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.

Unveiling the antifungal mechanisms of CTP, a new copper(II)-theophylline/1,10-phenanthroline complex, on drug-resistant non-albicans Candida species.

Candida species undeniably rank as the most prevalent opportunistic human fungal pathogens worldwide, with Candida albicans as the predominant representative. However, the emergence of non-albicans Candida species (NACs) has marked a significant shift, accompanied by rising incidence rates and concerning trends of antifungal resistance. The search for new strategies to combat antifungal-resistant Candida strains is of paramount importance. Recently, our research group reported the anti-Candida activity of a coordination compound containing copper(II) complexed with theophylline (theo) and 1,10-phenanthroline (phen), known as "CTP" - Cu(theo)2phen(H2O).5H2O. In the present work, we investigated the mechanisms of action of CTP against six medically relevant, antifungal-resistant NACs, including C. auris, C. glabrata, C. haemulonii, C. krusei, C. parapsilosis and C. tropicalis. CTP demonstrated significant efficacy in inhibiting mitochondrial dehydrogenases, leading to heightened intracellular reactive oxygen species production. CTP treatment resulted in substantial damage to the plasma membrane, as evidenced by the passive incorporation of propidium iodide, and induced DNA fragmentation as revealed by the TUNEL assay. Scanning electron microscopy images of post-CTP treatment NACs further illustrated profound alterations in the fungal surface morphology, including invaginations, cavitations and lysis. These surface modifications significantly impacted the ability of Candida cells to adhere to a polystyrene surface and to form robust biofilm structures. Moreover, CTP was effective in disassembling mature biofilms formed by these NACs. In conclusion, CTP represents a promising avenue for the development of novel antifungals with innovative mechanisms of action against clinically relevant NACs that are resistant to antifungals commonly used in clinical settings.

Decoding the antifungal resistance mechanisms in biofilms of emerging, ubiquitous and multidrug-resistant species belonging to the Scedosporium/Lomentospora genera.

The opportunistic filamentous fungi belonging to the Scedosporium and Lomentospora genera are highly tolerant to all classes of available antifungal drugs. Moreover, the mature biofilm formed by these fungi presents higher antifungal resistance when compared to planktonic cells. Nevertheless, the resistance mechanisms developed by the biofilm lifestyle are not completely elucidated. In the current study, we have investigated the mainly known resistance mechanisms to azoles (voriconazole and fluconazole) and polyenes (amphotericin B [AMB]) in S. apiospermum, S. minutisporum, S. aurantiacum, and L. prolificans (formerly S. prolificans) biofilms. Both classes of antifungals can physically bind to the extracellular matrix of mature biofilms, preventing the drugs from reaching their targets on biofilm-forming cells, which precludes their activity and toxicity. In addition, the activity of efflux pumps, measured by Rhodamine 6 G, was increased along with the maturation of the biofilm. The efflux pump's inhibition by L-Phe-L-Arg-ß-naphthylamide culminated in a 2- to 16-fold increase in azole susceptibility in conidial cells, but not in mature biofilms. Finally, we demonstrated by using specific inhibitors that in conidia, but not in biofilms, AMB induced the production of reactive oxygen species through the activity of the oxidative phosphorylation system (complex I-IV and alternative oxidases). However, the cellular redox imbalance caused by AMB was well-coped with the high activity of antioxidative enzymes, such as superoxide dismutase and catalase. Altogether, our results revealed that Scedosporium/Lomentospora biofilm resistance occurs through various mechanisms that operate concomitantly, which could explain the huge challenge in the clinical treatment of scedosporiosis/lomentosporiosis. LAY SUMMARY: Scedosporium/Lomentospora spp. are multidrug-resistant pathogens able to cause diverse types of infections with typical biofilm characteristics, which makes the treatment a hard issue. We deciphered the resistance mechanisms to classical antifungals developed in the biofilm formed by these fungi.

Differences in Charge Distribution in Leishmania tarentolae Leishmanolysin Result in a Reduced Enzymatic Activity.

