Differential antifungal activity of human and cryptococcal melanins with structural discrepancies

Indexación: Scopus.Melanin is a pigment found in all biological kingdoms, and plays a key role in protection against ultraviolet radiation, oxidizing agents, and ionizing radiation damage. Melanin exerts an antimicrobial activity against bacteria, fungi, and parasites. We demonstrated an antifungal activity of synthetic and human melanin against Candida sp. The members of the Cryptococcus neoformans and C. gattii species complexes are capsulated yeasts, which cause cryptococcosis. For both species melanin is an important virulence factor. To evaluate if cryptococcal and human melanins have antifungal activity against Cryptococcus species they both were assayed for their antifungal properties and physico-chemical characters. Melanin extracts from human hair and different strains of C. neoformans (n = 4) and C. gattii (n = 4) were investigated. The following minimum inhibitory concentrations were found for different melanins against C. neoformans and C. gattii were (average/range): 13.7/(7.8-15.6) and 19.5/(15.6-31.2) μg/mL, respectively, for human melanin; 273.4/(125- > 500) and 367.2/(125.5- > 500) μg/mL for C. neoformans melanin and 125/(62.5-250) and 156.2/(62-250) μg/mL for C. gattii melanin. Using Scanning Electron Microscopy we observed that human melanin showed a compact conformation and cryptococcal melanins exposed an amorphous conformation. Infrared spectroscopy (FTIR) showed some differences in the signals related to C-C bonds of the aromatic ring of the melanin monomers. High Performance Liquid Chromatography established differences in the chromatograms of fungal melanins extracts in comparison with human and synthetic melanin, particularly in the retention time of the main compound of fungal melanin extracts and also in the presence of minor unknown compounds. On the other hand, MALDI-TOF-MS analysis showed slight differences in the spectra, specifically the presence of a minor intensity ion in synthetic and human melanin, as well as in some fungal melanin extracts. We conclude that human melanin is more active than the two fungal melanins against Cryptococcus. Although some physico-chemical differences were found, they do not explain the differences in the antifungal activity against Cryptococcus of human and cryptococcal melanins. More detailed studies on the structure should be considered to associate structure and antifungal activity.https://www.frontiersin.org/articles/10.3389/fmicb.2017.01292/ful

Detecting Lactococcus lactis prophages by Mitomycin C-mediated induction coupled to flow cytometry analysis

Most analyzed Lactococcus lactis strains are predicted to harbor one or more prophage genomes within their chromosome; however, the true extent of the inducibility and functionality of such prophages cannot easily be deduced from sequence analysis alone. Chemical treatment of lysogenic strains with Mitomycin C is known to cause induction of temperate phages, though it is not always easy to clearly identify a lysogenic strain or to measure the number of released phage particles. Here, we report the application of flow cytometry as a reliable tool for the detection and enumeration of released lactococcal prophages using the green dye SYTO-9

Microbial community composition of transiently wetted Antarctic Dry Valley soils

During the summer months, wet (hyporheic) soils associated with ephemeral streams and lake edges in the Antarctic Dry Valleys (DVs) become hotspots of biological activity and are hypothesized to be an important source of carbon and nitrogen for arid DV soils. Recent research in the DV has focused on the geochemistry and microbial ecology of lakes and arid soils, with substantially less information being available on hyporheic soils. Here, we determined the unique properties of hyporheic microbial communities, resolved their relationship to environmental parameters and compared them to archetypal arid DV soils. Generally, pH increased and chlorophyll a concentrations decreased along transects from wet to arid soils (9.0 to ~7.0 for pH and ~0.8 to ~5 μg/cm3 for chlorophyll a, respectively). Soil water content decreased to below ~3% in the arid soils. Community fingerprinting-based principle component analyses revealed that bacterial communities formed distinct clusters specific to arid and wet soils; however, eukaryotic communities that clustered together did not have similar soil moisture content nor did they group together based on sampling location. Collectively, rRNA pyrosequencing indicated a considerably higher abundance of Cyanobacteria in wet soils and a higher abundance of Acidobacterial, Actinobacterial, Deinococcus/Thermus, Bacteroidetes, Firmicutes, Gemmatimonadetes, Nitrospira, and Planctomycetes in arid soils. The two most significant differences at the genus level were Gillisia signatures present in arid soils and chloroplast signatures related to Streptophyta that were common in wet soils. Fungal dominance was observed in arid soils and Viridiplantae were more common in wet soils. This research represents an in-depth characterization of microbial communities inhabiting wet DV soils. Results indicate that the repeated wetting of hyporheic zones has a profound impact on the bacterial and eukaryotic communities inhabiting in these areas

Detection and quantification of viable airborne bacteria and fungi using solid-phase cytometry

This protocol describes the use of solid-phase cytometry for the enumeration of airborne bacteria and fungi. In contrast with conventional methods, accurate results can be obtained in real time, especially for air samples with low numbers of microorganisms. Air samples are collected by impaction on a water-soluble polymer that is subsequently dissolved. Part of the sample can be filtered over two membrane filters with different pore sizes. One filter is used to obtain a total count of all viable microorganisms, and a second filter is used to determine the number of airborne fungi. Microorganisms present on the filter are labeled with a viability substrate and subsequently detected and quantified using a solid-phase cytometer. The detected spots are microscopically validated using an epifluorescence microscope to discriminate between bacteria, fungi and fluorescent particles. The whole procedure takes 5 h to complete and results in the accurate quantification of airborne bacteria and fungi for samples with a low or high microbial load

