Ricardo Sergio Almeida

Antifungal Activity of Condensed Tannins from Stryphnodendron adstringens: Effect on Candida tropicalis Growth and Adhesion Properties

Candida species are some of the most common causes of fungal infection worldwide. The limited efficacy of clinically available antifungals warrants the search for new compounds for treating candidiasis. This study evaluated the effect of condensed tannin-rich fraction (F2 fraction) of Stryphnodendron adstringens on in vitro and in vivo growth of Candida tropicalis, and on yeast adhesion properties. F2 exhibited a fungistatic effect with the minimum inhibitory concentration ranging from 0.5 to 8.0 μg/mL. A significant reduction in biofilm mass was observed after either pretreatment of planktonic cells for 2 h (mean reduction of 46.31±8.17%) or incubation during biofilm formation (mean reduction of 28.44±13.38%) with 4x MIC of F2. Prior exposure of planktonic cells to this F2 concentration also significantly decreased yeast adherence on HEp-2 cells (mean reduction of 43.13±14.29%), cell surface hydrophobicity (mean reduction of 25.89±10.49%) and mRNA levels of the genes ALST1-3 (2.9-, 1.8- and 1.8-fold decrease, respectively). Tenebrio molitor larvae, which are susceptible to C. tropicalis infection, were used for in vivo testing. Treatment with 128 and 256 μg/mL F2 significantly increased the survival of infected larvae. These results indicate a combined effect of F2 on inhibition of yeast growth and interference in yeast adhesion, which may contribute to the suppression of infection caused by C. tropicalis, thus reinforcing the potential of the condensed tannins from S. adstringens for the development of novel antifungal agents.

Analysis of Sporothrix schenckii sensu stricto and Sporothrix brasiliensis virulence in Galleria mellonella

The study of the host-pathogen interaction is essential to understand the mechanisms underlying adhesion, colonization and tissue damage by pathogens. This is usually achieved by performing in vivo studies using small mammals, such as rats, mice and guinea pigs. Nowadays, the mouse models of systemic or subcutaneous infection are the gold standard assays to analyze the virulence of members of the Sporothrix schenckii complex. There are, however, invertebrates that have been recently used as alternative hosts to assess the virulence of both bacteria and fungi, and among them, larvae of Galleria mellonella are popular because they are easy to breed, and require non-specialized facilities to maintain the colony. Here, we assessed the use of G. mellonella larvae to test the virulence of S. schenckii sensu stricto and Sporothrix brasiliensis strains, and found that infection with yeast-like cells, but not with conidia or germlings, reproduces the virulence data generated in the mouse model of infection. Furthermore, with this insect model we could classify the virulence of some strains as low, intermediate or high, in line with the observations in the mammalian model. Therefore, G. mellonella is suitable, and a new alternative, to test virulence of both S. schenckii sensu stricto and S. brasiliensis.

Phenotypic switching of Candida tropicalis is associated with cell damage in epithelial cells and virulence in Galleria mellonella model