Leishmania tarentolae is a non-pathogenic trypanosomatid isolated from lizards widely used for heterologous protein expression and extensively studied to understand the pathogenic mechanisms of leishmaniasis. The repertoire of leishmanolysin genes was reported to be expanded in L. tarentolae genome, but no proteolytic activity was detected. Here, we analyzed L. tarentolae leishmanolysin proteins from the genome to the structural levels and evaluated the enzymatic activity of the wild-type and overexpressing mutants of leishmanolysin. A total of 61 leishmanolysin sequences were retrieved from the L. tarentolae genome. Five of them were selected for phylogenetic analysis, and for three of them, we built 3D models based on the crystallographic structure of L. major ortholog. Molecular dynamics simulations of these models disclosed a less negative electrostatic potential compared to the template. Subsequently, L. major LmjF.10.0460 and L. tarentolae LtaP10.0650 leishmanolysins were cloned in a pLEXSY expression system into L. tarentolae. Proteins from the wild-type and the overexpressing parasites were submitted to enzymatic analysis. Our results revealed that L. tarentolae leishmanolysins harbor a weak enzymatic activity about three times less abundant than L. major leishmanolysin. Our findings strongly suggest that the less negative electrostatic potential of L. tarentolae leishmanolysin can be the reason for the reduced proteolytic activity detected in this parasite.

Anti-Leishmania braziliensis activity of 1,10-phenanthroline-5,6-dione and its Cu(II) and Ag(I) complexes.

Leishmaniasis, included in the priority list of the WHO, remains as a neglected disease caused by parasites of the Leishmania genus. There is no vaccine available for human leishmaniasis, and the current treatment is based on old drugs that cause serious side effects. Herein, we initially studied the cellular distribution of the virulence factor gp63, the major metallopeptidase, in a virulent strain of Leishmania braziliensis, and then we measured the inhibitory effects of 1,10-phenanthroline-5,6-dione (phendione), and its metal complexes, [Cu(phendione)3](ClO4)2.4H2O and [Ag(phendione)2]ClO4, on both cellular and extracellular metallopeptidases produced by promastigotes. The action of the three compounds on parasite viability and on parasite-macrophage interaction was also determined. Gp63 molecules were detected in several parasite compartments, including the cytoplasm, the membrane lining the cell body and flagellum, and in the flagellar pocket, which explains the presence of gp63 in the culture medium. The test compounds inhibited parasite metallopeptidases in a typical dose-dependent manner, and they also caused a significant and irreversible inhibition of parasite motility. Moreover, the pre-treatment of promastigotes with the test compounds induced a decrease in the association index with macrophages. Collectively, phendione and its Cu(II) and Ag(I) complexes are excellent prototypes for the development of new anti-L. braziliensis drugs.

Exploring Innovative Leishmaniasis Treatment: Drug Targets from Pre-Clinical to Clinical Findings.

Leishmaniasis is a group of tropical diseases caused by parasitic protozoa belonging to the genus Leishmania. The disease is categorized in cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL), and visceral leishmaniasis (VL). The conventional treatment is complex and can present high toxicity and therapeutic failures. Thus, there is a continuing need to develop new treatments. In this review, we focus on the novel molecules described in the literature with potential leishmanicidal activity, categorizing them in pre-clinical (in vitro, in vivo), drug repurposing and clinical research.

Surface, adhesiveness and virulence aspects of Candida haemulonii species complex.

