Protocols in Current Issue
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0 Q&A 233 Views Feb 5, 2024

The human pathogenic yeast Candida albicans can attach to epithelial cells or indwelling medical devices to form biofilms. These microbial communities are highly problematic in the clinic as they reduce both sensitivity to antifungal drugs and detection of fungi by the immune system. Amyloid structures are highly organized quaternary structures that play a critical role in biofilm establishment by allowing fungal cells to adhere to each other. Thus, fungal amyloids are exciting targets to develop new antifungal strategies. Thioflavin T is a specific fluorescent dye widely used to study amyloid properties of target proteins in vitro (spectrophotometry) and in vivo (epifluorescence/confocal microscopy). Notably, thioflavin T has been used to demonstrate the ability of Als5, a C. albicans adhesin, to form an amyloid fiber upon adhesion. We have developed a pipeline that allows us to study amyloid properties of target proteins using thioflavin T staining in vitro and in vivo, as well as in intact fungal biofilms. In brief, we used thioflavin T to sequentially stain (i) amyloid peptides, (ii) recombinant proteins, (iii) fungal cells treated or not with amyloid peptides, (iv) fungal amyloids enriched by cell fractionation, and (v) intact biofilms of C. albicans. Contrary to other methods, our pipeline gives a complete picture of the amyloid behavior of target proteins, from in vitro analysis to intact fungal biofilms. Using this pipeline will allow an assessment of the relevance of the in vitro results in cells and the impact of amyloids on the development and/or maintenance of fungal biofilm.

Key features

• Study of amyloid properties of fungal proteins.

• Visualization of the subcellular localization of fungal amyloid material using epifluorescence or confocal microscopy.

• Unraveling of the amyloid properties of target proteins and their physiological meaning for biofilm formation.

• Observation of the presence of amyloid structures with live-cell imaging on intact fungal biofilm using confocal microscopy.

Graphical overview

0 Q&A 362 Views Dec 5, 2023

Bio-hydrogen production is an eco-friendly alternative to commercial H2 production, taking advantage of natural systems. Microbial hydrogenases play a main role in biological mechanisms, catalyzing proton reduction to molecular hydrogen (H2) formation under ambient conditions. Direct determination is an important approach to screen bacteria with active hydrogenase and accurately quantify the amount of H2 production. Here, we present a detailed protocol for determining hydrogenase activity based on H2 production using methyl viologen (MV2+) as an artificial reductant, directly monitored by gas chromatography. Recombinant Escherichia coli is used as a hydrogenase-enriched model in this study. Even so, this protocol can be applied to determine hydrogenase activity in all biological samples.

Key features

• This protocol is optimized for a wide variety of biological samples; both purified hydrogenase (in vitro) and intracellular hydrogenase (in vivo) systems.

• Direct, quantitative, and accurate method to detect the amount of H2 by gas chromatography with reproducibility.

• Requires only 2 h to complete and allows testing various conditions simultaneously.

• Kinetic plot of H2 production allows to analyze kinetic parameters and estimate the efficiency of hydrogenase from different organisms.

Graphical overview

0 Q&A 314 Views Oct 5, 2023

Macrofungi, also known as mushrooms, can produce various bioactive compounds, including exopolysaccharides (EPS) with distinct biological properties and subsequent industrial applications in the preparation of cosmetics, pharmaceuticals, and food products. EPS are extracellular polymers with diverse chemical compositions and physical properties secreted by macrofungi in the form of capsules or biofilms into the cellular medium. Submerged cultivation is an industrially implemented biotechnological technique used to produce a wide variety of fungal metabolites, which are of economic and social importance due to their food, pharmaceutical, and agronomic applications. It is a favorable technique for cultivating fungi because it requires little space, minimal labor, and low production costs. Moreover, it allows for control over environmental variables and nutrient supply, essential for the growth of the fungus. Although this technique has been widely applied to yeasts, there is limited knowledge regarding optimal growth conditions for filamentous fungi. Filamentous fungi exhibit different behavior compared to yeast, primarily due to differences in cell morphology, reproductive forms, and the type of aggregates generated during submerged fermentation. Furthermore, various growing conditions can affect the production yield of metabolites, necessitating the development of new knowledge to scale up metabolite production from filamentous fungi. This protocol implements the following culture conditions: an inoculum of three agar discs with mycelium, agitation at 150 rpm, a temperature of 28 °C, an incubation time of 72 h, and a carbon source concentration of 40 g/L. These EPS are precipitated using polar solvents such as water, ethanol, and isopropanol and solubilized using water or alkaline solutions. This protocol details the production procedure of EPS using submerged culture; the conditions and culture medium used are described. A detailed description of the extraction is performed, from neutralization to lyophilization. The concentrations and conditions necessary for solubilization are also described.