Candida species are the most common causes of human fungal infection worldwide. The pathogenic status of Candida spp. is associated with modulation of virulence determinants in response to environmental changes, and impairment of host defenses. Among Candida putative virulence factors, phenotypic switching is associated with fungal adaptability to environmental changes during invasion of host organism. Switching is a biological event associated with generation of phenotypic heterogeneity that occurs in a small fraction of the population, is random, reversible and represents an epigenetic state. In yeast, phenotypic switching drives variable changes, leading to the emergence of colonies with altered morphologies. Our group has previously described a high-frequency switching of colony morphology in several clinical isolates of Candida tropicalis. The isolates could switch spontaneously, heritably and reversibly between at least six different phenotypes, not including the white-opaque transition. One of the identified switching system that include three colony phenotypes: smooth (parental phenotype) and two related switch variants (crepe and rough) was associated with in vitro changes in virulence traits, including biofilm formation, morphogenesis and hemolysis, as well as switching from itraconazole susceptibility to resistance. Soll et al. were the first to assess switch phenotypes in C. tropicalis during the course of a prolonged Candida infection in a compromised host. Further, a white-opaque phenotypic switch was described for C. tropicalis that shows some similarities to that in Candida albicans. Although these studies have begun to explore the mechanism of phenotypic switching in C. tropicalis, the role of switching in this species remains an open question. Porman et al. demonstrated that a white-opaque phenotypic switching in C. tropicalis regulates a cryptic program of sexual mating, potentially giving rise to strains with increased virulence. The white-opaque transition also is associated with sexual biofilm formation in C. tropicalis, where biofilms are formed exclusively by opaque cells. C. tropicalis together with the majority of the medically relevant Candida species belongs to the CTG clade and is genetically close to C. albicans. The frequency of infections caused by C. tropicalis has increased over the last years, particularly in tropical regions; in some settings, bloodstream infections due to C. tropicalis have been associated with higher mortality than other Candida species. In the present study, we address for the first time in the literature, if phenotypic switching affects virulence in C. tropicalis. To this end, we employed all three colony phenotypes (smooth, crepe and rough) described previously and revertants (strains that switched back from variant to parental phenotype) for the evaluation of the capacity of damaging epithelial cells and virulence in Galleria mellonella. The switched strains (crepe and rough variants) were obtained as individual subclones from a clinical isolate exhibiting a smooth colony dome. The crepe and rough variants exhibited an irregular and structured dome surface and were obtained at frequencies of 5£10¡3 and 3£10¡2, respectively. In the present study, we initially determined the reversibility of crepe and rough variants phenotypes to the smooth phenotype (parental phenotype) by visually scoring of four days old growth colonies on YPD agar medium (Gibco) at 28 C. To calculate the reversibility frequencies, a total of 6,000 colonies from each variant

Disruption of Protein Mannosylation Affects Candida guilliermondii Cell Wall, Immune Sensing, and Virulence

The fungal cell wall contains glycoproteins that interact with the host immune system. In the prominent pathogenic yeast Candida albicans, Pmr1 acts as a Golgi-resident ion pump that provides cofactors to mannosyltransferases, regulating the synthesis of mannans attached to glycoproteins. To gain insight into a putative conservation of such a crucial process within opportunistic yeasts, we were particularly interested in studying the role of the PMR1 homolog in a low-virulent species that rarely causes candidiasis, Candida guilliermondii. We disrupted C. guilliermondii PMR1 and found that loss of Pmr1 affected cell growth and morphology, biofilm formation, susceptibility to cell wall perturbing agents, mannan levels, and the wall composition and organization. Despite the significant increment in the amount of β1,3-glucan exposed at the wall surface, this positively influenced only the ability of the mutant to stimulate IL-10 production by human monocytes, suggesting that recognition of both mannan and β1,3-glucan, is required to stimulate strong levels of pro-inflammatory cytokines. Accordingly, our results indicate C. guilliermondii sensing by monocytes was critically dependent on the recognition of N-linked mannans and β1,3-glucan, as reported in other Candida species. In addition, chemical remotion of cell wall O-linked mannans was found to positively influence the recognition of C. guilliermondii by human monocytes, suggesting that O-linked mannans mask other cell wall components from immune cells. This observation contrasts with that reported in C. albicans. Finally, mice infected with C. guilliermondii pmr1Δ null mutant cells had significantly lower fungal burdens compared to animals challenged with the parental strain. Accordingly, the null mutant showed inability to kill larvae in the Galleria mellonella infection model. This study thus demonstrates that mannans are relevant for the C. guilliermondii-host interaction, with an atypical role for O-linked mannans.

Tenebrio molitor (Coleoptera: Tenebrionidae) as an alternative host to study fungal infections.