The emerging opportunistic pathogens comprising the Candida haemulonii complex (C. haemulonii [Ch], C. duobushaemulonii [Cd] and C. haemulonii var. vulnera[Chv]) are notable for their intrinsic antifungal resistance. Different clinical manifestations are associated with these fungal infections; however, little is known about their biology and potential virulence attributes. Herein, we evaluated some surface properties of 12 clinical isolates of Ch (n = 5), Cd (n = 4) and Chv (n = 3) as well as their virulence on murine macrophages and Galleria mellonella larvae. Scanning electron microscopy demonstrated the presence of homogeneous populations among the species of the C. haemulonii complex, represented by oval yeasts with surface irregularities able to form aggregates. Cell surface hydrophobicity was isolate-specific, exhibiting high (16.7%), moderate (25.0%) and low (58.3%) hydrophobicity. The isolates had negative surface charge, except for one. Mannose/glucose- and N-acetylglucosamine-containing glycoconjugates were evidenced in considerable amounts in all isolates; however, the surface expression of sialic acid was poorly detected. Cd isolates presented significantly higher amounts of chitin than Ch and Chv. Membrane sterol and lipid bodies, containing neutral lipids, were quite similar among all fungi studied. All isolates adhered to inert surfaces in the order: polystyrene > poly-L-lysine-coated glass > glass. Likewise, they interacted with murine macrophages in a quite similar way. Regarding in vivo virulence, the C. haemulonii species complex were able to kill at least 80% of the larvae after 120 hours. Our results evidenced the ability of C. haemulonii complex to produce potential surface-related virulence attributes, key components that actively participate in the infection process described in Candida spp.

ß-Cyclodextrin/Isopentyl Caffeate Inclusion Complex: Synthesis, Characterization and Antileishmanial Activity.

Isopentyl caffeate (ICaf) is a bioactive ester widely distributed in nature. Our patented work has shown promising results of this molecule against Leishmania. However, ICaf shows poor solubility, which limits its usage in clinical settings. In this work, we have proposed the development of an inclusion complex of ICaf in ß-cyclodextrin (ß-CD), with the aim to improve the drug solubility, and thus, its bioavailability. The inclusion complex (ICaf:ß-CD) was developed applying three distinct methods, i.e., physical mixture (PM), kneading (KN) or co-evaporation (CO) in different molar proportions (0.25:1, 1:1 and 2:1). Characterization of the complexes was carried out by thermal analysis, Fourier-transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM) and molecular docking. The ICaf:ß-CD complex in a molar ratio of 1:1 obtained by CO showed the best complexation and, therefore, was selected for further analysis. Solubility assay showed a marked improvement in the ICaf:ß-CD (CO, 1:1) solubility profile when compared to the pure ICaf compound. Cell proliferation assay using ICaf:ß-CD complex showed an IC50 of 3.8 and 2.7 µg/mL against L. amazonesis and L. chagasi promastigotes, respectively. These results demonstrate the great potential of the inclusion complex to improve the treatment options for visceral and cutaneous leishmaniases.

Protective outcomes of low-dose doxycycline on renal function of Wistar rats subjected to acute ischemia/reperfusion injury.

Renal ischemia-reperfusion injury (IRI) is a major cause of acute renal failure. Doxycycline (Dc) belongs to the tetracycline-class of antibiotics with demonstrated beneficial molecular effects in the brain and heart, mainly through matrix metalloproteinases inhibition (MMP). However, Dc protection of renal function has not been demonstrated. We determined whether low doses of Dc would prevent decreases in glomerular filtration rate (GFR) and maintain tubular Na+ handling in Wistar rats subjected to kidney I/R. Male Wistar rats underwent bilateral kidney ischemia for 30min followed by 24h reperfusion (I/R). Doxycycline (1, 3, and 10mg/kg, i.p.) was administered 2h before surgery. Untreated I/R rats showed a 250% increase in urine volume and proteinuria, a 60% reduction in GFR, accumulation of urea-nitrogen in the blood, and a 60% decrease in the fractional Na+ excretion due to unbalanced Na+ transporter activity. Treatment with Dc 3mg/kg maintained control levels of urine volume, proteinuria, GFR, blood urea-nitrogen, fractional Na+ excretion, and equilibrated Na+ transporter activities. The Dc protection effects on renal function were associated with kidney structure preservation and prevention of TGFß and fibronectin deposition. In vitro, total MMP activity was augmented in I/R and inhibited by 25 and 50µM Dc. In vivo, I/R augmented MMP-2 and -9 protein content without changing their activities. Doxycycline treatment downregulated total MMP activity and MMP-2 and -9 protein content. Our results suggest that treatment with low dose Dc protects from IRI, thereby preserving kidney function.

