Antimicrobial effect of farnesol, a Candida albicans quorum sensing molecule, on Paracoccidioides brasiliensis growth and morphogenesis

<p>Abstract</p> <p>Background</p> <p>Farnesol is a sesquiterpene alcohol produced by many organisms, and also found in several essential oils. Its role as a quorum sensing molecule and as a virulence factor of <it>Candida albicans </it>has been well described. Studies revealed that farnesol affect the growth of a number of bacteria and fungi, pointing to a potential role as an antimicrobial agent.</p> <p>Methods</p> <p>Growth assays of <it>Paracoccidioides brasiliensis </it>cells incubated in the presence of different concentrations of farnesol were performed by measuring the optical density of the cultures. The viability of fungal cells was determined by MTT assay and by counting the colony forming units, after each farnesol treatment. The effects of farnesol on <it>P. brasiliensis </it>dimorphism were also evaluated by optical microscopy. The ultrastructural morphology of farnesol-treated <it>P. brasiliensis </it>yeast cells was evaluated by transmission and scanning electron microscopy.</p> <p>Results</p> <p>In this study, the effects of farnesol on <it>Paracoccidioides brasiliensis </it>growth and dimorphism were described. Concentrations of this isoprenoid ranging from 25 to 300 μM strongly inhibited <it>P. brasiliensis </it>growth. We have estimated that the MIC of farnesol for <it>P. brasiliensis </it>is 25 μM, while the MLC is around 30 μM. When employing levels which don't compromise cell viability (5 to 15 μM), it was shown that farnesol also affected the morphogenesis of this fungus. We observed about 60% of inhibition in hyphal development following <it>P. brasiliensis </it>yeast cells treatment with 15 μM of farnesol for 48 h. At these farnesol concentrations we also observed a significant hyphal shortening. Electron microscopy experiments showed that, despite of a remaining intact cell wall, <it>P. brasiliensis </it>cells treated with farnesol concentrations above 25 μM exhibited a fully cytoplasmic degeneration.</p> <p>Conclusion</p> <p>Our data indicate that farnesol acts as a potent antimicrobial agent against <it>P. brasiliensis</it>. The fungicide activity of farnesol against this pathogen is probably associated to cytoplasmic degeneration. In concentrations that do not affect fungal viability, farnesol retards the germ-tube formation of <it>P. brasiliensis</it>, suggesting that the morphogenesis of this fungal is controlled by environmental conditions.</p

Activity of scorpion venom-derived antifungal peptides against planktonic cells of Candida spp. and Cryptococcus neoformans and Candida albicans Biofilms

The incidence of fungal infections has been increasing in the last decades, while the number of available antifungal classes remains the same. The natural and acquired resistance of some fungal species to available therapies, associated with the high toxicity of these drugs on the present scenario and makes an imperative of the search for new, more efficient and less toxic therapeutic choices. Antimicrobial peptides (AMPs) are a potential class of antimicrobial drugs consisting of evolutionarily conserved multifunctional molecules with both microbicidal and immunomodulatory properties being part of the innate immune response of diverse organisms. In this study, we evaluated 11 scorpion-venom derived non-disulfide-bridged peptides against Cryptococcus neoformans and Candida spp., which are important human pathogens. Seven of them, including two novel molecules, showed activity against both genera with minimum inhibitory concentration values ranging from 3.12 to 200 μM and an analogous activity against Candida albicans biofilms. Most of the peptides presented low hemolytic and cytotoxic activity against mammalian cells. Modifications in the primary peptide sequence, as revealed by in silico and circular dichroism analyses of the most promising peptides, underscored the importance of cationicity for their antimicrobial activity as well as the amphipathicity of these molecules and their tendency to form alpha helices. This is the first report of scorpion-derived AMPs against C. neoformans and our results underline the potential of scorpion venom as a source of antimicrobials. Further characterization of their mechanism of action, followed by molecular optimization to decrease their cytotoxicity and increase antimicrobial activity, is needed to fully clarify their real potential as antifungals

Murine Dendritic Cells Transcriptional Modulation upon Paracoccidioides brasiliensis Infection

