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.

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.

In vitro effects of the asymmetric peptidomimetic 157, containing l-tartaric acid core and valine/leucine substituents, on Leishmania amazonensis promastigotes and amastigotes.

The current treatments for leishmaniasis bump into several obstacles, including low efficacy, high costs, long monitoring, and several/severe side effects. Consequently, the search for promising compounds is a tangible need. Recently, we reported the anti-Leishmania amazonensis action of asymmetric peptidomimetic compounds containing tartaric acid as core, especially the 157 derivative that contains valine/leucine substituents in its structure. Herein, we decipher the multiple effects of 157 on the L. amazonensis physiology and on the interaction process with macrophages. The peptidomimetic 157 induced significant changes on the morphometric (internal granularity reduction as judged by flow cytometer) and on the ultrastructural (round-shaped parasites, presence of plasma membrane blebs and flagellum loss as visualized by scanning electron microscopy) aspects of treated promastigotes compared to untreated ones. The alteration on the plasma membrane permeability was confirmed by the passive incorporation of propidium iodide in 157-treated promastigotes. In parallel, the low viability of promastigotes was also associated to the perturbation of mitochondrial transmembrane electric potential. These combined results demonstrated that 157 induced irreversible metabolic damages that led to L. amazonensis death. The pre-treatment of promastigotes with 157 inhibited the association index with macrophages in a typically dose-dependent manner. Additionally, 157 significantly reduced the number of intramacrophage amastigotes after 72 h of drug contact, presenting an IC50 value of 30.2 µM. Under our experimental conditions, 157 showed higher toxicity to promastigotes and amastigotes when compared to RAW cells, resulting in good selective indexes. Therefore, 157 can be considered as an interesting candidate for further optimization, since its synthesis is simple and cheap.

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.

A new iron(III) complex-containing sulfadiazine inhibits the proliferation and induces cystogenesis of Toxoplasma gondii.

We have previously shown that metallocomplexes can control the growth of Toxoplasma gondii, the agent that causes toxoplasmosis. In order to develop new metallodrugs to treat this disease, we investigated the influence of the coordination of sulfadiazine (SDZ), a drug used to treat toxoplasmosis, on the biological activity of the iron(III) complex [Fe(HBPClNOL)Cl2]·H2O, 1, (H2BPClNOL=N-(2-hydroxybenzyl)-N-(2-pyridylmethyl)(3-chloro)(2-hydroxy)-propylamine). The new complex [(Cl)(SDZ)Fe(III)(µ-BPClNOL)2Fe(III)(SDZ)(Cl)]·2H2O, 2, which was obtained by the reaction between complex 1 and SDZ, was characterized using a range of physico-chemical techniques. The cytotoxic effect of the complexes and the ability of T. gondii to infect LLC-MK2 cells were assessed. It was found that both complexes reduced the growth of T. gondii while also causing low cytotoxicity in the host cells. After 48 h of treatment, complex 2 reduced the parasite's ability to proliferate by about 50% with an IC50 of 1.66 µmol/L. Meanwhile, complex 1 or SDZ alone caused a 40% reduction in proliferation, and SDZ displayed an IC50 of 5.3 µmol/L. In addition, complex 2 treatment induced distinct morphological and ultrastructural changes in the parasites and triggered the formation of cyst-like forms. These results show that the coordination of SDZ to the iron(III) complex is a good strategy for increasing the anti-toxoplasma activity of these compounds.

Extracellular vesicles and vesicle-free secretome of the protozoa Acanthamoeba castellanii under homeostasis and nutritional stress and their damaging potential to host cells.