Key features

• Production and extraction of EPS from submerged cultures of mycelial forms of macrofungi.

• Modification of the method described by Fariña et al. (2001), extending its application to submerged cultures of mycelial forms of the macrofungi.

• Determination of EPS production parameters in submerged cultures of mycelial forms of macrofungi.

• EPS solubilization using NaOH (0.1 N).

Graphical overview

0 Q&A 360 Views Aug 20, 2023

Various photoautotrophic cyanobacteria accumulate intracellular poly(3-hydroxybutyrate) (PHB) granules. This protocol can be used for determining the PHB contents of the cells as % PHB weight per dry cell weight using acid hydrolysis followed by high-performance liquid chromatography (HPLC). This HPLC analysis is rapid, with a running time of approximately 5 min per sample. The technique can accurately determine PHB concentrations in the range of 2–1,000 μg/mL PHB. However, this technique is not applicable for determining the contents of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in cyanobacteria.

0 Q&A 346 Views Jul 20, 2023

An efficient cell culture system for hepatitis B virus (HBV) is indispensable for research on viral characteristics and antiviral agents. Currently, for HBV infection assays in cell culture, HBV genome-integrated cell line–derived viruses are commonly used. However, these viruses are not suitable for the evaluation of polymorphism-dependent viral characteristics or resistant mutations against anti-viral agents. To detect the infection of cell culture–generated HBV (HBVcc) by the transient transfection of the HBV molecular clone, a large amount of purified viruses is needed, because such viruses exhibit limited infection efficiencies in cell culture. Here, we describe how to generate and purify HBVcc by the transient transfection of HBV molecular clones. This system provides a powerful tool for studying the infection and propagation of HBV and for developing anti-viral agents against HBV.

0 Q&A 260 Views Jul 5, 2023

Toxin–antitoxin (TA) systems are widespread bacterial immune systems that confer protection against various environmental stresses. TA systems have been classified into eight types (I–VIII) based on the nature and mechanism of action of the antitoxin. Type III TA systems consist of a noncoding RNA antitoxin and a protein toxin, forming a ribonucleoprotein (RNP) TA complex that plays crucial roles in phage defence in bacteria. Type III TA systems are present in the human gut microbiome and several pathogenic bacteria and, therefore, could be exploited for a novel antibacterial strategy. Due to the inherent toxicity of the toxin for E. coli, it is challenging to overexpress and purify free toxins from E. coli expression systems. Therefore, protein toxin is typically co-expressed and co-purified with antitoxin RNA as an RNP complex from E. coli for structural and biophysical studies. Here, we have optimized the co-expression and purification method for ToxIN type III TA complexes from E. coli that results in the purification of TA RNP complex and, often, free antitoxin RNA and free active toxin in quantities required for the biophysical and structural studies. This protocol can also be adapted to purify isotopically labelled (e.g., uniformly 15N- or 13C-labelled) free toxin proteins, free antitoxin RNAs, and TA RNPs, which can be studied using multidimensional nuclear magnetic resonance (NMR) spectroscopy methods.

Key features

• Detailed protocol for the large-scale purification of ToxIN type III toxin–antitoxin complexes from E. coli.

• The optimized protocol results in obtaining milligrams of TA RNP complex, free toxin, and free antitoxin RNA.

• Commercially available plasmid vectors and chemicals are used to complete the protocol in five days after obtaining the required DNA clones.

• The purified TA complex, toxin protein, and antitoxin RNA are used for biophysical experiments such as NMR, ITC, and X-ray crystallography.

