echinocandins are often the first-line treatment option for MDR Candida species.

Cell Aggregation Capability of Clinical Isolates from Candida auris and Candida haemulonii Species Complex.

The opportunistic fungal pathogens belonging to the Candida haemulonii complex and the phylogenetically related species Candida auris are well-known for causing infections that are difficult to treat due to their multidrug-resistance profiles. Candida auris is even more worrisome due to its ability to cause outbreaks in healthcare settings. These emerging yeasts produce a wide range of virulence factors that facilitate the development of the infectious process. In recent years, the aggregative phenotype has been receiving attention, as it is mainly associated with defects in cellular division and its possible involvement in helping the fungus to escape from the host immune responses. In the present study, we initially investigated the aggregation ability of 18 clinical isolates belonging to the C. haemulonii species complex (C. haemulonii sensu stricto, C. duobushaemulonii, and C. haemulonii var. vulnera) and C. auris. Subsequently, we evaluated the effects of physicochemical factors on fungal aggregation competence. The results demonstrated that cell-to-cell aggregation was a typically time-dependent event, in which almost all studied fungal isolates of both the C. haemulonii species complex and C. auris exhibited high aggregation after 2 h of incubation at 37 °C. Interestingly, the fungal cells forming the aggregates remained viable. The aggregation of all isolates was not impacted by pH, temperature, ß-mercaptoethanol (a protein-denaturing agent), or EDTA (a chelator agent). Conversely, proteinase K, trypsin, and sodium dodecyl sulfate (SDS) significantly diminished the fungal aggregation. Collectively, our results demonstrated that the aggregation ability of these opportunistic yeast pathogens is time-dependent, and surface proteins and hydrophobic interactions seem to mediate cell aggregation since the presence of proteases and anionic detergents affected the aggregation capability. However, further studies are necessary to better elucidate the molecular aspects of this intriguing phenomenon.

Chagas Disease Control-Many Approaches to Prospect.

Chagas disease is an emerging and neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi, estimated to infect 8 to 10 million people worldwide, according to the World Health Organization [...].

Fibronectin-binding molecules of Scedosporium apiospermum: focus on adhesive events.

Scedosporium apiospermum is a widespread, emerging, and multidrug-resistant filamentous fungus that can cause localized and disseminated infections. The initial step in the infection process involves the adhesion of the fungus to host cells and/or extracellular matrix components. However, the mechanisms of adhesion involving surface molecules in S. apiospermum are not well understood. Previous studies have suggested that the binding of fungal receptors to fibronectin enhances its ability to attach to and infect host cells. The present study investigated the effects of fibronectin on adhesion events of S. apiospermum. The results revealed that conidial cells were able to bind to both immobilized and soluble human fibronectin in a typically dose-dependent manner. Moreover, fibronectin binding was virtually abolished in trypsin-treated conidia, suggesting the proteinaceous nature of the binding site. Western blotting assay, using fibronectin and anti-fibronectin antibody, evidenced 7 polypeptides with molecular masses ranging from 55 to 17 kDa in both conidial and mycelial extracts. Fibronectin-binding molecules were localized by immunofluorescence and immunocytochemistry microscopies at the cell wall and in intracellular compartments of S. apiospermum cells. Furthermore, a possible function for the fibronectin-like molecules of S. apiospermum in the interaction with host lung cells was assessed. Conidia pre-treated with soluble fibronectin showed a significant reduction in adhesion to either epithelial or fibroblast lung cells in a classically dose-dependent manner. Similarly, the pre-treatment of the lung cells with anti-fibronectin antibodies considerably diminished the adhesion. Collectively, the results demonstrated the presence of fibronectin-binding molecules in S. apiospermum cells and their role in adhesive events.

Silver(I) and Copper(II) 1,10-Phenanthroline-5,6-dione Complexes as Promising Antivirulence Strategy against Leishmania: Focus on Gp63 (Leishmanolysin).

