Candida parapsilosis: heterogeneous and strain-specific expression of secreted aspartic proteases (Sapp1 and Sapp2).

The increasing prevalence of Candida parapsilosis as a causative agent of fungal infections underscores the need to comprehensively understand its virulence factors. Secreted aspartic proteases (Saps) play a significant role in adhesion events, promoting biofilm formation, causing tissue damage and evading the host immune response. The present study investigates the production dynamics of Sapp1 and Sapp2 across 10 clinical isolates of C. parapsilosis using various approaches. Each fungal isolate demonstrated the capability to utilize bovine serum albumin (BSA) as the sole nitrogen source, as evidenced by its degradation in cell-free culture medium, forming low molecular mass polypeptides. Interestingly, the degradation of different proteinaceous substrates, such as BSA, human serum albumin (HSA), gelatin and hemoglobin, was typically isolate-dependent. Notably, higher proteolysis of HSA compared to BSA, gelatin and hemoglobin was observed. A quantitative assay revealed that the cleavage of a peptide fluorogenic substrate (cathepsin D) was isolate-specific, ranging from 44.15 to 270.61 FAU, with a mean proteolysis of 150.7 FAU. The presence of both Sapp1 and Sapp2 antigens on the cell surface of these fungal isolates was confirmed through immunological detection employing specific anti-Sapp1 and anti-Sapp2 antibodies. The surface levels of Sapp1 were consistently higher, up to fourfold, compared to Sapp2. Similarly, higher levels of Sapp1 than Sapp2 were detected in fungal secretions. This study provides insights into the dynamic expression and regulation of Sapps in C. parapsilosis, highlighting a known virulence factor that is considered a potential target for drug development against this increasingly prominent pathogen.

The fungal pathogen Candida parapsilosis can secrete aspartic proteases (Sapps) as part of its arsenal of virulence factors. We demonstrated that Sapps were able to cleave key host proteins, and the production of Sapp1 and Sapp2 antigens was typically dependent on the fungal isolate when grown in both planktonic- and biofilm-forming cells.

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.

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.

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.

Histoplasma capsulatum-induced extracellular DNA trap release in human neutrophils.

Neutrophils are leukocytes that are capable of eliminating both intra- and extracellular pathogens by mechanisms such as phagocytosis, degranulation, and release of neutrophil extracellular traps (NETs). Histoplasma capsulatum var. capsulatum (H. capsulatum) is a dimorphic fungus with a global distribution that causes histoplasmosis, a disease that is endemic in different geographic areas and is spreading worldwide. The release of NETs has been described as an important host defense mechanism against different fungi; however, there are no reports demonstrating that this process is implicated in neutrophil response to H. capsulatum infection. Therefore, the aim of this work is to investigate whether isolated human neutrophils release NETs in response to H. capsulatum and the potential mechanisms involved, as well as delineate the NETs antifungal activity. Using both confocal fluorescence and scanning electron microscopy techniques, we determined that NETs are released in vitro in response to H. capsulatum via an oxidative mechanism that is downstream of activation of the Syk and Src kinase pathways and is also dependent on CD18. NETs released in response to H. capsulatum yeasts involve the loss of neutrophil viability and are associated with elastase and citrullinated histones, however also can occur in a PAD4 histone citrullination independent pathway. This NETs also presented fungicidal activity against H. capsulatum yeasts. Our findings may contribute to the understanding of how neutrophils recognize and respond as immune effector cells to H. capsulatum, which may lead to better knowledge of histoplasmosis pathophysiology and treatment.

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.

Biofilms formed by Scedosporium and Lomentospora species: focus on the extracellular matrix.

In the present study, the composition of the extracellular matrix (ECM) of the biofilm formed by Scedosporium apiospermum, S. aurantiacum, S. minutisporum and Lomentospora prolificans on a polystyrene surface was investigated. Confocal laser scanning microscopy revealed a dense mycelial mass, with an ECM covering/interspersing the fungal cells and containing carbohydrate-rich molecules (e.g. glycoproteins) and extracellular DNA. The ECMs that were chemically extracted from mature biofilms formed by each of these fungi was predominantly composed of polysaccharides, followed by proteins, nucleic acids and sterols. In general, the amount of biofilm ECM was significantly greater in S. minutisporum and S. aurantiacum than in S. apiospermum and L. prolificans. Corroborating these results, the disarticulation of mature biofilms with enzymes, sodium metaperiodate and chelating agents occurred mainly in S. minutisporum and S. aurantiacum. Collectively, these results have revealed for the first time the composition of the ECM of the biofilms formed by Scedosporium/Lomentospora species and the role it plays in their architecture.

Aspartic peptidase of Phialophora verrucosa as target of HIV peptidase inhibitors: blockage of its enzymatic activity and interference with fungal growth and macrophage interaction.