Limited information is available regarding the modulation of genes involved in the innate host response to Paracoccidioides brasiliensis, the etiologic agent of paracoccidioidomycosis. Therefore, we sought to characterize, for the first time, the transcriptional profile of murine bone marrow-derived dendritic cells (DCs) at an early stage following their initial interaction with P. brasiliensis. DCs connect innate and adaptive immunity by recognizing invading pathogens and determining the type of effector T-cell that mediates an immune response. Gene expression profiles were analyzed using microarray and validated using real-time RT-PCR and protein secretion studies. A total of 299 genes were differentially expressed, many of which are involved in immunity, signal transduction, transcription and apoptosis. Genes encoding the cytokines IL-12 and TNF-α, along with the chemokines CCL22, CCL27 and CXCL10, were up-regulated, suggesting that P. brasiliensis induces a potent proinflammatory response in DCs. In contrast, pattern recognition receptor (PRR)-encoding genes, particularly those related to Toll-like receptors, were down-regulated or unchanged. This result prompted us to evaluate the expression profiles of dectin-1 and mannose receptor, two other important fungal PRRs that were not included in the microarray target cDNA sequences. Unlike the mannose receptor, the dectin-1 receptor gene was significantly induced, suggesting that this β-glucan receptor participates in the recognition of P. brasiliensis. We also used a receptor inhibition assay to evaluate the roles of these receptors in coordinating the expression of several immune-related genes in DCs upon fungal exposure. Altogether, our results provide an initial characterization of early host responses to P. brasiliensis and a basis for better understanding the infectious process of this important neglected pathogen

Antibiotic development challenges: the various mechanisms of action of antimicrobial peptides and of bacterial resistance

Antimicrobial peptides (AMPs) are natural antibiotics produced by various organisms such as mammals, arthropods, plants, and bacteria. In addition to antimicrobial activity, AMPs can induce chemokine production, accelerate angiogenesis, and wound healing and modulate apoptosis in multicellular organisms. Originally, their antimicrobial mechanism of action was thought to consist solely of an increase in pathogen cell membrane permeability, but it has already been shown that several AMPs do not modulate membrane permeability in the minimal lethal concentration. Instead, they exert their effects by inhibiting processes such as protein and cell wall synthesis, as well as enzyme activity, among others. Although resistance to these molecules is uncommon several pathogens developed different strategies to overcome AMPs killing such as surface modification, expression of efflux pumps, and secretion of proteases among others. This review describes the various mechanisms of action of AMPs and how pathogens evolve resistance to them

Effect of mannan and laminarin on cytokine secretion by murine dendritic cells infected with <i>P. brasiliensis</i>.

<p>Murine bone marrow-derived DCs were incubated with mannan (Man) or laminarin (Lam) at 100 and 200 µg/ml, respectively, for 30 min. Subsequently, <i>P. brasiliensis</i> yeast cells (Pb) were added to DCs at a ratio of 1∶1, and the co-culture was incubated for 6 h. Culture supernatants were harvested and secreted protein levels were measured using ELISA. Data are reported as the mean ± standard deviation. * P<0.05 compared to the DCs+Pb group.</p

Effect of mannan and laminarin on the accumulation of selected immune-related transcripts in dendritic cells infected with <i>P. brasiliensis</i>.

<p>Murine bone marrow-derived DCs were incubated with mannan (Man) or laminarin (Lam) at 100 and 200 µg/ml, respectively, for 30 min. Subsequently, <i>P. brasiliensis</i> yeast cells (Pb) were added to DCs at a ratio of 1∶1, and the co-culture was incubated for 6 h. Total RNA was extracted from DCs and used in qRT-PCR assays. Fold-change values were determined after each gene was normalized to <i>RPS9</i> using the comparative threshold method. Data are reported as the mean ± standard deviation. * P<0.05 compared to the DCs+Pb/DCs group.</p

Real-time PCR validation of microarray data.

<p>*Fold-change values were determined after normalization to <i>Rps9</i> using the comparative threshold method. Values indicate mean fold change ± SD of two independent experiments.</p><p>NM: not significantly modulated, as shown by microarray.</p

Selected genes up-regulated in murine dendritic cells after 6 h of infection with <i>P. brasiliensis</i>.

<p>*cDNA clones were obtained from the Soares mouse thymus 2NbMT normalized library, available from the IMAGE Consortium (<a href="http://image.hudsonalpha.org/" target="_blank">http://image.hudsonalpha.org/</a>).</p

Relative quantification of dectin-1 and mannose receptor transcripts in dendritic cells infected with <i>P. brasiliensis</i>.

<p>BALB/c bone marrow-derived DCs were cultured for 6 h with or without <i>P. brasiliensis</i> yeast cells (Pb). Then total RNA was extracted from the DCs and used in qRT-PCR assays. Fold change values were determined after each gene was normalized to the constitutively expressed <i>RPS9</i> gene using the comparative threshold method. Data are reported as the mean ± standard deviation. * P<0.05.</p

Selected genes down-regulated in murine dendritic cells after 6 h of infection with <i>P. brasiliensis</i>.

<p>*cDNA clones were obtained from the Soares mouse thymus 2NbMT normalized library, available at the IMAGE Consortium (<a href="http://image.hudsonalpha.org/" target="_blank">http://image.hudsonalpha.org/</a>).</p