Leishmaniasis, caused by protozoa of the genus Leishmania, encompasses a group of neglected diseases with diverse clinical and epidemiological manifestations that can be fatal if not adequately and promptly managed/treated. The current chemotherapy options for this disease are expensive, require invasive administration and often lead to severe side effects. In this regard, our research group has previously reported the potent anti-Leishmania activity of two coordination compounds (complexes) derived from 1,10-phenanthroline-5,6-dione (phendione): [Cu(phendione)3].(ClO4)2.4H2O and [Ag(phendione)2].ClO4. The present study aimed to evaluate the effects of these complexes on leishmanolysin (gp63), a virulence factor produced by all Leishmania species that plays multiple functions and is recognized as a potential target for antiparasitic drugs. The results showed that both Ag-phendione (-74.82 kcal/mol) and Cu-phendione (-68.16 kcal/mol) were capable of interacting with the amino acids comprising the active site of the gp63 protein, exhibiting more favorable interaction energies compared to phendione alone (-39.75 kcal/mol) or 1,10-phenanthroline (-45.83 kcal/mol; a classical gp63 inhibitor) as judged by molecular docking assay. The analysis of kinetic parameters using the fluorogenic substrate Z-Phe-Arg-AMC indicated Vmax and apparent Km values of 0.064 µM/s and 14.18 µM, respectively, for the released gp63. The effects of both complexes on gp63 proteolytic activity were consistent with the in silico assay, where Ag-phendione exhibited the highest gp63 inhibition capacity against gp63, with an IC50 value of 2.16 µM and the lowest inhibitory constant value (Ki = 5.13 µM), followed by Cu-phendione (IC50 = 163 µM and Ki = 27.05 µM). Notably, pretreatment of live L. amazonensis promastigotes with the complexes resulted in a significant reduction in the expression of gp63 protein, including the isoforms located on the parasite cell surface. Both complexes markedly decreased the in vitro association indexes between L. amazonensis promastigotes and THP-1 human macrophages; however, this effect was reversed by the addition of soluble gp63 molecules to the interaction medium. Collectively, our findings highlight the potential use of these potent complexes in antivirulence therapy against Leishmania, offering new insights for the development of effective treatments for leishmaniasis.

Scedosporium/Lomentospora Species Induce the Production of Siderophores by Pseudomonas aeruginosa in a Cystic Fibrosis Mimic Environment.

Over the last years, the interkingdom microbial interactions concerning bacteria and fungi cohabiting and/or responsible for human pathologies have been investigated. In this context, the Gram-negative bacterium Pseudomonas aeruginosa and fungal species belonging to the Scedosporium/Lomentospora genera are widespread, multidrug-resistant, emergent, opportunistic pathogens that are usually co-isolated in patients with cystic fibrosis. The available literature reports that P. aeruginosa can inhibit the in vitro growth of Scedosporium/Lomentospora species; however, the complex mechanisms behind this phenomenon are mostly unknown. In the present work, we have explored the inhibitory effect of bioactive molecules secreted by P. aeruginosa (3 mucoid and 3 non-mucoid strains) on S. apiospermum (n = 6 strains), S. minutisporum (n = 3), S. aurantiacum (n = 6) and L. prolificans (n = 6) under cultivation in a cystic fibrosis mimic environment. It is relevant to highlight that all bacterial and fungal strains used in the present study were recovered from cystic fibrosis patients. The growth of Scedosporium/Lomentospora species was negatively affected by the direct interaction with either mucoid or non-mucoid strains of P. aeruginosa. Moreover, the fungal growth was inhibited by the conditioned supernatants obtained from bacteria-fungi co-cultivations and by the conditioned supernatants from the bacterial pure cultures. The interaction with fungal cells induced the production of pyoverdine and pyochelin, 2 well-known siderophores, in 4/6 clinical strains of P. aeruginosa. The inhibitory effects of these four bacterial strains and their secreted molecules on fungal cells were partially reduced with the addition of 5-flucytosine, a classical repressor of pyoverdine and pyochelin production. In sum, our results demonstrated that distinct clinical strains of P. aeruginosa can behave differently towards Scedosporium/Lomentospora species, even when isolated from the same cystic fibrosis patient. Additionally, the production of siderophores by P. aeruginosa was induced when co-cultivated with Scedosporium/Lomentospora species, indicating competition for iron and deprivation of this essential nutrient, leading to fungal growth inhibition.

In Vitro Effects of Aminopyridyl Ligands Complexed to Copper(II) on the Physiology and Interaction Process of Trypanosoma cruzi.