Phialophora verrucosa causes several fungal human diseases, mainly chromoblastomycosis, which is extremely difficult to treat. Several studies have shown that human immunodeficiency virus peptidase inhibitors (HIV-PIs) are attractive candidates for antifungal therapies. This work focused on studying the action of HIV-PIs on peptidase activity secreted by P. verrucosa and their effects on fungal proliferation and macrophage interaction. We detected a peptidase activity from P. verrucosa able to cleave albumin, sensitive to pepstatin A and HIV-PIs, especially lopinavir, ritonavir and amprenavir, showing for the first time that this fungus secretes aspartic-type peptidase. Furthermore, lopinavir, ritonavir and nelfinavir reduced the fungal growth, causing remarkable ultrastructural alterations. Lopinavir and ritonavir also affected the conidia-macrophage adhesion and macrophage killing. Interestingly, P. verrucosa had its growth inhibited by ritonavir combined with either itraconazole or ketoconazole. Collectively, our results support the antifungal action of HIV-PIs and their relevance as a possible alternative therapy for fungal infections.

Naringenin-Functionalized Multi-Walled Carbon Nanotubes: A Potential Approach for Site-Specific Remote-Controlled Anticancer Delivery for the Treatment of Lung Cancer Cells.

Multi-walled carbon nanotubes functionalized with naringenin have been developed as new drug carriers to improve the performance of lung cancer treatment. The nanocarrier was characterized by Transmission Electron Microscopy (TEM), Fourier-Transform Infrared Spectroscopy (FTIR), X-ray photoelectron spectroscopy, Raman Spectroscopy, and Differential Scanning Calorimetry (DSC). Drug release rates were determined in vitro by the dialysis method. The cytotoxic profile was evaluated using the MTT assay, against a human skin cell line (hFB) as a model for normal cells, and against an adenocarcinomic human alveolar basal epithelial (A569) cell line as a lung cancer in vitro model. The results demonstrated that the functionalization of carbon nanotubes with naringenin occurred by non-covalent interactions. The release profiles demonstrated a pH-responsive behavior, showing a prolonged release in the tumor pH environment. The naringenin-functionalized carbon nanotubes showed lower cytotoxicity on non-malignant cells (hFB) than free naringenin, with an improved anticancer effect on malignant lung cells (A549) as an in vitro model of lung cancer.

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.

The serine peptidase inhibitor TPCK induces several morphophysiological changes in the opportunistic fungal pathogen Candida parapsilosis sensu stricto.

Candida parapsilosis sensu stricto (C. parapsilosis) has emerged as the second/third commonest Candida species isolated from hospitals worldwide. Candida spp. possess numerous virulence attributes, including peptidases that play multiple roles in both physiological and pathological events. So, fungal peptidases are valid targets for new drugs development. With this premise in mind, we have evaluated the effect of serine peptidase inhibitors (SPIs) on both cell biology and virulence aspects of C. parapsilosis. First, five different SPIs, phenylmethylsulfonyl fluoride, benzamidine, 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, N-α-tosyl-L-lysine chloromethyl ketone hydrochloride, and N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) were tested, and TPCK showed the best efficacy to arrest fungal growth. Subsequently, the ability of TPCK to modulate physiopathological processes was investigated. Overall, TPCK was able to (i) inhibit the cell-associated serine peptidase activities, (ii) promote morphometric and ultrastructural alterations, (iii) induce an increase in the intracellular oxidation level, which culminates in a vigorous lipid peroxidation and accumulation of neutral lipids in cytoplasmic inclusions, (iv) modulate the expression/exposition of surface structures, such as mannose/glucose-rich glycoconjugates, N-acetylglucosamine-containing molecules, chitin, polypeptides and surface aspartic peptidases, (v) reduce the adhesion to either polystyrene or glass surfaces as well as to partially disarticulate the mature biofilm, (vi) block the fungal interaction with macrophages, and (vii) protect Galleria mellonella from fungal infection, enhancing larvae survivability. Altogether, these results demonstrated that TPCK induced several changes over fungal biology besides the interference with aspects associated to C. parapsilosis virulence and pathogenesis, which indicates that SPIs could be novel promising therapeutic agents in dealing with candidiasis.

Participation of Trypanosoma cruzi gp63 molecules on the interaction with Rhodnius prolixus.

Trypanosoma cruzi is the causative agent of Chagas disease, a vector-borne disease. The parasite molecules involved in vector interaction have been little investigated. Metallopeptidases and gp63 molecules have been implicated in parasite adhesion of several trypanosomatids to the insect midgut. Although gp63 homologues are highly expanded in the T. cruzi genome, and are implicated in parasite-mammalian host interaction, its role in the insect vector has never been explored. Here, we showed that divalent metal chelators or anti-Tcgp63-I antibodies impaired T. cruzi adhesion to Rhodnius prolixus midgut. Parasites isolated after insect colonization presented a drastic enhancement in the expression of Tcgp63-I. These data highlight, for the first time, that Tcgp63-I and Zn-dependent enzymes contribute to the interaction of T. cruzi with the insect vector.