Acanthamoeba castellanii (Ac) are ubiquitously distributed in nature, and by contaminating medical devices such as heart valves and contact lenses, they cause a broad range of clinical presentations to humans. Although several molecules have been described to play a role in Ac pathogenesis, including parasite host-tissue invasion and escaping of host-defense, little information is available on their mechanisms of secretion. Herein, we describe the molecular components secreted by Ac, under different protein availability conditions to simulate host niches. Ac extracellular vesicles (EVs) were morphologically and biochemically characterized. Dynamic light scattering analysis of Ac EVs identified polydisperse populations, which correlated to electron microscopy measurements. High-performance thin liquid chromatography of Ac EVs identified phospholipids, steryl-esters, sterol and free-fatty acid, the last two also characterized by GC-MS. Secretome composition (EVs and EVs-free supernatants) was also determined and proteins biological functions classified. In peptone-yeast-glucose (PYG) medium, a total of 179 proteins were identified (21 common proteins, 89 exclusive of EVs and 69 in EVs-free supernatant). In glucose alone, 205 proteins were identified (134 in EVs, 14 common and 57 proteins in EVs-free supernatant). From those, stress response, oxidative and protein and amino acid metabolism proteins prevailed. Qualitative differences were observed on carbohydrate metabolism enzymes from Krebs cycle and pentose phosphate shunt. Serine proteases and metalloproteinases predominated. Analysis of the cytotoxicity of Ac EVs (upon uptake) and EVs-free supernatant to epithelial and glioblastoma cells revealed a dose-dependent effect. Therefore, the Ac secretome differs depending on nutrient conditions, and is also likely to vary during infection.

Assessment of biofilm formation by Scedosporium apiospermum, S. aurantiacum, S. minutisporum and Lomentospora prolificans.

Reported herein is the ability of Scedosporium apiospermum, S. aurantiacum, S. minutisporum and Lomentospora prolificans conidia to adhere, differentiate into hyphae and form biofilms on both polystyrene and lung epithelial cells. To different degrees, all of the fungi adhered to polystyrene after 4 h, with a predominance of those with germinated conidia. Prolonged fungi-polystyrene contact resulted in the formation of a monolayer of intertwined mycelia, which was identified as a typical biofilm structure due to the presence of a viable mycelial biomass, extracellular matrix and enhanced antifungal resistance. Ultrastructural details were revealed by SEM and CLSM, showing the dense compaction of the mycelial biomass and the presence of channels within the organized biofilm. A similar biofilm structure was observed following the co-culture of each fungus with A549 cells, revealing a mycelial trap covering all of the lung epithelial monolayer. Collectively, these results highlight the potential for biofilm formation by these clinically relevant fungal pathogens.

Effect of 3-bromopyruvate and atovaquone on infection during in vitro interaction of Toxoplasma gondii and LLC-MK2 cells.

Toxoplasma gondii infection can be severe during pregnancy and in immunocompromised patients. Current therapies for toxoplasmosis are restricted to tachyzoites and have little or no effect on bradyzoites, which are maintained in tissue cysts. Consequently, new therapeutic alternatives have been proposed as the use of atovaquone has demonstrated partial efficacy against tachyzoites and bradyzoites. This work studies the effect of 3-bromopyruvate (3-BrPA), a compound that is being tested against cancer cells, on the infection of LLC-MK2 cells with T. gondii tachyzoites, RH strain. No effect of 3-BrPA on host cell proliferation or viability was observed, but it inhibited the proliferation of T. gondii. The incubation of cultures with lectin Dolichos biflorus agglutinin (DBA) showed the development of cystogenesis, and an ultrastructural analysis of parasite intracellular development confirmed morphological characteristics commonly found in tissue cysts. Moreover, the presence of degraded parasites and the influence of 3-BrPA on endodyogeny were observed. Infected cultures were alternatively treated with a combination of this compound plus atovaquone. This resulted in a 73% reduction in intracellular parasites after 24 h of treatment and a 71% reduction after 48 h; cyst wall formation did not occur in these cultures. Therefore, we conclude that the use of 3-BrPA may serve as an important tool for the study of (i) in vitro cystogenesis; (ii) parasite metabolism, requiring a deeper understanding of the target of action of this compound on T. gondii; (iii) the alternative parasite metabolic pathways; and (iv) the molecular/cellular mechanisms that trigger parasite death.

Spontaneous cystogenesis of Toxoplasma gondii in feline epithelial cells in vitro.

Toxoplasma gondii Nicolle et Manceaux, 1908 is an obligate intracellular parasite with the ability to infect mammals and birds. The only definitive hosts for T. gondii are felids, as the parasites form immature oocysts that are shed in the faeces. Here we introduce cat cells as a model for the study of experimental toxoplasmosis. We selected epithelial cells derived from cat kidneys (CRFK) as a target to determine the intracellular fate ofbradyzoites of the T. gondii ME49 strain. In parallel, we compared this infection using epithelial cells from the rat intestine (IEC-6), considering the enteroepithelial development that occurs in the cat. Different ratios of parasites to host cells were assayed over the course of a 14-day-infection. The intracellular development of T. gondii was dependent on the source of the epithelial cells and also on the parasite/host cell ratio. Cystogenesis was well established in the CRFK cell line at a ratio of 1:10 after 10-14 days of infection. This cellular model system opens a new field of investigation into the molecular aspects of the interactions between T. gondii and feline epithelial cells. The CRFK cell line appears to be a potential cellular model for large scale cyst production in vitro, which would allow a reduction in the number of animals used and/or replacement of animals by in vitro cultures.