Lactococcal 949 group phages recognize a carbohydrate receptor on the host cell surface

Lactococcal bacteriophages represent one of the leading causes of dairy fermentation failure and product inconsistencies. A new member of the lactococcal 949 phage group, named WRP3, was isolated from cheese whey from a Sicilian factory in 2011. The genome sequence of this phage was determined, and it constitutes the largest lactococcal phage genome currently known, at 130,008 bp. Detailed bioinformatic analysis of the genomic region encoding the presumed initiator complex and baseplate of WRP3 has aided in the functional assignment of several open reading frames (ORFs), particularly that for the receptor binding protein required for host recognition. Furthermore, we demonstrate that the 949 phages target cell wall phospho-polysaccharides as their receptors, accounting for the specificity of the interactions of these phages with their lactococcal hosts. Such information may ultimately aid in the identification of strains/strain blends that do not present the necessary saccharidic target for infection by these problematic phages

Chemoenzymatic Synthesis of Cryptophycin Anticancer Agents by an Ester Bond-Forming Non-ribosomal Peptide Synthetase Module

Cryptophycins (Crp) are a group of cyanobacterial depsipeptides with activity against drug-resistant tumors. Although they have been shown to be promising, further efforts are required to return these highly potent compounds to the clinic through a new generation of analogues with improved medicinal properties. Herein, we report a chemosynthetic route relying on themultifunctional enzyme CrpD-M2 that incorporates a 2-hydroxy acid moiety (unit D) into Crp analogues. CrpD-M2 is a unique nonribosomal peptide synthetase (NRPS) module comprised of condensation-adenylation-ketoreduction-thiolation (C-A-KR-T) domains. We interrogated A-domain 2-keto and 2-hydroxy acid activation and loading, and KR domain activity in the presence of NADPH and NADH. The resulting 2-hydroxy acid was elongated with three synthetic Crp chain elongation intermediate analogues through ester bond formation catalyzed by CrpD-M2 C domain. Finally, the enzyme-bound seco-Crp products were macrolactonized by the Crp thioesterase. Analysis of these sequential steps was enabled through LC-FTICR-MS of enzyme-bound intermediates and products. This novel chemoenzymatic synthesis of Crp involves four sequential catalytic steps leading to the incorporation of a 2-hydroxy acid moiety in the final chain elongation intermediate. The presented work constitutes the first example where a NRPS-embedded KR domain is employed for assembly of a fully elaborated natural product, and serves as a proof-of-principle for chemoenzymatic synthesis of new Crp analogues

Similar Microbial Communities Found on Two Distant Seafloor Basalts.

The oceanic crust forms two thirds of the Earth's surface and hosts a large phylogenetic and functional diversity of microorganisms. While advances have been made in the sedimentary realm, our understanding of the igneous rock portion as a microbial habitat has remained limited. We present the first comparative metagenomic microbial community analysis from ocean floor basalt environments at the Lō'ihi Seamount, Hawai'i, and the East Pacific Rise (EPR; 9°N). Phylogenetic analysis indicates the presence of a total of 43 bacterial and archaeal mono-phyletic groups, dominated by Alpha- and Gammaproteobacteria, as well as Thaumarchaeota. Functional gene analysis suggests that these Thaumarchaeota play an important role in ammonium oxidation on seafloor basalts. In addition to ammonium oxidation, the seafloor basalt habitat reveals a wide spectrum of other metabolic potentials, including CO2 fixation, denitrification, dissimilatory sulfate reduction, and sulfur oxidation. Basalt communities from Lō'ihi and the EPR show considerable metabolic and phylogenetic overlap down to the genus level despite geographic distance and slightly different seafloor basalt mineralogy

First human isolate of Mycobacterium madagascariense in the sputum of a patient with tracheobronchitis

Reports on the isolation and identification of unusual Mycobacteria species in humans, animals and plants have increased considerably recently due, largely, to the implementation of molecular biology methods which have higher discriminative powers than the classical phenotype-based techniques

Structure and Assembly of TP901-1 Virion Unveiled by Mutagenesis

International audienceBacteriophages of the Siphoviridae family represent the most abundant viral morphology in the biosphere, yet many molecular aspects of their virion structure, assembly and associated functions remain to be unveiled. In this study, we present a comprehensive mutational and molecular analysis of the temperate Lactococcus lactis-infecting phage TP901-1. Fourteen mutations located within the structural module of TP901-1 were created; twelve mutations were designed to prevent full length translation of putative proteins by nonsense mutations, while two additional mutations caused aberrant protein production. Electron microscopy and Western blot analysis of mutant virion preparations, as well as in vitro assembly of phage mutant combinations, revealed the essential nature of many of the corresponding gene products and provided information on their biological function(s). Based on the information obtained, we propose a functional and assembly model of the TP901-1 Siphoviridae virion

Phoenix dactylifera L. sap enhances wound healing in Wistar rats : Phytochemical and histological assessment

Acknowledgment Financial support of the Tunisian Ministry of Higher Education and Scientific Research is gratefully acknowledged.Peer reviewedPostprin