Graphical overview

0 Q&A 378 Views Jun 20, 2023

Export of type 3 secretion (T3S) substrates is traditionally evaluated using trichloroacetic acid (TCA) precipitation of cultured cell supernatants followed by western blot analysis of the secreted substrates. In our lab, we have developed β-lactamase (Bla), lacking its Sec secretion signal, as a reporter for the export of flagellar proteins into the periplasm via the flagellar T3S system. Bla is normally exported into the periplasm through the SecYEG translocon. Bla must be secreted into the periplasm in order to fold into an active conformation, where it acts to cleave β-lactams (such as ampicillin) to confer ampicillin resistance (ApR) to the cell. The use of Bla as a reporter for flagellar T3S allows the relative comparison of translocation efficiency of a particular fusion protein in different genetic backgrounds. In addition, it can also be used as a positive selection for secretion.

Graphical overview

Utilization of β-lactamase (Bla) lacking its Sec secretion signal and fused to flagellar proteins to assay the secretion of exported flagellar substrates, into the periplasm, through the flagellar T3S system. A. Bla is normally transported into the periplasm space through the Sec secretion pathway, where it folds into an active conformation and allows resistance to ampicillin (ApR). B. Bla, lacking its Sec secretion signal, is fused to flagellar proteins to assay the secretion of exported flagellar proteins into the periplasm through the flagellar T3S system.

0 Q&A 844 Views Mar 20, 2023

The envelope of Gram-negative bacteria consists of an outer membrane (OM), a peptidoglycan cell wall, and an inner membrane (IM). The OM and IM have different components of proteins and lipids. Separating the IM and OM is a basic biochemical procedure to further study lipids and membrane proteins in different locations. Sucrose gradient ultracentrifugation of lysozyme/EDTA-treated total membrane is the most widely used method to separate the IM and OM of Gram-negative bacteria. However, EDTA is often harmful to protein structure and function. Here, we describe a relatively simple sucrose gradient ultracentrifugation method to separate the IM and OM of Escherichia coli. In this method, the cells are broken by a high-pressure microfluidizer, and the total cell membrane is collected by ultracentrifugation. The IM and OM are then separated on a sucrose gradient. Because EDTA is not used, this method is beneficial for subsequent membrane protein purification and functional study.

0 Q&A 764 Views Nov 5, 2022

Ubiquitination is a post-translational modification conserved across eukaryotic species. It contributes to a variety of regulatory pathways, including proteasomal degradation, DNA repair, and cellular differentiation. The ubiquitination of substrate proteins typically requires three ubiquitination enzymes: a ubiquitin-activating E1, a ubiquitin-conjugating E2, and an E3 ubiquitin ligase. Cooperation between E2s and E3s is required for substrate ubiquitination, but some ubiquitin-conjugating E2s are also able to catalyze by themselves the formation of free di-ubiquitin, independently or in cooperation with a ubiquitin E2 variant. Here, we describe a method for assessing (i) di-ubiquitin formation by an E1 together with an E2 and an E2 variant, and (ii) the cooperation of an E3 with an E1 and E2 (with or without the E2 variant). Reaction products are assessed using western blotting with one of two antibodies: the first detects all ubiquitin conjugates, while the second specifically recognizes K63-linked ubiquitin. This allows unambiguous identification of ubiquitinated species and assessment of whether K63 linkages are present. We have developed these methods for studying ubiquitination proteins of Leishmania mexicana, specifically the activities of the E2, UBC2, and the ubiquitin E2 variant UEV1, but we anticipate the assays to be applicable to other ubiquitination systems with UBC2/UEV1 orthologues.

0 Q&A 957 Views Oct 20, 2022

The human immunodeficiency virus 1 (HIV-1) consists of a viral membrane surrounding the conical capsid. The capsid is a protein container assembled from approximately 1,500 copies of the viral capsid protein (CA), functioning as a reaction and transport chamber for the viral genome after cell entry. Transmission electron microscopy (TEM) is a widely used technique for characterizing the ultrastructure of isolated viral capsids after removal of the viral membrane, which otherwise hinders negative staining of structures inside the viral particle for TEM. Here, we provide a protocol to permeabilize the membrane of HIV-1 particles using a pore-forming toxin for negative staining of capsids, which are stabilized with inositol hexakisphosphate to prevent premature capsid disassembly. This approach revealed the pleomorphic nature of capsids with a partially intact membrane surrounding them. The permeabilization strategy using pore-forming toxins can be readily applied to visualize the internal architecture of other enveloped viruses using TEM.

Graphical abstract:

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