In silico approaches in the identification of Cryptococcus neoformans chemoreceptors

Cryptococcus neoformans, a pathogenic, yeast-like fungus, typically forms biofilms on medical devices that prevents the efficient penetration of antifungal drugs. As quorum sensing (QS)-related chemoreceptors play a crucial role in the pathogenesis of Cryptococcus neoformans particularly in biofilm formation, studying them would enable a clearer understanding on the host-pathogen interaction. However, information regarding C. neoformans QS-related chemoreceptors such as the encoding DNA sequences, protein structures and the binding ligands remain unknown till today. Here, we provide information to facilitate researchers to comprehend the basic views on gene homology and its various applications in the identification of novel chemoreceptors in microorganism especially the novel QS-related chemoreceptors for future use.Keywords: Cryptococcus neoformans, homology-based transfer, quorum sensing, chemoreceptor

Cytotoxicity Mechanism of a Fungal Inhibitor from a Soil-Derived Streptomyces SP

Effective fungal growth inhibitors are important to drive the development of antifungal compound. In the search for fungal inhibitors, actinomycete H7372 was isolated from a mangrove soil sample from Sabah. Subsequently, in a yeast cell-based screening system, the crude acetone extract prepared from the fermentative culture of H7372 was found to inhibit the growth of the yeasts. The purposes of this study were to establish the phylogenetic position of H7372 and to isolate, characterize and examine the toxicity mechanism of the active fungal growth inhibitor produced by H7372. The partial sequence of 16S rRNA gene was amplified from H7372 for phylogenetic analysis. An active compound was isolated from crude acetone extract of mannitol-soybean fermentation culture and its structure was elucidated. Antifungal properties of the isolated active compound against Candida spp and Aspergillus spp were characterised by minimum inhibitory concentration and time-kill kinetic studies. The consequences of C. glabrata treatment with the active compound were examined by electron microscopy and cDNA microarray. Phylogenetic analysis placed H7372 to its closest relative, S. kasugaensis M338-M1.The isolated active compound, designated as J5, was determined to be the natural (11S, 13S, 9S, 8R)-cycloheximide. All Candida species tested (except C. albicans) and only A. niger were sensitive to J5, with MIC at 24H ranging from 0.313 to 40 μg/ml. The degree of susceptibility shown by some species of Candida, from the most to the least susceptible, were C. krusei, C. glabrata, C. rugosa and C. parapsilosis. J5 is a fungistatic compound which showed total fungicidal effect at 12 times of its MIC when applied to C. glabrata. Treatment with J5 demonstrated profound intracellular and cell surface modifications, such as by marked cell wall thickening, confused cytoplasm, mitochondria loss, and irregular plasma membrane invaginations with detachment of the protoplast from the cell wall. cDNA microarray revealed a total of 60 genes affected by J5 treatment, corresponding to genes involved in protein synthesis, plasma membrane and H+ pumps, mitochondria maintenance and nutrient metabolism. In conclusion, H7372 is a Streptomyces sp. which is closely related to S. kasugaensis M338-M1. The active compound, J5, is a cis-cycloheximide. Comprehensive susceptibility profiles of Candida and Aspergillus species toward J5 were established for the first time and generated new MIC readings for C. krusei (0.313 μg/ml), C. rugosa (0.625 μg/ml), C. glabrata (2.5 μg/ml), C. parapsilosis (2.5 μg/ml), C. tropicalis (5 μg/ml) and A. niger (40 μg/ml). Ultrastructures of J5-treated C. glabrata revealed new evidence on the toxicity mechanisms of cycloheximide on plasma membranes and mitochondria. The gene expression profiles for J5, the cycloheximide treatment, were revealed for the first time in yeast

Antifungal compound and its production

The invention relates to a compound for the preparation of a medicament, and methods of obtaining the compound a product of the microorganism. In particularly, the present invention relates to the anti-fungal effects pharmaceutical composition, medicament or method of treatment related to said compound of the invention are for the treatment of diseases associated with fungus or yeast

In Vitro Analysis of Metabolites Secreted during Infection of Lung Epithelial Cells by Cryptococcus neoformans.

Cryptococcus neoformans is an encapsulated basidiomycetous yeast commonly associated with pigeon droppings and soil. The opportunistic pathogen infects humans through the respiratory system and the metabolic implications of C. neoformans infection have yet to be explored. Studying the metabolic profile associated with the infection could lead to the identification of important metabolites associated with pulmonary infection. Therefore, the aim of the study was to simulate cryptococcal infection at the primary site of infection, the lungs, and to identify the metabolic profile and important metabolites associated with the infection at low and high multiplicity of infections (MOI). The culture supernatant of lung epithelial cells infected with C. neoformans at MOI of 10 and 100 over a period of 18 hours were analysed using gas chromatography mass spectrometry. The metabolic profiles obtained were further analysed using multivariate analysis and the pathway analysis tool, MetaboAnalyst 2.0. Based on the results from the multivariate analyses, ten metabolites were selected as the discriminatory metabolites that were important in both the infection conditions. The pathways affected during early C. neoformans infection of lung epithelial cells were mainly the central carbon metabolism and biosynthesis of amino acids. Infection at a higher MOI led to a perturbance in the β-alanine metabolism and an increase in the secretion of pantothenic acid into the growth media. Pantothenic acid production during yeast infection has not been documented and the β-alanine metabolism as well as the pantothenate and CoA biosynthesis pathways may represent underlying metabolic pathways associated with disease progression. Our study suggested that β-alanine metabolism and the pantothenate and CoA biosynthesis pathways might be the important pathways associated with cryptococcal infection

Growth curve of <i>C</i>. <i>neoformans</i> H99 after the addition of discriminant metabolites.

<p>There was a significant increase (P ≤ 0.01) in growth after addition of pantothenic acid (16 μg/ml and 128 μg/ml). * represents P ≤ 0.05; ** represents P ≤ 0.01</p

CAP model detailing the metabolites secreted during infection of MOI10.

<p>There was a distinct spread between the metabolites of the different time points. This allowed for the grouping of specific metabolites based on the time points.</p

PCA model results for the metabolites secreted during infection of MOI100.

<p>Data show difference between 36 samples (4 biological replicates, 3 technical replicates of samples co-incubated for 6, 12 and 18 hours)</p

PLS-DA model results for the metabolites secreted during infection of MOI10.

<p>Data show difference between 36 samples, but the Q²Y values are too low for PLS-DA to be used as the sole mode of identification of discriminant metabolites.</p

Results of the pathway analysis of the MOI100 samples.

<p>Results of the pathway analysis of the MOI100 samples.</p