Cytotoxic and Antimicrobial Constituents from the Essential Oil of Lippia alba (Verbenaceae).

Backgroud:Lippia alba (Verbenaceae) is a plant widely used in folk medicine to treat various diseases. The present work deals with the chemical composition of the crude essential oil extracted from leaves of L. alba and evaluation of its antimicrobial and cytotoxic activities. Methods: Leaves of L. alba were extracted by hydrodistillation and analyzed by gas chromatography/mass spectrometry (GC/MS) as well as by nuclear magnetic resonance (NMR) spectroscopy. Cytotoxic and antimicrobial activities of crude essential oil were evaluated in vitro using MTT and broth microdilution assays, respectively. Results: Chemical analysis afforded the identification of 39 substances corresponding to 99.45% of the total oil composition. Concerning the main compounds, monoterpenes nerol/geraniol and citral correspond to approximately 50% of crude oil. The cytotoxic activity of obtained essential oil against several tumor cell lines showed IC50 values ranging from 45 to 64 µg/mL for B16F10Nex2 (murine melanoma) and A549 (human lung adenocarcinoma). In the antimicrobial assay, was observed that all tested yeast strains, except C. albicans, were sensitive to crude essential oil. MIC values were two to four-folds lower than those determined to bacterial strains. Conclusion: Analysis of chemical composition of essential oils from leaves of L. alba suggested a new chemotype nerol/geraniol and citral. Based in biological evidences, a possible application for studied oil as an antifungal in medicine, as well as in agriculture, is described

Chemical Composition and In Vitro Cytotoxic and Antimicrobial Activities of the Essential Oil from Leaves of Zanthoxylum monogynum St. Hill (Rutaceae).

Background: The Zanthoxylum monogynum species belongs to the family Rutaceae and is found in Southeast, Midwest, and Northeast Brazil. For this genus several biological activities have been described. Methods: The essential oil (EO) was obtained from the leaves of Zanthoxylum monogynum by hydro-distillation and was analyzed by gas chromatograph and gas chromatograph/mass spectrometry (GC and GC/MS). Also the EO of Z. monogynum was evaluated for in vitro cytotoxic activity against six tumor cell lines and for antimicrobial activity, performing disk diffusion and MIC assays with yeast and bacterial strains. Results: The chemical analysis afforded the identification of 18 components (99.0% of the EO). The major components were found to be citronellol (43.0%) and farnesol (32.0%). The in vitro cytotoxic activity against tumor cell lines, resulted in IC50 values ranging from 11-65 µg/mL against all tested cell lines. Antimicrobial activity of the essential oil was also tested and oil was effective, especially against Cryptococcus sp. yeast. All the tested yeast strains showed at least 90% growth inhibition. Conclusions: the essential oil from leaves of Z. monogynum has a different qualitative and quantitative composition when compared to the composition previously described. Also this EO has significant cytotoxic activity and moderate activity against Cryptococcus sp. and Saccharomyces cereviseae yeasts

The role of the de novo pyrimidine biosynthetic pathway in Cryptococcus neoformans high temperature growth and virulence

Fungal infections are often difficult to treat due to the inherent similarities between fungal and animal cells and the resulting host toxicity from many antifungal compounds. Cryptococcus neoformans is an opportunistic fungal pathogen of humans that causes life-threatening disease, primarily in immunocompromised patients. Since antifungal therapy for this microorganism is limited, many investigators have explored novel drug targets aim at virulence factors, such as the ability to grow at mammalian physiological temperature (37 degrees C). To address this issue, we used the Agrobacterium tumefaciens gene delivery system to create a random insertion mutagenesis library that was screened for altered growth at elevated temperatures. Among several mutants unable to grow at 37 degrees C, we explored one bearing an interruption in the URA4 gene. This gene encodes dihydroorotase (DHOase) that is involved in the de nova synthesis of pyrimidine ribonucleotides. Loss of the C. neoformans Ura4 protein, by targeted gene interruption, resulted in an expected uracil/uridine auxotrophy and an unexpected high temperature growth defect. in addition, the ura4 mutant displayed phenotypic defects in other prominent virulence factors (melanin, capsule and phospholipase) and reduced stress response compared to wild type and reconstituted strains. Accordingly, this mutant had a decreased survival rate in macrophages and attenuated virulence in a murine model of cryptococcal infection. Quantitative PCR analysis suggests that this biosynthetic pathway is induced during the transition from 30 degrees C to 37 degrees C, and that transcriptional regulation of de nova and salvage pyrimidine pathway are under the control of the Ura4 protein. (C) 2014 the Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-SA license (http://creativecommons.org/licenses/by-nc-sa/3.0/).Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)NIHUniversidade Federal de São Paulo, Dept Ciencias Biol, BR-09972270 Diadema, SP, BrazilUniv Brasilia, Fac Ceilandia, Brasilia, DF, BrazilDuke Univ, Sch Med, Dept Med, Durham, NC 27706 USAUniversidade Federal de São Paulo, Dept Ciencias Biol, BR-09972270 Diadema, SP, BrazilFAPESP: 2007/50536-3FAPESP: 2011/50953-9NIH: AI050128NIH: AI074677Web of Scienc

Metagenomic analysis of a tropical composting operation at the São Paulo Zoo Park reveals diversity of biomass degradation functions and organisms.