In vitro selection of Phytomonas serpens cells resistant to the calpain inhibitor MDL28170: alterations in fitness and expression of the major peptidases and efflux pumps.

The species Phytomonas serpens is known to express some molecules displaying similarity to those described in trypanosomatids pathogenic to humans, such as peptidases from Trypanosoma cruzi (cruzipain) and Leishmania spp. (gp63). In this work, a population of P. serpens resistant to the calpain inhibitor MDL28170 at 70 µ m (MDLR population) was selected by culturing promastigotes in increasing concentrations of the drug. The only relevant ultrastructural difference between wild-type (WT) and MDLR promastigotes was the presence of microvesicles within the flagellar pocket of the latter. MDLR population also showed an increased reactivity to anti-cruzipain antibody as well as a higher papain-like proteolytic activity, while the expression of calpain-like molecules cross-reactive to anti-Dm-calpain (from Drosophila melanogaster) antibody and calcium-dependent cysteine peptidase activity were decreased. Gp63-like molecules also presented a diminished expression in MDLR population, which is probably correlated to the reduction in the parasite adhesion to the salivary glands of the insect vector Oncopeltus fasciatus. A lower accumulation of Rhodamine 123 was detected in MDLR cells when compared with the WT population, a phenotype that was reversed when MDLR cells were treated with cyclosporin A and verapamil. Collectively, our results may help in the understanding of the roles of calpain inhibitors in trypanosomatids.

Surface properties, adhesion and biofilm formation on different surfaces by Scedosporium spp. and Lomentospora prolificans.

In the present work, some surface properties of the fungi Scedosporium apiospermum, S. aurantiacum, S. minutisporum, and Lomentospora prolificans and their capability to adhere to and form a biofilm on diverse surfaces were evaluated. All four species had high conidial surface hydrophobicity and elevated electronegative zeta potentials. Abundant quantities of melanin were detected at the conidial surface, whereas sialic acid was absent. The numbers of non-germinated and germinated conidia adhered to poly-L-lysine-covered slides was higher than on glass after 4 h of fungi-surface contact. Additionally, after 72 h of interaction a typical biofilm structure had formed. Mature biofilms were also observed after 72 h on a nasogastric catheter (made from polyvinyl chloride), a late bladder catheter (siliconized latex), and a nasoenteric catheter (polyurethane). Interestingly, biofilm biomass increased considerably when the catheters had previously been incubated with serum. These results confirm that Scedosporium/Lomentospora spp. are capable of forming biofilms on diverse abiotic surfaces.

Planktonic growth and biofilm formation profiles in Candida haemulonii species complex.

Candida haemulonii species complex have emerged as multidrug-resistant yeasts able to cause fungemia worldwide. However, very little is known regarding their physiology and virulence factors. In this context, planktonic growth and biofilm formation of Brazilian clinical isolates of Candida haemulonii (n = 5), Candida duobushaemulonii (n = 4), and Candida haemulonii var. vulnera (n = 3) were reported. Overall, the fungal planktonic growth curves in Sabouraud dextrose broth reached the exponential phase in 48 h at 37°C. All the clinical isolates formed biofilm on polystyrene in a time-dependent event, as judged by the parameters evaluated: biomass (crystal violet staining), metabolic activity (XTT reduction), and extracellular matrix (safranin incorporation). No statistically significant differences were observed when the average measurements among the three Candida species were compared regarding both planktonic and biofilm lifestyles; however, typical isolate-specific differences were clearly noticed in fungal growth kinetics.

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.

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 [...].

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.

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.

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.

Antileishmanial Efficacy of the Calpain Inhibitor MDL28170 in Combination with Amphotericin B.