Chagas disease is derived from the infection by the protozoan Trypanosoma cruzi. In many countries, benznidazole is the only drug approved for clinical use despite several side effects and the emergence of resistant parasite strains. In this context, our group has previously pointed out that two novel aminopyridine derivatives complexed with Cu2+, namely, cis-aquadichloro(N-[4-(hydroxyphenyl)methyl]-2-pyridinemethamino)copper (3a) and its glycosylated ligand cis-dichloro (N-{[4-(2,3,4,6-tetra-O-acetyl-ß-D-glucopyranosyloxy)pheny]lmethyl}-2-pyridinemethamino)copper (3b), are effective against T. cruzi trypomastigote forms. With this result in mind, the present work aimed to investigate the effects of both compounds on trypomastigotes physiology and on the interaction process with host cells. Apart from loss of plasma membrane integrity, an increased generation of reactive oxygen species (ROS) and decreased mitochondrial metabolism were observed. Pretreatment of trypomastigotes with these metallodrugs inhibited the association index with LLC-MK2 cells in a typical dose-dependent manner. Both compounds showed low toxicity on mammalian cells (CC50 > 100 µM), and the IC50 values calculated for intracellular amastigotes were determined as 14.4 µM for 3a and 27.1 µM for 3b. This set of results demonstrates the potential of these aminopyridines complexed with Cu2+ as promising candidates for further antitrypanosomal drug development.

Cell dispersion during biofilm formation by Scedosporium apiospermum, Scedosporium aurantiacum, Scedosporium minutisporum and Lomentospora prolificans.

Dispersion is an essential step in the lifecycle of biofilms, since it enables the dissemination of microbial cells and, consequently, the potential colonization of new sites. Filamentous fungi belonging to the Scedosporium/Lomentospora genera are opportunistic human pathogens able to form multidrug-resistant biofilms on surfaces of different chemical compositions, environments and nutritional conditions. Despite the rising understanding of how biofilms are formed by Scedosporium/Lomentospora species, the cell dispersal step has not yet been explored. In the present study, the cell dispersion was investigated during biofilm formation by S. apiospermum, S. minutisporum, S. aurantiacum and L. prolificans cells. The results revealed that conidia were the major type of dispersed cells, which were detected throughout biofilm development (from 24 to 72 h). Dispersion was not influenced by increased glucose concentration (the main source for energetic metabolism) neither the presence of voriconazole (the most common antifungal used to treat scedosporiosis); however, the presence of mucin (a component of mucous, present in the lungs of cystic fibrosis patients, who are usually affected by these filamentous fungi) triggered cell dispersion. Contrarily, a poor nutritional environment (e.g., phosphate-buffered saline) inhibited this step. Overall, our study reveals new insights into the biofilm development of Scedosporium/Lomentospora species.

Peptidases Are Potential Targets of Copper(II)-1,10-Phenanthroline-5,6-dione Complex, a Promising and Potent New Drug against Trichomonas vaginalis.

Trichomonas vaginalis is responsible for 156 million new cases per year worldwide. When present asymptomatically, the parasite can lead to serious complications, such as development of cervical and prostate cancer. As infection increases the acquisition and transmission of HIV, the control of trichomoniasis represents an important niche for the discovery and development of new antiparasitic molecules. This urogenital parasite synthesizes several molecules that allow the establishment and pathogenesis of infection. Among them, peptidases occupy key roles as virulence factors, and the inhibition of these enzymes has become an important mechanism for modulating pathogenesis. Based on these premises, our group recently reported the potent anti-T. vaginalis action of the metal-based complex [Cu(phendione)3](ClO4)2.4H2O (Cu-phendione). In the present study, we evaluated the influence of Cu-phendione on the modulation of proteolytic activities produced by T. vaginalis by biochemical and molecular approaches. Cu-phendione showed strong inhibitory potential against T. vaginalis peptidases, especially cysteine- and metallo-type peptidases. The latter revealed a more prominent effect at both the post-transcriptional and post-translational levels. Molecular Docking analysis confirmed the interaction of Cu-phendione, with high binding energy (-9.7 and -10.7 kcal·mol-1, respectively) at the active site of both TvMP50 and TvGP63 metallopeptidases. In addition, Cu-phendione significantly reduced trophozoite-mediated cytolysis in human vaginal (HMVII) and monkey kidney (VERO) epithelial cell lineages. These results highlight the antiparasitic potential of Cu-phendione by interaction with important T. vaginalis virulence factors.