The calpain inhibitor MDL28170 induces the expression of apoptotic markers in Leishmania amazonensis promastigotes.

BACKGROUND: Human cutaneous leishmaniasis is caused by distinct species, including Leishmania amazonensis. Treatment of cutaneous leishmaniasis is far from satisfactory due to increases in drug resistance and relapses, and toxicity of compounds to the host. As a consequence for this situation, the development of new leishmanicidal drugs and the search of new targets in the parasite biology are important goals. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we investigated the mechanism of death pathway induced by the calpain inhibitor MDL28170 on Leishmania amazonensis promastigote forms. The combined use of different techniques was applied to contemplate this goal. MDL28170 treatment with IC50 (15 µM) and two times the IC50 doses induced loss of parasite viability, as verified by resazurin assay, as well as depolarization of the mitochondrial membrane, which was quantified by JC-1 staining. Scanning and transmission electron microscopic images revealed drastic alterations on the parasite morphology, some of them resembling apoptotic-like death, including cell shrinking, surface membrane blebs and altered chromatin condensation pattern. The lipid rearrangement of the plasma membrane was detected by Annexin-V labeling. The inhibitor also induced a significant increase in the proportion of cells in the sub-G0/G1 phase, as quantified by propidium iodide staining, as well as genomic DNA fragmentation, detected by TUNEL assay. In cells treated with MDL28170 at two times the IC50 dose, it was also possible to observe an oligonucleossomal DNA fragmentation by agarose gel electrophoresis. CONCLUSIONS/SIGNIFICANCE: The data presented in the current study suggest that MDL28170 induces apoptotic marker expression in promastigotes of L. amazonensis. Altogether, the results described in the present work not only provide a rationale for further exploration of the mechanism of action of calpain inhibitors against trypanosomatids, but may also widen the investigation of the potential clinical utility of calpain inhibitors in the chemotherapy of leishmaniases.

Phosphatidylserine exposure and surface sugars in two Leishmania (Viannia) braziliensis strains involved in cutaneous and mucocutaneous leishmaniasis.

BACKGROUND: Phosphatidylserine (PS) and surface carbohydrates (SC) are known as virulence factors that may contribute to the different clinical symptoms ranging from self-healing cutaneous leishmaniasis lesions to fatal visceral disease. Leishmania (Viannia) braziliensis causes localized cutaneous leishmaniasis (LCL) and mucocutaneous leishmaniasis (MCL). METHODS: We analyzed PS exposure and SC expression associated with 2 primary L. braziliensis isolates from patients with LCL or MCL. The role of PS exposure was also addressed during promastigotes phagocytosis by macrophages. RESULTS: We observed higher PS exposure on the surface of late stationary growth phase promastigotes from patients with LCL, compared with those from patients with MCL, and both strains were alive during PS display. Reduction in the infectivity index was observed during macrophage interaction with late stationary growth phase promastigotes in which PS was blocked by annexin V. The major surface carbohydrates detected on LCL and MCL promastigotes were α-Man, α-Glc, and α-Gal. However, α-ß-GalNAc, although observed on the surface of the LCL strain during the late stationary growth phase was highly expressed on the surface of early stationary growth phase promastigotes. CONCLUSIONS: Our results suggest that PS and SC can modulate interactions between Leishmania organisms and host cells and may be important for the outcome of the clinical course of diseases caused by L. braziliensis.

Melanin in Fonsecaea pedrosoi: a trap for oxidative radicals.

