Luciano Aparecido Panagio

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

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.

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.

Accurate and sensitive real-time PCR assays using intergenic spacer 1 region to differentiate Cryptococcus gattii sensu lato and Cryptococcus neoformans sensu lato.

In this work, two accurate and sensitive real-time polymerase chain reaction (PCR) assays to differentiate pathogenic Cryptococcus gattii sensu lato (s.l.) and C. neoformans sensu lato (s.l.) targeting the intergenic spacer 1 (IGS1) region from rDNA locus were developed. Specific primers were designed based on their IGS1 sequence analyses and the optimal real-time PCR assays showed that the dissociation curves generated two different melting peaks, at 82.8 and 84.2ºC for C. gattii s.l. and C. neoformans s.l., respectively. No amplifications were observed in the negative template control. The minimum limit of detection of both primers was 100 plasmid copies per reaction, and they were highly specific when tested with a range of fungal DNAs. Overall, the results showed that the designed primers completely differentiated C. gattii s.l. and C. neoformans s.l. from clinical and environmental sources with great accuracy when compared to phenotypic identification, with no cross-reactivity to other fungal DNA.