Silver(I) 1,10-Phenanthroline Complexes Are Active against Fonsecaea pedrosoi Viability and Negatively Modulate Its Potential Virulence Attributes.

The genus Fonsecaea is one of the etiological agents of chromoblastomycosis (CBM), a chronic subcutaneous disease that is difficult to treat. This work aimed to evaluate the effects of copper(II), manganese(II) and silver(I) complexes coordinated with 1,10-phenanthroline (phen)/1,10-phenanthroline-5,6-dione (phendione) on Fonsecaea spp. Our results revealed that most of these complexes were able to inhibit F. pedrosoi, F. monophora and F. nubica conidial viability with minimum inhibitory concentration (MIC) values ranging from 0.6 to 100 µM. The most effective complexes against F. pedrosoi planktonic conidial cells, the main etiologic agent of CBM, were [Ag(phen)2]ClO4 and [Ag2(3,6,9-tdda)(phen)4].EtOH, (tdda: 3,6,9-trioxaundecanedioate), displaying MIC values equal to 1.2 and 0.6 µM, respectively. These complexes were effective in reducing the viability of F. pedrosoi biofilm formation and maturation. Silver(I)-tdda-phen, combined with itraconazole, reduced the viability and extracellular matrix during F. pedrosoi biofilm development. Moreover, both silver(I) complexes inhibited either metallo- or aspartic-type peptidase activities of F. pedrosoi as well as its conidia into mycelia transformation and melanin production. In addition, the complexes induced the production of intracellular reactive oxygen species in F. pedrosoi. Taken together, our data corroborate the antifungal action of metal-phen complexes, showing they represent a therapeutic option for fungal infections, including CBM.

SARS-CoV-2 Post-Infection and Sepsis by Saccharomyces cerevisiae: A Fatal Case Report-Focus on Fungal Susceptibility and Potential Virulence Attributes.

The pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been responsible for approximately 6.8 million deaths worldwide, threatening more than 753 million individuals. People with severe coronavirus disease-2019 (COVID-19) infection often exhibit an immunosuppression condition, resulting in greater chances of developing co-infections with bacteria and fungi, including opportunistic yeasts belonging to the Saccharomyces and Candida genera. In the present work, we have reported the case of a 75-year-old woman admitted at a Brazilian university hospital with an arterial ulcer in the left foot, which was being prepared for surgical amputation. The patient presented other underlying diseases and presented positive tests for COVID-19 prior to hospitalization. She received antimicrobial treatment, but her general condition worsened quickly, leading to death by septic shock after 4 days of hospitalization. Blood samples collected on the day she died were positive for yeast-like organisms, which were later identified as Saccharomyces cerevisiae by both biochemical and molecular methods. The fungal strain exhibited low minimal inhibitory concentration values for the antifungal agents tested (amphotericin B, 5-flucytosine, caspofungin, fluconazole and voriconazole), and it was able to produce important virulence factors, such as extracellular bioactive molecules (e.g., aspartic peptidase, phospholipase, esterase, phytase, catalase, hemolysin and siderophore) and biofilm. Despite the activity against planktonic cells, the antifungals were not able to impact the mature biofilm parameters (biomass and viability). Additionally, the S. cerevisiae strain caused the death of Tenebrio molitor larvae, depending on the fungal inoculum, and larvae immunosuppression with corticosteroids increased the larvae mortality rate. In conclusion, the present study highlighted the emergence of S. cerevisiae as an opportunistic fungal pathogen in immunosuppressed patients presenting several severe comorbidities, including COVID-19 infection.

The Anti-Leishmania amazonensis and Anti-Leishmania chagasi Action of Copper(II) and Silver(I) 1,10-Phenanthroline-5,6-dione Coordination Compounds.