Models of host–pathogen interactions are crucial for the analysis of microbial pathogenesis. In this context, invertebrate hosts, including Drosophila melanogaster (fruit fly), Caenorhabditis elegans (nematode) and Galleria mellonella (moth), have been used to study the pathogenesis of fungi and bacteria. Each of these organisms offers distinct benefits in elucidating host–pathogen interactions. In this study,we present a newinvertebrate infection model to study fungal infections: the Tenebrio molitor (beetle) larvae. Here we performed T. molitor larvae infection with one of two important fungal human pathogens, Candida albicans or Cryptococcus neoformans, and analyzed survival curves and larva infected tissues.We showed that increasing concentrations of inoculum of both fungi resulted in increased mortality rates, demonstrating the efficiency of the method to evaluate the virulence of pathogenic yeasts. Additionally, following 12 h post-infection, C. albicans formsmycelia, spreading its hyphae through the larva tissue,whilst GMS stain enabled the visualization of C. neoformans yeast and theirmelanin capsule. These larvae are easier to cultivate in the laboratory than G. mellonella larvae, and offer the same benefits. Therefore, this insect model could be a useful alternative tool to screen clinical pathogenic yeast strainswith distinct virulence traits or different mutant strains.

Pravastatin and simvastatin inhibit the adhesion, replication and proliferation of Toxoplasma gondii (RH strain) in HeLa cells.

The conventional treatment for toxoplasmosis with pyrimethamine and sulfadiazine shows toxic effects to the host, and it is therefore necessary to search for new drugs. Some studies suggest the use of statins, which inhibit cholesterol synthesis in humans and also the initial processes of isoprenoid biosynthesis in the parasite. Thus, the objective of this study was to evaluate the activity of the statins pravastatin and simvastatin in HeLa cells infected in vitro with the RH strain of T. gondii. HeLa cells (1×105) were infected with T. gondii tachyzoites (5×105) following two different treatment protocols. In the first protocol, T. gondii tachyzoites were pretreated with pravastatin (50 and 100μg/mL) and simvastatin (1.56 and 3.125μg/mL) for 30min prior to infection. In the second, HeLa cells were first infected (5×105) with tachyzoites and subsequently treated with pravastatin and simvastatin for 24h at the concentrations noted above. Initially, we evaluated the cytotoxicity of drugs by the MTT assay, number of tachyzoites adhered to cells, number of infected cells, and viability of tachyzoites by trypan blue exclusion. The supernatant of the cell cultures was collected post-treatment for determination of the pattern of Th1/Th2/Th17 cytokines by cytometric bead array. There was no cytotoxicity to HeLa cells with 50 and 100μg/mL pravastatin and 1.56 and 3.125μg/mL simvastatin. There was no change in the viability of tachyzoites that received pretreatment. Regarding the pre- and post-treatment of the cells with pravastatin and simvastatin alone, there was a reduction in adhesion, invasion and proliferation of cells to T. gondii. As for the production of cytokines, we found that IL-6 and IL-17 were significantly reduced in cells infected with T. gondii and treated with pravastatin and simvastatin, when compared to control. Based on these results, we can infer that pravastatin and simvastatin alone possess antiproliferative effects on tachyzoites forms of T. gondii, giving these drugs new therapeutic uses.

Immunological and histopathological characterization of cutaneous candidiasis

Chronic mucocutaneous candidiasis constitutes a heterogeneous group of syndromes, characterized by non-invasive infection of the skin, nails and mucosal membranes by the fungus Candida spp. Although symptoms are heterogeneous, in all cases there is a reduction in protective cytokines, favouring the development of disease. The normal role of cytokines in skin lesions is not well understood. The present study aimed to investigate the progression of disease, understand specific cellular and molecular components involved in immunity to Candida albicans and determine the balance between pro- and anti-inflammatory cytokines over the course of cutaneous infection in immunocompetent mice. BALB/c mice (five per group) were inoculated with 5 × 10(6)C. albicans pseudohyphae in the deep dermis of the paw and analysed over 1-14  days post-infection. The contralateral paws were used for negative controls. Haematoxylin and eosin staining of skin sections during C. albicans infection was performed to analyse structural modifications to the epidermis such as hyperplasia, and infiltration of neutrophils and fibroblasts in the dermis. The cytokine populations were determined by capture ELISA using popliteal lymph node tissue. Pro-inflammatory cytokines (IL-6, TNF-α, IL-12, IFN-γ and IL-17) were detected at significant levels during the initial phase of cutaneous infection and correlated with the rapid elimination of C. albicans. Anti-inflammatory cytokines (IL-13, IL-4, IL-10 and transforming growth factor-β) were detected on day 4 post-infection, and prevented exacerbation of inflammation and participated in healing of lesions. Thus, a balance between pro- and anti-inflammatory cytokines was fundamental for the resolution of infection. Importantly, these findings broaden our understanding of the immune mechanisms involved in chronic cutaneous candidiasis.