The necessity of drug combinations to treat leishmaniasis came to the surface mainly because of the toxicity of current treatments and the emergence of resistant strains. The calpain inhibitor MDL28170 has previously shown anti-Leishmania activity, therefore its use in association with standard drugs could provide a new alternative for the treatment strategy against leishmaniasis. In this study, we analyzed the potential of the combination of MDL28170 and the antileishmanial drug amphotericin B against Leishmania amazonensis and Leishmania chagasi. The compounds were tested in the combination of the ½ × IC50 value of MDL28170 plus the » × IC50 value of amphotericin B, which led to an increment in the anti-promastigote activity when compared to the single drug treatments. This drug association revealed several and severe morphophysiological changes on parasite cells, such as loss of plasma membrane integrity, reduced size of flagellum, and depolarization of mitochondrial membrane potential besides increased reactive oxygen species production. In addition, the combination of both drugs had a deleterious effect on the Leishmania-macrophage interaction, reflecting in a significant anti-amastigote action, which achieved a reduction of 50% in the association index. These results indicate that the combination treatment proposed here may represent a new alternative for leishmaniasis chemotherapy.

Analysis of the mechanisms of action of isopentenyl caffeate against Leishmania.

Leishmaniasis is a neglected parasitic disease for which the conventional treatment can be considered inefficient and extremely aggressive, generating several and severe side effects. Therefore, the discovery of new drug candidates is important for the improvement in the quality of life of patients. Previously, we reported the promising results of isopentyl caffeate (ICaf) against Leishmania chagasi (agent of visceral leishmaniasis) and Leishmania amazonensis (agent of cutaneous leishmaniasis) promastigotes, displaying IC50 of 1.56 and 1.71 µM, respectively. Herein, we aimed to decipher the mechanisms of anti-Leishmania action of ICaf. Light and scanning electron microscopy assays showed relevant morphological changes in promastigotes when treated with ICaf, including rounding of the parasite body, shortening of the flagellum, blebs on the plasma membrane and cellular aggregation. The parasite mitochondrion was targeted by ICaf, resulting in a significant reduction in its metabolic activity and electric membrane potential followed by an increase in the production of reactive oxygen species, which culminated in the loss of plasma membrane integrity and parasite death. Relevantly, ICaf also had a potent anti-amastigote action. The IC50 values calculated for intracellular amastigotes of L. amazonensis were 3.27, 1.60 and 1.52 µM, while for L. chagasi the values were 2.48, 1.84 and 1.60 µM, respectively, after treating the infected macrophages with ICaf for 24, 48 and 72 h. ICaf was well tolerated by THP-1 macrophages, which gave rise to excellent selectivity indexes considering both Leishmania species. The current results suggest that ICaf may emerge as a chemotherapeutic alternative for the treatment of leishmaniasis.

Doxycycline treatment reestablishes renal function of Wistar rats in experimental envenomation with Bothrops jararacussu venom.

Acute kidney injury is one of the main complications of ophidian accidents and the leading cause of death in patients who survive the initial damage effects of venom. The hypothesis proposed in this investigation is that the pharmacological repositioning of doxycycline (doxy) attenuates renal injury provoked by Bothrops jararacussu (Bj) venom. Male Wistar rats were subjected or not (control) to experimental envenomation with Bj venom (3.5 mg/kg, im). Doxy (3 mg/kg, ip) was administered 2 h after envenoming. Envenomation with Bj venom promoted tissue damage in the renal cortex (moderate degree, score 3) in 24 h associated with decreased glomerular and tubular function, which promoted proteinuria and polyuria. Doxy treatment prevented the increase in urinary volume in 3 times, the increase in plasma creatinine in 33%, the increase in blood urea-nitrogen accumulation in 65%, the increase in urinary Na+ excretion in 2 times, marked proteinuria and kidney cortex injury induced by Bj envenomation. Bj venom promoted increase in protein content (66%) and reduction of 45% (Na++K+)-ATPase activity in the renal cortex. The enzyme was detected mainly in the luminal membrane. Doxy treatment was effective in preventing the mentioned alterations, maintaining (Na++K+)-ATPase in the basolateral membranes.