Composting operations are a rich source for prospection of biomass degradation enzymes. We have analyzed the microbiomes of two composting samples collected in a facility inside the Sao Paulo Zoo Park, in Brazil. All organic waste produced in the park is processed in this facility, at a rate of four tons/day. Total DNA was extracted and sequenced with Roche/454 technology, generating about 3 million reads per sample. To our knowledge this work is the first report of a composting whole-microbial community using high-throughput sequencing and analysis. The phylogenetic profiles of the two microbiomes analyzed are quite different, with a clear dominance of members of the Lactobacillus genus in one of them. We found a general agreement of the distribution of functional categories in the Zoo compost metagenomes compared with seven selected public metagenomes of biomass deconstruction environments, indicating the potential for different bacterial communities to provide alternative mechanisms for the same functional purposes. Our results indicate that biomass degradation in this composting process, including deconstruction of recalcitrant lignocellulose, is fully performed by bacterial enzymes, most likely by members of the Clostridiales and Actinomycetales orders.FAPESP 2009/52030-5RCNPqCAPE

A Rac Homolog Functions Downstream of Ras1 To Control Hyphal Differentiation and High-Temperature Growth in the Pathogenic Fungus Cryptococcus neoformans

The Cryptococcus neoformans Ras1 protein serves as a central regulator for several signaling pathways. Ras1 controls the induction of the mating pheromone response cascade as well as a distinct signaling pathway that allows this pathogenic fungus to grow at human physiological temperature. To characterize elements of the Ras1-dependent high-temperature growth pathway, we performed a multicopy suppressor screen, identifying genes whose overexpression allows the ras1 mutant to grow at 37°C. Using this genetic technique, we identified a C. neoformans gene encoding a Rac homolog that suppresses multiple ras1 mutant phenotypes. Deletion of the RAC1 gene does not affect high-temperature growth. However, a rac1 mutant strain demonstrates a profound defect in haploid filamentation as well as attenuated mating. In a yeast two-hybrid assay, Rac1 physically interacts with the PAK kinase Ste20, which similarly regulates hyphal formation in this fungus. Similar to Rac1, overexpression of the STE20α gene also restores high-temperature growth to the ras1 mutant. These results support a model in which the small G protein Rac1 acts downstream of Ras proteins and coordinately with Ste20 to control high-temperature growth and cellular differentiation in this human fungal pathogen

The Role of Amino Acid Permeases and Tryptophan Biosynthesis in <i>Cryptococcus neoformans</i> Survival

<div><p>Metabolic diversity is an important factor during microbial adaptation to different environments. Among metabolic processes, amino acid biosynthesis has been demonstrated to be relevant for survival for many microbial pathogens, whereas the association between pathogenesis and amino acid uptake and recycling are less well-established. <i>Cryptococcus neoformans</i> is an opportunistic fungal pathogen with many habitats. As a result, it faces frequent metabolic shifts and challenges during its life cycle. Here we studied the <i>C</i>. <i>neoformans</i> tryptophan biosynthetic pathway and found that the pathway is essential. RNAi indicated that interruptions in the biosynthetic pathway render strains inviable. However, auxotroph complementation can be partially achieved by tryptophan uptake when a non preferred nitrogen source and lower growth temperature are applied, suggesting that amino acid permeases may be the target of nitrogen catabolism repression (NCR). We used bioinformatics to search for amino acid permeases in the <i>C</i>. <i>neoformans</i> and found eight potential global permeases (<i>AAP</i>1 to <i>AAP</i>8). The transcriptional profile of them revealed that they are subjected to regulatory mechanisms which are known to respond to nutritional status in other fungi, such as (i) quality of nitrogen (<u>N</u>itrogen <u>C</u>atabolism <u>R</u>epression, NCR) and carbon sources (<u>C</u>arbon <u>C</u>atabolism <u>R</u>epression, CCR), (ii) amino acid availability in the extracellular environment (SPS-sensing) and (iii) nutritional deprivation (<u>G</u>lobal <u>A</u>mino <u>A</u>cid <u>C</u>ontrol, GAAC). This study shows that <i>C</i>. <i>neoformans</i> has fewer amino acid permeases than other model yeasts, and that these proteins may be subjected to complex regulatory mechanisms. Our data suggest that the <i>C</i>. <i>neoformans</i> tryptophan biosynthetic pathway is an excellent pharmacological target. Furthermore, inhibitors of this pathway cause <i>Cryptococcus</i> growth arrest <i>in vitro</i>.</p></div