The Effect of Phenazine-1-Carboxylic Acid on Mycelial Growth of Botrytis cinerea Produced by Pseudomonas aeruginosa LV Strain

One of the most important postharvest plant pathogens that affect strawberries, grapes and tomatoes is Botrytis cinerea, known as gray mold. The fungus remains in latent form until spore germination conditions are good, making infection control difficult, causing great losses in the whole production chain. This study aimed to purify and identify phenazine-1-carboxylic acid (PCA) produced by the Pseudomonas aeruginosa LV strain and to determine its antifungal activity against B. cinerea. The compounds produced were extracted with dichloromethane and passed through a chromatographic process. The purity level of PCA was determined by reversed-phase high-performance liquid chromatography semi-preparative. The structure of PCA was confirmed by nuclear magnetic resonance and electrospray ionization mass spectrometry. Antifungal activity was determined by the dry paper disk and minimum inhibitory concentration (MIC) methods and identified by scanning electron microscopy and confocal microscopy. The results showed that PCA inhibited mycelial growth, where MIC was 25 μg mL-1. Microscopic analysis revealed a reduction in exopolysaccharide (EPS) formation, showing distorted and damaged hyphae of B. cinerea. The results suggested that PCA has a high potential in the control of B. cinerea and inhibition of EPS (important virulence factor). This natural compound is a potential alternative to postharvest control of gray mold disease.

Galleria mellonella hemocytes: A novel phagocytic assay for Leishmania (Viannia) braziliensis.

Galleria mellonella is an excellent invertebrate model for the study of diseases that involve interactions with cells from the innate immune system, since they have an innate immune system capable of recognizing the pathogens. Here we present for the first time, an alternative model for an in vitro phagocytic assay using hemocytes of G. mellonella larvae to study infection by Leishmania (Viannia) braziliensis. We showed that the insect phagocytic cells were able to engulf promastigotes. Furthermore, this infective form differentiated into the amastigote form inside those cells. However, the cells in this model seem resistant to the parasite, since amastigotes were depleted after 24h and NO levels were maintained after infection. Our model opens an avenue of possibilities for new investigations regarding other Leishmania species, mechanisms of invasion and evasion, receptors involved, release of signaling molecules and, above all, it is a novel infection model using invertebrate animals.

Concanavalin‐A induces IL‐17 production during the course of Candida albicans infection

In a previous study, our group verified that 100% of mice survived to a lethal dose of Candida albicans following pretreatment with concanavalin-A (Con-A) for 3 days. This work proposed to investigate whether treatment could mediate an adaptative immune response involving T(H) 17 cells. A significant increase in IL-17 levels at 6 h postinfection was observed and was maintained up to 18 h in the Con-A group, whereas in control mice, a reduction in this cytokine was verified. In addition, T(H) 17 cells develop in the presence of TGF-β, IL-1 β, and IL-6 that were increased significantly 2 h postinfection in Con-A-treated mice. Macrophages were involved in the process, engulfing greater numbers of yeast cells, and were activated through TNF-α and interferon-γ produced at significant levels at 2 h postinfection. A significant increase in IL-12 levels was also observed at 2 h postinfection. Thus, activated macrophages were probably more capable of killing and processing Candida antigens, signalizing an adaptative immune response. Macrophages from controls did not prevent yeast-to-hyphae transition and were partially destroyed, as shown in scanning microscopy. These results suggest that treatment with Con-A facilitated the triggering of T(H) 17 and T(H) 1 responses via IL-17 and IFN-γ production, leading to the resolution of C. albicans infection.