Chymotrypsin- and trypsin-like activities secreted by the multidrug-resistant yeasts forming the Candida haemulonii complex.

Candida haemulonii complex (C. haemulonii, C. haemulonii var. vulnera and C. duobushaemulonii) consists of emergent multidrug-resistant pathogens that cause bloodstream and deep-seated infections. However, little is known about their virulence factors. Herein, we evaluated the presence of extracellular serine peptidases in this fungal complex. Serine peptidase activity was measured by spectrophotometry using chromogenic peptide substrates to the S1 family. Chymotrypsin-, trypsin- and elastase-like activities were detected in all fungal isolates. Since higher chymotrypsin- and trypsin-like activities were observed from the cleavage of N-succinyl-Ala-Ala-Pro-Phe-pNa and N-benzoyl-Phe-Val-Arg-pNa, respectively, these substrates were selected for further experiments. Overall, pHs 7.0 and 9.0 were those in which higher chymotrypsin- and trypsin-like activities were observed, respectively, displaying higher hydrolytic activities at 37-45°C. Additionally, the serine peptidases produced by C. haemulonii complex were inhibited by PMSF and AEBSF in a typically concentration-dependent manner. Although the Michaelis constant (Km) values obtained for chymotrypsin-like peptidases were similar, greater differences were observed for trypsin-like enzymes secreted by the different fungal isolates. This is the first time that peptidases belonging to the S1 family are described in the C. haemulonii species complex. Thus, these data open the doors for more detailed studies into potential roles of these peptidases in fungal virulence.

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.

Virulence of Candida haemulonii complex in Galleria mellonella and efficacy of classical antifungal drugs: a comparative study with other clinically relevant non-albicans Candida species.

Candida haemulonii complex has emerged as notorious yeasts causing invasive infections with high rates of treatment failures. Since there is a particular interest in the development of non-mammalian host models to study microbial virulence, with the aim to evade the ethical impact of animal tests, herein we compared the virulence of C. haemulonii, C. duobushaemulonii and C. haemulonii var. vulnera with non-albicans Candida species (C. tropicalis, C. krusei and C. lusitaniae) on Galleria mellonella and the efficacy of antifungal drugs. All these fungi induced a dose-dependent effect on larvae killing, a decrease in hemocyte density and fungi were phagocytozed by hemocytes in equal proportions. Fungal inoculation caused early larvae melanization after some minutes of injection, followed by an augmented pigmentation after 24 h. Differences among species virulence can be explained, in part, by differences in growth rate and production of hydrolytic enzymes. First-line antifungals were tested with equivalent therapeutic doses and MIC profile in vitro was correlated with in vivo antifungal efficacy. Additionally, fungal burden increased in infected larvae along time and only caspofungin reduced the number of CFUs of C. haemulonii species complex. So, G. mellonella offers a simple and feasible model to study C. haemulonii complex virulence and drug efficacy.

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.

Lopinavir, an HIV-1 peptidase inhibitor, induces alteration on the lipid metabolism of Leishmania amazonensis promastigotes.

The anti-leishmania effects of HIV peptidase inhibitors (PIs) have been widely reported; however, the biochemical target and mode of action are still a matter of controversy in Leishmania parasites. Considering the possibility that HIV-PIs induce lipid accumulation in Leishmania amazonensis, we analysed the effects of lopinavir on the lipid metabolism of L. amazonensis promastigotes. To this end, parasites were treated with lopinavir at different concentrations and analysed by fluorescence microscopy and spectrofluorimetry, using a fluorescent lipophilic marker. Then, the cellular ultrastructure of treated and control parasites was analysed by transmission electron microscopy (TEM), and the lipid composition was investigated by thin-layer chromatography (TLC). Finally, the sterol content was assayed by gas chromatography-mass spectrometry (GC/MS). TEM analysis revealed an increased number of lipid inclusions in lopinavir-treated cells, which was accompanied by an increase in the lipophilic content, in a dose-dependent manner. TLC and GC-MS analysis revealed a marked increase of cholesterol-esters and cholesterol. In conclusion, lopinavir-induced lipid accumulation and affected lipid composition in L. amazonensis in a concentration-response manner. These data contribute to a better understanding of the possible mechanisms of action of this HIV-PI in L. amazonensis promastigotes. The concerted action of lopinavir on this and other cellular processes, such as the direct inhibition of an aspartyl peptidase, may be responsible for the arrested development of the parasite.