Leishmaniasis is a neglected disease caused by protozoa belonging to the Leishmania genus. Notably, the search for new, promising and potent anti-Leishmania compounds remains a major goal due to the inefficacy of the available drugs used nowadays. In the present work, we evaluated the effects of 1,10-phenanthroline-5,6-dione (phendione) coordinated to silver(I), [Ag(phendione)2]ClO4 (Ag-phendione), and copper(II), [Cu(phendione)3](ClO4)2·4H2O (Cu-phendione), as potential drugs to be used in the chemotherapy against Leishmania amazonensis and Leishmania chagasi. The results showed that promastigotes treated with Ag-phendione and Cu-phendione presented a significant reduction in the proliferation rate. The IC50 values calculated to Ag-phendione and Cu-phendione, respectively, were 7.8 nM and 7.5 nM for L. amazonensis and 24.5 nM and 20.0 nM for L. chagasi. Microscopical analyses revealed several relevant morphological changes in promastigotes, such as a rounding of the cell body and a shortening/loss of the single flagellum. Moreover, the treatment promoted alterations in the unique mitochondrion of these parasites, inducing significant reductions on both metabolic activity and membrane potential parameters. All these cellular perturbations induced the triggering of apoptosis-like death in these parasites, as judged by the (i) increased percentage of annexin-positive/propidium iodide negative cells, (ii) augmentation in the proportion of parasites in the sub-G0/G1 phase and (iii) DNA fragmentation. Finally, the test compounds showed potent effects against intracellular amastigotes; contrarily, these molecules were well tolerated by THP-1 macrophages, which resulted in excellent selective index values. Overall, the results highlight new selective and effective drugs against Leishmania species, which are important etiological agents of both cutaneous (L. amazonensis) and visceral (L. chagasi) leishmaniasis in a global perspective.

Extracellularly Released Molecules by the Multidrug-Resistant Fungal Pathogens Belonging to the Scedosporium Genus: An Overview Focused on Their Ecological Significance and Pathogenic Relevance.

The multidrug-resistant species belonging to the Scedosporium genus are well recognized as saprophytic filamentous fungi found mainly in human impacted areas and that emerged as human pathogens in both immunocompetent and immunocompromised individuals. It is well recognized that some fungi are ubiquitous organisms that produce an enormous amount of extracellular molecules, including enzymes and secondary metabolites, as part of their basic physiology in order to satisfy their several biological processes. In this context, the molecules secreted by Scedosporium species are key weapons for successful colonization, nutrition and maintenance in both host and environmental sites. These biologically active released molecules have central relevance on fungal survival when colonizing ecological places contaminated with hydrocarbons, as well as during human infection, particularly contributing to the invasion/evasion of host cells and tissues, besides escaping from the cellular and humoral host immune responses. Based on these relevant premises, the present review compiled the published data reporting the main secreted molecules by Scedosporium species, which operate important physiopathological events associated with pathogenesis, diagnosis, antimicrobial activity and bioremediation of polluted environments.

Biofilm Formation by Chromoblastomycosis Fungi Fonsecaea pedrosoi and Phialophora verrucosa: Involvement with Antifungal Resistance.

Patients with chromoblastomycosis (CBM) suffer chronic tissue lesions that are hard to treat. Considering that biofilm is the main growth lifestyle of several pathogens and it is involved with both virulence and resistance to antimicrobial drugs, we have investigated the ability of CBM fungi to produce this complex, organized and multicellular structure. Fonsecaea pedrosoi and Phialophora verrucosa conidial cells were able to adhere on a polystyrene abiotic substrate, differentiate into hyphae and produce a robust viable biomass containing extracellular matrix. Confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) showed the tridimensional architecture of the mature biofilms, revealing a dense network of interconnected hyphae, inner channels and amorphous extracellular polymeric material. Interestingly, the co-culture of each fungus with THP-1 macrophage cells, used as a biotic substrate, induced the formation of a mycelial trap covering and damaging the macrophages. In addition, the biofilm-forming cells of F. pedrosoi and P. verrucosa were more resistant to the conventional antifungal drugs than the planktonic-growing conidial cells. The efflux pump activities of P. verrucosa and F. pedrosoi biofilms were significantly higher than those measured in conidia. Taken together, the data pointed out the biofilm formation by CBM fungi and brought up a discussion of the relevance of studies about their antifungal resistance mechanisms.