BACKGROUND: The pathogenic fungus Fonsecaea pedrosoi constitutively produces the pigment melanin, an important virulence factor in fungi. Melanin is incorporated in the cell wall structure and provides chemical and physical protection for the fungus.We evaluated the production of nitric oxide (NO) in macrophages, the oxidative burst and the inducible nitric oxide synthase (i-NOS) activity in interactions between activated murine macrophages and F. pedrosoi. Experiments were carried out with or without tricyclazole (TC) treatment, a selective inhibitor of the dihydroxynaphthalene (DHN)-melanin biosynthesis pathway in F. pedrosoi. The paramagnetisms of melanin and the TC-melanin were analysed by electron spin resonance. The fungal growth responses to H2O2 and to S-nitroso-N-acetylpenicillamine (SNAP), a nitric oxide donor, were also evaluated. RESULTS: Melanised F. pedrosoi cells were more resistant to both H2O2 and NO. Nitrite was not detected in the supernatant of macrophages incubated with melanised fungal cells. However, i-NOS expression was unaffected by the presence of either untreated control F. pedrosoi or TC-treated F. pedrosoi. In addition, the inhibition of the DHN-melanin pathway by TC improved the oxidative burst capability of the macrophages. CONCLUSION: The NO-trapping ability of F. pedrosoi melanin is an important mechanism to escape the oxidative burst of macrophages.

Trypanosoma cruzi exposes phosphatidylserine as an evasion mechanism.

Chagas disease is caused by Trypanosoma cruzi and affects 18 million people in Central and South America. Here we analyzed the exposure of phosphatidylserine by the different forms of the parasite life cycle. Only the infective trypomastigotes, but not the epimastigotes or intracellular amastigotes, expose this phospholipid. This triggers a transforming growth factor beta signaling pathway, based on phosphorylated Smad 2 nuclear translocation, leading to iNOS disappearance in infected macrophages. This macrophage deactivation favors the survival of this intracellular parasite. Thus, phosphatidylserine exposure may be used by T. cruzi to evade innate immunity and be a common feature of obligate intracellular parasites that have to deal with activated macrophages.

Toxoplasma gondii exposes phosphatidylserine inducing a TGF-beta1 autocrine effect orchestrating macrophage evasion.

Toxoplasmosis is a worldwide disease caused by Toxoplasma gondii. Activated macrophages control T. gondii growth by nitric oxide (NO) production. However, T. gondii active invasion inhibits NO production, allowing parasite persistence. Here we show that the mechanism used by T. gondii to inhibit NO production persisting in activated macrophages depends on phosphatidylserine (PS) exposure. Masking PS with annexin-V on parasites or activated macrophages abolished NO production inhibition and parasite persistence. NO production inhibition depended on a transforming growth factor-beta1 (TGF-beta1) autocrine effect confirmed by the expression of Smad 2 and 3 in infected macrophages. TGF-beta1 led to inducible nitric oxide synthase (iNOS) degradation, actin filament (F-actin) depolymerization, and lack of nuclear factor-kappaB (NF-kappaB) in the nucleus. All these features were reverted by TGF-beta1 neutralizing antibody treatment. Thus, T. gondii mimics the evasion mechanism used by Leishmania amazonensis and also the anti-inflammatory response evoked by apoptotic cells.

Toxoplasma gondii partially inhibits nitric oxide production of activated murine macrophages.

Activated macrophages produce nitric oxide (NO) and as such are able to control the multiplication of Toxoplasma gondii. Until now, no reports have described a possible modulation of NO production of macrophages after T. gondii infection. To investigate this possibility, murine blood monocyte-derived and peritoneal macrophages were activated in vitro with interferon-gamma and lipopolysaccharide and infected with T. gondii and Trypanosoma cruzi, and NO production was evaluated. NO was produced by monocyte-derived macrophages only if cultured in the presence of macrophage-colony-stimulating factor. Monocyte-derived or peritoneal macrophages infected with T. gondii presented a significant reduction in NO production. NO production inhibition was not detected after T. cruzi infection. Macrophages infected with higher T. gondii/macrophage ratios presented lower NO production. Furthermore, only viable T. gondii could cause partial inhibition of NO production. In macrophages activated 24 h before the interaction, partial inhibition was detected after 3 h of infection and continued for 48 h. In macrophages activated immediately after the interaction, partial inhibition was not detected at 12 h, but was observed at 24 h. T. gondii-infected macrophages present lower inducible nitric oxide synthase expression as assayed by immunofluorescence. T. gondii did not develop in monocyte-derived macrophages producing NO, but were not totally eliminated. These results demonstrate that T. gondii infection partially inhibits NO production by murine macrophages, suggesting that a deactivating macrophage mechanism may be used for better survival into phagocytic cells.